CN1620499A

CN1620499A - Igamete recruitment and developmental competence in mammals by inhibiting the de novo sterol biosynthesis and/or promoting sterol efflux

Info

Publication number: CN1620499A
Application number: CNA028280792A
Authority: CN
Inventors: M·巴尔特森
Original assignee: Kobenhavns Universitet
Current assignee: Kobenhavns Universitet
Priority date: 2001-12-21
Filing date: 2002-12-20
Publication date: 2005-05-25
Also published as: AU2002358468A1; EP1463804A1; WO2003055991A1

Abstract

The present invention relates to methods for increasing the developmental competence of at least one mammalian germ cell, gamete, zygote, early embryo, implanted blastocyst and/or embryo by administrating a compound which is capable of inhibiting the de novo biosynthesis of sterols and thereby establishing cellular conditions that improve their development and survival. The invention also relates to methods for increasing the sterol efflux prior to fertilisation from at least one mammalian ovary, oocyte, female gamete, or ovary derived cell surrounding an oocyte by administrating a compound which is capable of promoting the sterol efflux and thereby reducing the phopholipid/sterol ratio of said cells.

Description

Mammiferous igamete recruitment and developmental potency-by suppressing sterol from the beginning biosynthesizing and/or the outflow of promotion sterol

Invention field

The present invention relates to suppress the from the beginning biosynthetic compound of sterol by using, set up thus and improve the blastocyst of at least one Mammals sexual cell, gamete, zygote, body early embryo, implantation and/or the cell condition of fetal development and existence, improve the method for their developmental potency.The present invention also relates to the compound that can promote sterol outflow by using, the sterol of the ovary derived cell around improving at least one Mammalian Ovary, ovocyte, megagamete or ovocyte prefecundation outflows, and reduces the method for the phosphatide/sterol ratio of cell thus.

Background of invention

In the past during the decade, sterile therapeutic treatment has experienced very long course, and the possibility of child-bearing is provided for infertility or sterile Mr. and Mrs by medical intervention.But, a large amount of Mr. and Mrs do not obtain any benefit from well known to a person skilled in the art in the serial infertility treatment.Thereby the chosen process of the medical practice of fertility treatment is normally made us puzzled and is expended the experience of emotion.

All are sterile about 40% owing to the women among the mankind, and 40% owing to the male sex, and in 20% case, spouse both sides have caused this problem jointly.The sterile age according to the women is variously defined.Sterile standard definition is when the women is lower than 35 years old, and the unshielded sexual intercourse of spouse can not realize gestation after 1 year.Parallel therewith, the fertility demonstration began to descend from about 34 years old age, in surpassing 42 years old women, unless use the donor ovum, otherwise almost fall to 0.Thereby, need the sterile improvement method of treatment, because average pregnant woman age is raising.

New mammiferous growth fundamentally originates in the fertilizability sexual cell that tends to participate in fertilization process, the i.e. growth of gamete.The cell of two eggcases, promptly sperm and ovocyte engage and produce zygote, by this process, can produce new individuality successively.In preparing possible prefecundation, male and female sex cell system all experiences the many variations that comprise nuclear and kytoplasm.The purpose of these variations is dual: 1) that homologous chromosomes is right chromosome number (as representative in the somatocyte) (diploid) reduces by half (monoploid), and 2) change the shape and the biochemical characteristics of the sexual cell of preparing fertilization.

The individuality of ovarian follicle and ovocyte takes place

Female sexual cell progenitor cell is so-called primary oocyte.Primary oocyte duplicates its DNA and enters first meiotic prophase.This moment, cell degradation began, and many primary oocyte lockings are also dead.But, the primary oocyte of all survivals enters first meiotic prophase, and they are at this moment by one deck epithelial bodies cell: primordial follicle surrounds separately.

During birth, human primary oocyte is prevented at meiosis prophase, and does not enter mid-term.In Mammals, primary oocyte does not continue reduction division, up to pubescence (Fig. 1).Have only a little before pubescence, the part primordial follicle just can be grown, via hormone (gonad-stimulating hormone, i.e. follicular stimulating hormone FSH and the prolan B LH) separate phases of supposition, arrive the gonad-stimulating hormone dependency stage, further growth depends on hormonal stimulation since then.After gonad-stimulating hormone dependency growth in, the ovarian follicle of most Mammalss (comprising the mankind) develops into the topping up chamber, promptly so-called follicular cavity is referred to as hole shape or Graafian follicles from ovarian follicle after this.The circulation peak of gonad-stimulating hormone once more (mainly being LH) is caused ovocyte and is continued reduction division after a while, from ovarian follicle eruption and ovulation.

The primordial follicle that has only a certain number is from tranquillization storehouse initial growth.A large amount of ovarian follicles is also dead from the experience locking between the hole growth period in early stage of primordial follicle phase.Even from this stage, most hole vesicular ovarian follicles also can experience locking and dead, causes significant ovocyte to run off.These chosen processs guarantee to get rid of the sexual cell that can not educate or grow by mistake jointly.But, regulatory mechanism has also determined only to have the ovarian follicle of certain number to realize ovulation in the ovary.The average number of the ovum of discharging is a species specificity, and relevant with the breeding strategy of discussion species, and irrelevant with the sexual cell that eliminating can not be educated or mistake is grown.The mechanism of guaranteeing the ovocyte of enough numbers it is believed that with circulate gonad-stimulating hormone level and hole before in the ovarian follicle on the somatocyte compartment number of gonadotropin receptor relevant.

In so-called follicular phase (during little hole vesicular ovarian follicles develop into preovulatory follicle), stimulate with external source gonad-stimulating hormone and to grow up femalely, cause and can regain the viable eggs parent cell that increases number from the preovulatory follicle that increases number.Remain complicated based on this clinical treatment, and success fully in many cases.

During human IVF treatment and cattle breeding, the number of the ovocyte alive of withdrawal still usually is the conceived successful limiting factor of back.In addition, hormonotherapy has obvious defects, that is, the hCG hCG that increases circulation gonad-stimulating hormone (especially LH) or functional equivalent may cause side effect to the women.

The individuality of sperm takes place

Male sexual cell progenitor cell is a spermatogonium.Prespermatogonia is surrounded by somatocyte, and in utero or after the birth is being enclosed in soon in the so-called seminiferous cord.This has represented main structural similarity in the gamogenesis cell development.Peripheral cell is referred to as sustenticular cell and exercises the function of nurse cell, is used for the growth of sexual cell, is similar to the granulosa cell that nourishes ovocyte.

Main difference female and male sex-cell system is reduction division round-robin selection of time and process.In male, the mitotic division that the diploid Prespermatogonia is divided into stem spermatogonium tranquillization storehouse and spermatogonium in some way enlivens the storehouse.The latter's division produces still can mitotic A-spermatogonium and be destined to enter maiotic B-spermatogonium.Male sex-cell is after reduction division occurs over just pubarche, but prevents the diplotene stage that is different from female sex cell system, continues after it to grow up to from single B-spermatogonium generation 4 monoploid sexual cell (Fig. 1) with preventing.Twice maiotic product is spermatocyte (1 ' and 2 '), and second spermatocyte further is divided into spermatid and is divided into the zoosperm that will be discharged in the little tube cavity of testicular spermatogenic after a while.

After being discharged in the inner chamber, sperm enters epididymis, many Biochemical processes here takes place make sperm can experience so-called capacitation, and this is that essence-ovum merges and the prerequisite of so-called acrosomal reaction between receptive period.At occurring in nature, capacitation occurs in the female road, uterus, and except that other possible factor, also relates to cytolemma cholesterol losing of environment towards periphery.

The number of male sex-cell and quality depend on many factors, comprise hormone, cell processing, environmental factor and coerce.On the principle, the number of male sex-cell is unlimited, because there is stem spermatogonium, it will produce splitted B-spermatogonium and mature sperm afterwards.Yet, in fact, nourish in the testis the sophisticated sustenticular cell of male sex-cell numerical limitations this number.

The mature sperm of low number causes male infertility and sterile, low-quality ejaculation sperm or both to have both at the same time." low sperm quality " be a generalized operational term, can cover heredity, biological chemistry and morphology composition, and or even the factor of environmental induction.

The sterol biosynthesizing

Sterol is the important component of all metazoan cytolemma.Cholesterol is the most general sterol, and the natural biological synthetic end point in the biosynthesizing of Mammals sterol.Sterol is defined as containing the material of perhydrocyclopentanophenanthrene tetra-atomic ring system, has the side chain and the C3 beta-hydroxy substituting group (Fig. 4) of carbon 17 (C17) replacement at least.In the context of the invention, the notion of sterol can be limited to the structure consistent with these structural requirements, and shows as the intermediate (for example referring to Fig. 2) from the lanosterol to the cholesterol under physiological environment.

Mammiferous sterol biosynthesizing fundamentally originates in carbon source.Acetate is the from the beginning biosynthetic precursor of cholesterol.But, this is not equal to acetate and only can be used for the biosynthetic notion of cholesterol, because there are many branches in the acetate metabolism path, the branch that wherein causes cholesterol to be produced is one of them (see figure 3).The biosynthesizing degree of any all depends on the physiology potential energy in biosynthesizing path and the regulation and control model that can encourage some ramose advantage with respect to other branch in these products.Particularly, have recognized that for many years that in the biosynthesizing of prenylation albumen, dolichol, ubiquinone, Cytoheme side chain and cholesterol, the step that the 3-hydroxy-3-methyl glutaryl is converted into mevalonic acid is a rate-limiting step.This biosynthesizing step is by 3-hydroxy-3-methyl glutaryl-coenzyme A-reductase enzyme (HMG-CoA-reductase enzyme) catalysis (Fig. 3).Acetate relates to the different enzyme of kind more than 20 with biosynthesizing path between cholesterol.The HMG-CoA-reductase enzyme occurs in early days in this path, and is sterol and other downstream factor and metabolite synthetic rate-limiting enzyme (Goldstein etc., 1990).

Sterol in the sexual cell and sterol kinetics

Sterol is biomembranous important component, and has constituted a large amount of lipid matters of all membrane structures in the cell jointly with phosphatide.Sterol is with free form, and promptly the sterol of non-covalent modification appears in the film, but is housed in the subcellular compartment with sterol-ester.In Mammals, cholesterol is dominant sterol material, and the quantitative appearance of this sterol has determined the important physical character of film.The variable effect membrane permeability of C/P ratio, film transhipment performance, cytogamy, enzymic activity and membrane fluidity in the plasma membrane.

Sperm

Mammalian sperm has unique sterol and forms, in the hamster sperm, find desmosterol and cholest--7 as in the rhesus monkey sperm, finding desmosterol, 24-diene-3 β-alcohol (Awano etc., 1989), and find that in the human sperm testis reduction division activates sterol (T-MAS) (Baltsen etc., 1998) like that.Generally speaking, cholesterol main sterol in the mammalian sperm seemingly.But, in some sperm stage of some species, the sterol of non-cholesterol may account for the sterol total amount (Awano etc., 1989) up to 90%.

Between mating season, sperm capacitation between the delivery period in uterus and uterine tube.On the physiology, acrosome reaction of spermatozoa by cytogamy before sperm combine with zona pellucida (chorion) on the ovocyte and (cumulus cell that also is the ovarian cumulus-ovocyte complex body of being discharged by ovulation recently in the uterine tube produces) progesterone induced.Sperm can make medicament that cholesterol runs off and capacitation by being exposed to external in film, and external source cholesterol and desmosterol suppress reply (Cross, 1996) of acrosome to progesterone.Sterol content or and the content of the sterol molecule of keeping in balance thereby be the key parameter of male sex-cell between the growth period.Ratio between known for a long time free cholesterol and the phosphatide changes (Davis, 1981) during capacitation.Proved that free cholesterol exercises restraining effect to capacitation, these opposite with its esterification counterpart (Davis, 1980).Confirmed operatively that cholesterol is vital from the ability that the loss of plasma membrane fertilizes an egg for sperm.In some species, be the removal of sterol in the plasmalemmae of sperms lipid bilayer before the sperm acrosome reaction.Experimentally, the sterol in the sperm is marked at sperm by significantly descend during the epididymis (Lopez etc., 1991).And the external source cholesterol suppresses the capacitation (Go etc., 1985) of mouse sperm.

Though it has been generally acknowledged that sperm can be from acetate de novo synthesis cholesterol (Gunasegaram etc., 1995), it receives the cholesterol (Cross, 1998) from sustentacular tissue.Yet this statement is thoroughly studied, and the actual sterol metabolism in the male sex-cell maturation is known little about it.The long-term diet that is rich in cholesterol of using is handled rabbit, compares with the rabbit of the control diet of feeding, and causes lower sperm count and sperm motility (Yamamoto etc., 1999).But, as if long-term man with statin treatment hypercholesterolemia does not influence plasma testosterone, total sperm count, sperm morphology or sperm motility (Bernini etc., 1998).Thereby the sterol in the uncertain reproductive tract cell is sent or whether sterol content is disturbed the influence of the systematic treating of the dynamic (dynamical) medicament of sterol.

The expression degree of some important surface protein when the sterol content that has proved sperm membrane has determined essence-ovum identification (1993a such as Benoff, b).It is relevant with the rising of pH in the increase of sperm protein tyrosine phosphorylation and the born of the same parents that cholesterol discharges also, the two acrosome is discharged and the after fertilization incident very important.Causing in the external sperm of capacitation cholesterol to discharge can be by realizing with many known material incubations that combine cholesterol.

The cholesterol total amount of each sperm may change more than 10 times between individuality, but the fertility relevant with cholesterol level seems main and the cholesterol of the latter stage of the ejaculating ability of losing is correlated with (Benhoff etc., 1993b).The absolute sterol content of sperm may be important not as the ratio between free cholesterol and phosphatide.Compare with normally educating the man, the not clear sterile man and the man of asthenospermia may have free cholesterol/phosphatide (C/PL) ratio of rising, but the clinical importance of cholesterol levels seemingly indefinite (Sugkraroek etc., 1991; Huacuja etc., 1981).Handle the human sperm with phospholipid preparations, be accompanied by the decline of sperm C/PL ratio, sperm is improved (Gamzu etc., 1987) with combining of egg vitellary membrane.

The lipid content of sperm and motility parameter correlation (Connor etc., 1997).And, C/PL ratio and motility (Hamamah etc., 1995) and sperm amalgamation negative correlation.Although C/PL is than being important for sperm quality, the absolute magnitude of sterol remains open question.

Ovocyte

Cholesterol level, cholesterol biosynthesizing and the dynamic (dynamical) any content of cholesterol in the at present unexposed relevant mammal ovocyte.This fact can be used for analyzing owing to the tissue that has only low amount probably.

Statin

Statin is competitive and reversibly suppresses the common name of the compound of HMG-CoA-reductase enzyme.When using in vivo, statin mediates the decline of serum low-density LP (LDL) and the rising of serum high-density LP (HDL) by the cholesterol output that suppresses health self.In this application, the term statin has covered by suppressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-Co-reductase enzyme) and has suppressed the from the beginning biosynthetic any compound of cholesterol.

Epidemiological study has proved the blood cholesterol levels of rising, and perhaps more specifically, the LDL-cholesterol levels reason of rising ground is relevant with the coronary heart disease danger that improves.Also there be the total cholesterol levels and LDL-cholesterol levels of reduction can reduce the pathogenetic a large amount of evidences of coronary disease.Because this effect, the further investigation in past 10 years has produced the multiple compound that can suppress the HMG-CoA-reductase enzyme.It is generally acknowledged that these compounds are safety and generally avirulent.Only there is considerably less report to relate to the harm of statin to reproduction.A relevant human toxic reports quoted as saying: in 4 months II type hyperlipoproteinemia patient of the oral 40mg of acceptance lovastatin meter every day, with compare during the independent dietary control, sperm motility has the little still decline (Hazardous substances database, SigmaAldrich Norway) of statistical significance.

Summary of the invention

The invention provides the number of the organism of improving female and male sex-cell and being produced by this class sexual cell fertilization and the simple and new method of developmental potency.As the method for a brilliance, it is realized the improvement of fertility and not have the side effect of applied hormonotherapy in the methods of great majority uses at present.In addition, the present invention also can with present used clinically in vitro method combined utilization, for example maturation in vitro (IVM) and in vitro fertilization-embryo shift (IFV-ET) technology, application that external source gonad-stimulating hormone in various degree stimulates and sexual cell cultivation.The time relevant with being used for the treatment of low birthrate or sterile ex vivo technique carries out order and describes in Figure 12.As replenishing of these ordinary methods, present method reduces possible side effect.

The present invention relates to improve Mammals gamete and embryo's number and method for quality.Described method can body in or external application, itself or suppress the from the beginning biosynthesizing of sterol of the sexual cell and the flanking cell thereof of two kinds of sexes, perhaps promote the prefecundation sterol from the outflow of female sex cell, perhaps two aspects have both at the same time.Expection can be by the process prescription of material of the present invention operation in Figure 11.Working mechanism is considered to relate to and reduces the ratio between free (non-deutero-) sterol and phosphatide in Mammals sexual cell and/or the gamete, but may also relate to other the relevant mechanism of utilization with the product of mevalonic acid and mevalonic acid back.

In one aspect, the present invention use the pharmacology scheme by the compound using or add 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme in antagonism sexual cell and the sexual cell sustentacular tissue (HMG-CoA reductase enzyme) in vivo and/or in the from the beginning biosynthesizing of vitro inhibition endogenous cholesterol.But, this use of HMG-CoA-reductase inhibitor is carried out with pulse mode (pulsate manner), and the common use when this is used for the atherosclerosis prevention with these compounds is different.And concentration height used usually when the working concentration of HMG-CoA-reductase inhibitor can prevent than atherosclerosis is quite a lot of.This treatment mediation born of the same parents internal effect is saved otherwise can not be educated or the sexual cell of fertilizability not, and makes this type of sexual cell can educate and be easy to fertilization.In fact, this treatment causes the sexual cell survival rate to raise, and promotes the sexual cell maturation.And this treatment makes sexual cell be easier to maturation and participates in the ripe back incident of influence fertilization and early embryonic development, and this is likely by influencing ratio realization between sterol and the phosphatide.The number that can be used for the sexual cell of fertilization after a while is improved thus, this success raising that causes becoming pregnant, and offspring's number increases in each female each reproductive cycle of growing up.

The present invention relates to the non-hormone pharmacological treatment in follicular phase, its ovocyte number that causes regaining increases, and the ovocyte quality is improved with regard to morphology and fertilization ability.As the technology of a brilliance, can operate method of the present invention and do not have the possible danger of side effects of conventional hormonotherapy.But, when the method for needs improvement increases the number of the sexual cell of regaining and improve the ovocyte quality with regard to fertility and fertilization ability, this new non-hormone pharmacological method also can be united conventional women IVF treatment plan and the general utilisation technology in domestic animal or the improvement of breed, uses under the condition that ex vivo technique is used.

Detailed Description Of The Invention

Aspect generalized, the present invention relates to suppress the from the beginning biosynthetic method of sterol, its effect is to reduce the sterol content of sexual cell, and might reduce the ratio that dissociates at least one Mammals sexual cell and/or the gamete between sterol and the phosphatide, improve at least one Mammals sexual cell thus, gamete, zygote, body early embryo, blastocyst, blastocyst of implanting and/or embryo's developmental potency, and/or Mammals gamete, zygote, body early embryo, the blastocyst of implanting, the number of embryo and/or fetus, described method comprise to this administration of needs can suppress the from the beginning biosynthetic compound of sterol.

In yet another aspect, the present invention relates to improve the method for the sterol outflow of the ovary derived cell around at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or the ovocyte, improve the developmental potency of described at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side thus, described method comprises uses the compound or the compound combination that can promote the sterol outflow in the Mammals of needs.

In the context of the present invention, term " sexual cell " relates to any mammalian cell that can produce gamete, and " gamete " is defined as having the ripe male or female sex cell of haploid chromosomes group, its by with the formation of can the be initial new diploid individuality of the gametogamy of relative sexuality, this term thereby comprise ovocyte (ovocyte and cumulus cell) and the isolating ovocyte that surrounds as secondary spermatocyte, spermatid, sperm, ovarian cumulus.

In the present context, term " Mammals " relates to the guiding principle of Vertebrata highest ranking, comprise all animals of bringing up its little son with the milk of mammary gland secretion, for example the mankind, horse, ox, rat, mouse, pig, sheep, goat, camel (llama), dog, cat and ermine.In preferred embodiments, the present invention relates to the mankind.In another embodiment preferred, the present invention relates to because human agricultural and pet industry and animal for example mentioned above industrial available Mammals.

Used term " zygote " is to describe by the cell of two gametes in conjunction with formation in the context of the invention, and its experience protokaryon takes place and former nuclear fusion is grown, and initial cell fission first, perhaps more broadly, is meant by individual in the gametogenic growth.That term " body early embryo " is defined as producing from zygote in the present context, up to two cells or the multicellular organisms of blastocyst stage, it is the blastocelic body early embryo stage that blastocyst is defined as having occurred the topping up chamber.Term " blastocyst of implantation " relate in the present context begun by around chorion be that zona pellucida is hatched and just begun to penetrate the endometrial blastocyst of bringing up parent.Embryo in this context is defined as Mammals from the etap that implantation process stops and growth and differentiation commitment begin, with cell fission, standard weave draft and primitive organ and tract form feature.Human individual growth when this term especially refers in implanting to finish to 8 weeks of back of becoming pregnant, this distinguishes embryo and fetus.

Growth from " sexual cell " to " fetus " has constituted the successive processes that Mammals grows in the present context, and any intermediate stage that does not allow to be not suitable for one of above-mentioned definition takes place.

Term " developmental potency " is defined as developing into the ability of specific unicellular or multicellular stage, and comprise any cytodifferentiation, growth and maturation, fertilization in the present context, and the after fertilization process that covers early embryonic development, blastocyst formation, implantation and embryo and fetal growth.

In the most preferred embodiment of method of the present invention, sexual cell is human reproduction's cell, for example ovocyte, the immature egg parent cell of regaining by ovarian follicle (pre-antral follicle) before the suction hole, by suction immature egg parent cell little or that medium sized hole vesicular ovarian follicles (antral follicle) is regained and/or cultivate at least one sperm that is in immature egg parent cell that the primordial follicle phase obtains behind the hole prematurity ovarian follicle in earlier stage, ejaculation, from prematurity sperm, spermatid or the spermatocyte of arrenotoky road withdrawal.Term " prematurity " relates to sexual cell and the gamete that can not become pregnant in its present stage under the nature situation.

Be ready to use in method of the present invention and can suppress the compounds that the from the beginning biosynthetic compound of sterol is preferably selected from antagonism 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (HMG-CoA reductase enzyme), for example with HMG-CoA competitiveness or noncompetitive ground with the HMG-CoA-reductase enzyme is reversible or irreversible fixation and reduce any material of the molar weight of the mevalonic acid that produces in every mole of HMG-CoA-reductase enzyme unit time.Can utilize one or more compounds, promptly compound can use independently, and perhaps two or more compounds can be united use.

Relevant HMG-CoA-reductase inhibitor in this context can be identified qualification by the method for testing of the microsomal fraction that relates to purifying dissolved HMG-CoA-reductase enzyme, freezing thawing dissolved HMG-CoA-reductase enzyme, obtained by Mammals sexual gland or sexual gland cell.This type of cell or subcellular fraction are referred to as " HMG-CoA-reductase enzyme goods " hereinafter.

In relating to HMG-CoA that Cf is 20 μ M, incubation medium in the experiment in vitro of high 10mg/ml " HMG-CoA-reductase enzyme goods ", be in the air balance and added cofactor NADH and the physiological buffer of NADPH and oxygen regeneration system rapidly in 37 ℃ of incubations, the molar weight of the mevalonic acid that produces in every mole of HMG-CoA-reductase enzyme unit time between steady state period is:

Control value, V _cMevalonic acid mole number/mg enzyme preparation/the second of=generation.

Make that producing Cf is this material (Cf is the concentration that freely spreads rather than be attached to the material on the macromole entity) of 100 μ M if add the material of certain density inhibition HMG-CoA-reductase enzyme under identical experiment is provided with, then the molar weight of every mole of interior mevalonic acid that produces of HMG-CoA-reductase enzyme unit time is under the stable state:

Inhibiting value, V _{I (100 μ M)}Mevalonic acid mole number/mg enzyme preparation/the second of=generation.

If V _{I (100 μ M)}≤ 0.9 * V _c

Then this material make the molar weight of the mevalonic acid that produces in every mole of HMG-CoA-reductase enzyme unit time reduce to control value 90% or lower, wherein control value is the transformation efficiency when not applying inhibitor.Suppress at least 10% condition if satisfy when adding the material of 100 μ M, this material is qualified as this contextual HMG-CoA-reductase inhibitor.

Preferably, described amount is less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%.In preferred embodiments, described compound causes 3-hydroxy-3-methylglutaryl-coenzyme A almost to descend up hill and dale to the transformation efficiency of mevalonic acid under these conditions.

Can reduce from the beginning biosynthetic other compound of sterol and can be any compound of the enzyme (Fig. 2 and 3) of biological synthesis path before suppressing to participate in originating in acetyl-CoA and ending at the sterol of lanosterol.The example of enzyme to be suppressed has Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium mevalonic acid decarboxylase, isopentenylpyrophosphate isomerase, Dimethylallyltransferase, geranyl transferring enzyme, squalene synthase, squalene monooxygenase, oxidation shark alkene lanosterol cyclase and lanosterol synthetic enzyme.

The inhibition potentiality of this type of medicine should be by them in the sexual gland tissue or tissue block of incubation, and the incubation when not existing with potential inhibitor is compared, and reduces the merit rating of the enzyme product amount in every mole of enzyme time per unit.Therefore experiment is provided with should be similar to the condition that HMG-CoA-reductase inhibitor material is tested in mentioned above being used to---and the natural substrate of the enzyme discussed except using substitutes the HMG-CoA, and wherein this substrate should use with the Cf of the Km (rice Man constant) that equals enzyme-substrate reactions.Be similar under the inhibiting condition of the above-mentioned HMG-CoA-of being used for reductase enzyme (promptly with 100 μ M concentration application of substances), the inhibition effect of described material should be at least 10%, so that qualified as the sterol synthetic inhibitor.

This type of tabulation will include but not limited to, zaragozic acid, diazonium squalene (azasqualene), diphosphonate (suberyl aminomethylene-1 for example, the two phosphonic acids of 1-, (3-(1-pyrrolidyl)-1-hydroxy propylidene-1, the two phosphonic acids of 1-) and PHPBP (3-(1-piperidino-(1-position only))-1-hydroxy propylidene-1,1-pair of phosphonic acids)) and other squalestatin (squalene synthase inhibitor), glyoxaline compound, ammonia hydroxy-diphosphonic acid salt, sodium Alendronate (alendronate) and oxysterol.

Generally speaking, the present invention relates to be applied in and to suppress from the beginning biosynthesizing and cause the content of free sterol in Mammals epididymal sperm or ovocyte and the ovarian cumulus-ovocyte complex body to descend thus of sterol during itself and the sexual gland tissue interaction, and may finally cause sterol in the sexual cell sustenticular cell/phosphatide than any compound that reduces.The present invention relates to any combination of compound and method of application, this combination will by and the preceding path of sterol (being the biosynthesizing path between acetate and the lanosterol) in the inhibition of the enzyme that relates to interact, cause that total free sterol descends in Mammals epididymal sperm or ovocyte and ovarian cumulus-ovocyte complex body.

The present invention relates to above-mentioned any can causing compares with the mammiferous situation of not accepting this type of processing, the free sterol scheme of at least 10% (as at least 5%) that descends in Mammals epididymal sperm or ovocyte and ovarian cumulus-ovocyte complex body.The present invention also relates to sterol from the beginning biosynthesizing suppress scheme and the known technology of using for propagation sexual cell in the Mammals of needs, for example IVM and IVF-ET technology, stimulate and/or any combination of other technology with external source gonad-stimulating hormone.

In the present context, term " gonad-stimulating hormone " relates to the Mammals peptide hormone that contains two peptide subunit α and β that is produced by hypophysis or placenta, and wherein the α subunit is total for them, and the β subunit has different chemical property and biological property.Hitherto known gonad-stimulating hormone comprises follicle stimulating hormone (FSH), prolan B (LH) and chorionic-gonadotropin hormone (CG).

In the present embodiment preferred of the present invention, being used to suppress the from the beginning biosynthetic compound of sterol is statin.There are several common names in statin, thereby following tabulation is not intended to limit the scope of the invention.In preferred embodiments, statin is selected from holder and cuts down his spit of fland, simvastatin, Dalvastatin (RG 12561), Fluvastatin, BAY W 62, Pravastatin, lovastatin (in the past being referred to as Mevacor or lovastatin K), 28, HR 780, Pravastatin, simvastatin, mevastatin, methyl-mevastatin, Mevastatin (in the past being referred to as mevastatin or ML-236B), ML-236A, ML-236C, the dihydro mevastatin, lovastatin J, lovastatin L, lovastatin M, lovastatin X, dihydro lovastatin L, Mevacor, the single sodium salt of 3 beta-hydroxy mevastatins acid (Pravastatin, CS-514), Simvastatin, simvastatin and nystatin/Triamcinolone Acetonide.

In the present context, can promote the compound (SBTS=sterol in conjunction with or transhipment material) of sterol outflow thus be between incubation tissue or cell stage, will cause the sterol increase to be diffused into causing in the medium described sterol outside biomaterial, to flow at first not by the saturated sterol acceptor of sterol, any material on serum albumin or other albumen, carbohydrate or the matrix material for example.

Sterol outflow can be measured by the radiolabeled condition Fu5AH rat hepatocytes of incubation oncocyte in the EagleShi minimum essential medium of the sterol combination of the deduction that is supplemented with 3mg/mL (3 ‰ w/v) human serum albumin and is concerned about and transhipment material.The condition cultivation of cell and the cell density between mark and incubation period must with (Moya etal., 1994, Arterioscler.Thromb., 14 (7): document description unanimity 1056-1065) such as Moya.If Fu5AH rat hepatocytes oncocyte is under these conditions in 37 ℃ of incubations 4 hours, behind incubation subsequently by centrifugation exhausted substratum, washed cell in new substratum (being supplemented with 3mg/mL human serum albumin's EagleShi minimum essential medium), and in these two fractions, measure radioactivity, then:

Radioactivity=RA in the cell _Cell

Radioactivity=RA in the substratum _Substratum

RA wherein _CellRepresent the gross activity in the cell fraction, and RA _SubstratumRepresented the gross activity that comprises from the substratum fraction of the substratum of 3 continuous washing.If there is not other material except that the 3mg/mL serum albumin in cultivating, then:

Radioactivity in the cell (standard)=RA _Cell ^Standard

Radioactivity in the substratum (standard)=RA _Substratum ^Standard

If add SBTS, then:

Radioactivity in the cell (SBTS)=RA _Cell ^SBTS

Radioactivity in the substratum (SBTS)=RA _Substratum ^SBTS

If material is qualified as the SBTS in this context, then at 37 ℃ of incubations after 4 hours, from cell ³H-cholesterol fraction discharges and improves 50% or more, that is:

(RA _Substratum ^SBTS)/(RA _Cell ^SBTS) 〉=150% * (RA _Substratum ^Standard)/(RA _Cell ^Standard)

The SBTS amount of adding in substratum in preferred embodiments, causes the osmolarity of substratum to increase 0.001-10%.The SBTS amount of adding in substratum in another preferred embodiment, is in the scope of 0.001-10mM.The SBTS amount of adding in substratum again in another preferred embodiment, is in the scope of 0.01-50 ‰ (w/w), such as but not limited to 0.5-4 ‰.

The interesting compound that can promote the sterol outflow can be a cyclodextrin, the cyclodextrin of chemically modified, such as but not limited to sulfated cyclodextrin, high-density lipoprotein (HDL) (HDL), from the apoprotein of HDL, sterol carrier protein I and II or can dissolve any other albumen of sterol.

In one embodiment, can promote the compound of sterol outflow to be selected from cyclodextrin, the cyclodextrin of chemically modified, such as but not limited to sulfated cyclodextrin, high-density lipoprotein (HDL) (HDL), from the apoprotein of HDL, and sterol carrier protein I and II.

In the most preferred embodiment of the present invention, can promote that the compound of sterol outflow is a cyclodextrin.

In the present context, term " cyclodextrin " relates to one group of ring-type dextran homologue that the glucose unit by α-1,4 bonding that the effect of starch or similar substrate obtained by cyclodextrin glucanotrasferase enzyme constitutes.The material of cyclodextrin and broad variety forms bag and replies zoarium by letter, thus make otherwise in water or damping fluid insoluble or low molten material molten altogether.Structurally there is Glucopyranose units purpose difference each other in cyclodextrin.The mother body cyclodextrin that contains 6,7 and 8 Glucopyranose units is called as alpha (α), beta (β) and gamma (γ) cyclodextrin respectively.As an example, Figure 13 has shown the universal architecture (R=H) of sulfated cyclodextrin.If R=CH2CH (OH) is CH3, then this structure is represented hereinafter used hydroxypropyl-beta-cyclodextrin among the embodiment 8.

Should maybe can suppress to the statin of the administration of needs sterol from the beginning the amount of biosynthetic compound depend on selected specific statin and/or compound, and between every day every kg body weight 0.01-100mg, for example every day every kg body weight 0.05-90mg, every day every kg body weight 0.1-80mg, every day every kg body weight 1-50mg, every day every kg body weight 1-25mg, every day every kg body weight 1-10mg, every day every kg body weight 1.0-10mg, every day every kg body weight 2.0-10mg, every day every kg body weight 2.5-10mg, every day every kg body weight 3.0-10mg, every day every kg body weight 4.0-10mg, every day every kg body weight 5.0-10mg, every day every kg body weight 6.0-10mg, every day every kg body weight 7.0-10mg, every day every kg body weight 8.0-10mg, between every day every kg body weight 9.0-10mg or every day every kg body weight 1.0-9.0mg, every day every kg body weight 2.0-8.0mg, every day every kg body weight 2.5-7.5mg, every day every kg body weight 4.0-6.0mg or every day every kg body weight 4.5-5.5mg.

Should depend on selected specific compound to the amount of the compound that can promote the sterol outflow of the administration of needs, but may be between every day every kg body weight 0.01-1000mg, for example every day every kg body weight 0.5-900mg, every day every kg body weight 1-800mg, every day every kg body weight 10-500mg, every day every kg body weight 10-250mg, every day every kg body weight 10-100mg, every day every kg body weight 20-100mg, every day every kg body weight 25-100mg, every day every kg body weight 30-100mg, every day every kg body weight 40-100mg, every day every kg body weight 50-100mg, every day every kg body weight 600-100mg, every day every kg body weight 70-100mg, every day every kg body weight 80-100mg, between every day every kg body weight 90-100mg or every day every kg body weight 10-90mg, every day every kg body weight 20-80mg, every day every kg body weight 25-75mg, every day every kg body weight 40-60mg or every day every kg body weight 45-55mg.

The reproduction state of female mammal is a round-robin, and complex interactions causes ovulation process between hypothalamus, prepituitary gland and the ovary.In the mankind, this circulation is to be (scope 21-35 days) repetition about 28 days average period.Menstrual period fs continues 3-5 days.Human round-robin first day was exactly first day of described fs, first day of bleeding of menstruation just.Human ovarian's second follicular phase is corresponding to endometrial proliferative phase, and 5-16 days at least (being alterable height).Then being the onset of ovulation (36 hours), is luteal phase at last, and it is corresponding to the endometrial secretory phase, and more constant usually, is about 14 days.In the present context, ovulation constantly is exactly the time of ovum from ovary release.

Be necessary to use in each ovulatory cycle, calculate the period of treatment plan of the present invention.Ovulatory cycle is different between each species of Mammals, and therefore the best period that need use should preferably be calculated according to its ovulatory cycle with regard to each species.In several Mammalss, the time length of ovulatory cycle is highly different with the human cycle.The multiple mammiferous average ovulatory cycle time length provides in following table 1:

Table 1

Mammals	The people	Horse	Ox	Goat	Pig	Sheep	Hamster	Mouse	Rat	Rabbit	Ermine	Dog	Cat
Mammals	The people	Horse	Ox	Goat	Pig	Sheep	Hamster	Mouse	Rat	Rabbit	Ermine	Dog	Cat	Mean length (fate)	28	21	21	20	21	16	4	5	5	16	8	15	18

The method according to this invention, use to the jenny of needs and can suppress from the beginning biosynthesizing and/or promote the time of the compound of sterol outflow during preceding 0 hour-12 days, for example to ovulate preceding 0 hour-11 days, 0 hour-10 days, 0 hour-9 days, 0 hour-8 days, 0 hour-7 days, 0 hour-6 days, 0 hour-5 days, 0 hour-4 days, 0 hour-3 days, 0 hour-2 days or 0 hour-24 hours of sterol in ovulation.

Can suppress from the beginning biosynthesizing and/or promote the using of compound of sterol outflow to be no more than 3 days after can being extended down to ovulation of sterol, for example ovulate back 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours or 72 hours.

Important aspect of the present invention relates to pharmaceutical composition, and it comprises can suppress from the beginning biosynthetic compound of sterol and the combination of compounds that can improve sterol outflow, with pharmaceutically acceptable carrier randomly.

In a particularly preferred embodiment according to the invention, because having two various process of influence, pair cell sterol content takes place simultaneously in time, so can obtain the synergistic effect of statin and cyclodextrin, the effect of generation surpasses that two schemes use separately adding and, preferably at least 3 times.

The preferred present composition is the combination that comprises statin and cyclodextrin.In another embodiment, the present invention relates to statin and cyclodextrin are used to prepare together with pharmaceutically acceptable carrier the medicine of the mammiferous fertility of raising needs.

Somatomedin just is used for clinical for a long time, thereby in preferred embodiments, the present invention relates to further comprise the composition that contains statin and cyclodextrin of somatomedin.This type of somatomedin can be gonad-stimulating hormone (FSH, LH, CG), rhIGF-1 (IGF), Urogastron (EGF), Regular Insulin, tethelin, interleukin or other peptide hormone.

In preferred embodiments, somatomedin is selected from gonad-stimulating hormone (FSH, LH, CG), rhIGF-1 (IGF), Urogastron (EGF), Regular Insulin, tethelin, interleukin and other peptide hormone.

Another important aspect of the present invention relates to pharmaceutical composition, and it comprises the combination that can suppress from the beginning biosynthetic compound of sterol and somatomedin.In preferred embodiments, described compound is a statin.

Again another important aspect, the present invention relates to pharmaceutical composition, it comprises the compound that can promote the sterol outflow and the combination of somatomedin.In preferred embodiments, described compound is a cyclodextrin.

In the preferred embodiment of the invention, one day to administration compound and/or arbitrary composition mentioned above 1-10 time, for example one day 1 time, one day 2 times, one day 3 times, one day 4 times, one day 5 times, one day 6 times, one day 7 times, one day 8 times, one day 9 times or one day 10 times.

Compound and/or composition also can extremely be used for 1 time several times, perhaps use continuously by organizing pumping installation in this section period.Compound and/or composition can be used 1 time to several times by organizing pumping installation continuously in this section period.Compound and/or composition can be used with the dosage form that can delay to discharge in its body.This type of preparation makes can be in period the last period of aforementioned stages such as 0-30 days administered compounds and/or composition, and realizes the tissue exposure by sustained release profile in vivo test on the very identical time period.This type of use with by this to the growth of sexual cell produce to cause body immediately in the similar desired effects of application program that discharges.

The present invention also relates to arbitrary composition mentioned above is used for preparing the pharmaceutical composition that the arbitrary method of the present invention is used.

Ovum as well known to those skilled in the art is the ovum that experience karyomit(e) reduces thereby can prepare to be fertilized, and the form that it takes big relatively non-motility gamete provides most of kytoplasm of zygote.Even these ovum discharge with form ripe and that prepare to be fertilized, still may lack the ability of finishing fertilization process to a certain extent, this can suppress the from the beginning biosynthetic compound of sterol by interpolation and get help.

In an embodiment, prove Mevacor ^(by suppressing the statin class medicament production that the from the beginning biosynthetic crucial regulation and control step HMG-CoA-reductase enzyme of cholesterol works) causes serum cholesterol to descend, and, influence organ and resemble free content of cholesterol (embodiment 3) in liver and the ovary as proving here.Corresponding therewith, ovary is replied and is produced a large amount of fertilizable ovocytes and/or improve the developmental potency of these dominant discharge ovum---this by with normal (being untreated) male mice post-coitum uterine tube in the increase of 2-cell number be confirmed (embodiment 1).

By embodiment 4 proofs, it shows to substratum adds statin (mevastatin, Sigma) reduction division maturation in the body of mediation ovocyte to statin to the sophisticated improvement of reduction division.As and if this effect can add up with the ripe effect of the reduction division of known gonad-stimulating hormone (FSH herein).

Comprised the from the beginning metabolite data of dependency between the biosynthesizing of proof oocyte maturation and cholesterol herein.Replace Mevacor at the external use mevastatin ^Be necessary, because Mevacor ^, lovastatin activeconstituents activity is only just arranged after the bio-transformation in liver.Compare with more natural ovulation initiator hCG, statin confirms by embodiment 3 enhancement that cholesterol reduces.The oocyte of mouse of hormone induction grow and between the onset of ovulation the overall decline of free cholesterol be confirmed by the data that embodiment 5 provides.

Statin shows by embodiment 7 improvement of fertilization, and (mevastatin Sigma) improves the sperm fertilization of ovocyte to add statin when its proof is used IVF in the IVM substratum.Prove and contrasted incubation and compared the oocyte number purpose raising of being fertilized behind the existence of the mevastatin mediation IVF during the IVM with the incubation of FSH.The ratio of the ovocyte of degenerating keeps identical (Fig. 8).Surprisingly, compare with adding FSH, statin has the enhanced effect.In Fig. 9, the result shows with above-mentioned two groups that are used for comparison and compares that the cell of handling with Mevacor arrives more progressive state after cultivating in 72 hours behind the IVF.Degeneration ratio after cultivating in 72 hours in the Mevacor treatment group is compared low with above-mentioned two groups that are used for comparison, this may conform in the fact that shows sightless height cumulus expansion in vitro during microscope inspection behind the IVM with the ovocyte of cultivating when having Mevacor.Cumulus expansion in vitro is a prerequisite for suitable oocyte maturation and fertilization preparation.

With the hydroxypropyl-beta-cyclodextrin is example, and sterol combination and transhipment material prove by embodiment 8 that to the improvement of fertilization it shows that applying hydroxypropyl-beta-cyclodextrin in the IVM substratum improves the sperm fertilization of ovocyte when using IVF, as shown in figure 10.Ovocyte degeneration ratio is not subjected to the influence of the cyclodextrin in this scope.

Background of the present invention is that relevant free sterol is the existing knowledge of the final correct sophisticated prerequisite of mammalian sperm from the loss of plasma membrane.But, the present invention is based on three new main thoughts.

1) decline of film sterol is the Biochemical processes that occur in ovocyte final prefecundation of ripening period equally.Still do not have so far relevant ovary or ovocyte prefecundation the run off understanding of sterol be published in scientific literature or other places.

2) loss of sterol and/or with born of the same parents in sterol content reduce relevant process, can by apply sterol from the beginning the inhibitor in biosynthesizing path in the sexual cell of two kinds of sexes, strengthened.This inhibition can not cause sexual cell or sexual cell environment, and for example lipoprotein is taken in or sterol compensation is completely carried out in the hydrolysis of raising sterol ester by improving.This is likely because produce near the original position under natural condition of most sterol in the sexual cell environment before the fertilization.Sterol biosynthesizing restraining effect will cause the remarkable decline of film sterol content, and this natural sterol that can replace hormonal stimulation runs off.And natural sterol runs off and will be strengthened by the from the beginning biosynthetic inhibition of sterol.

3) from the beginning biosynthetic inhibition not only mediates important ripening process itself as the reduction division maturation to sterol, and is a kind of mechanism that also mediates the committed step of sexual cell growth except that the mediation capacitation.And the film sterol runs off and mediates important ripening process.

The application provides and has utilized statin with the negative experiment of handling the free sterol levels in ovocyte and the ovocyte environment.Relevant with the present invention experimental results show that strengthening natural sterol during final oocyte maturation runs off and cause the raising of ovocyte viability and fertility, the locking that can become thereby this is likely by saving many realizes at hormonal dependent " edge " ovocyte that runs off vegetative period.And, can consider in the male mice body, to give statin and cause from the beginning biosynthesizing decline and/or phosphatide/sterol ratio decline of sterol in the epididymis swarm cell (eupyrene sperm).

The present invention proves that (Fig. 5) handles the preadolescence female mice than using the hormonal stimulation same mouse separately with hormonal stimulation and statin, produces many 2-cells of average about 70%.We think by adjustment statin dosage and the processing cycle can obtain even higher per-cent.This example proof applies the developmental potency that statin can improve its gamete to female mammal.

The present invention's proof can be used statin in the body during the follicular phase of follicle maturity, reach the effect that strengthens ripe incident.This effect depends on probably with the from the beginning biosynthetic animal of sterol is taken place and compares, the decline of total free sterol content in the sexual cell, and this causes the decline of sterol and phosphatide ratio in the plasma membrane, thereby makes sexual cell be more prone to participate in fertilization process.And external oocyte maturation and fertilization also will have benefited from suppressing the from the beginning biosynthetic processing of sterol, cause the raising and the sophisticated enhancing of postmeiotic of reduction division speed.

In the known sperm body or the sterol loss process during the external capacitation never with born of the same parents in the sterol de novo synthesis connect.Cholesterol is present in cell interior and the outer membrane, and according to the character of born of the same parents' external environment, the stable loss of cholesterol to extracellular space takes place in all cells.And cholesterol is used as the substrate of synthesis example such as bile acide and steroid.On the other hand, cell obtains free cholesterol from inner cholesterol-ester storage, outside lipoprotein source and de novo synthesis.Suppress one of these processes and cause one of other process to strengthen usually, to obtain constant cytolemma and organoid sterol levels.

These processes are guaranteed the stable state of cholesterol in the cell jointly, and the single cholesterol delivery process of unimaginable inhibition can change the overview image of cholesterol stable state and depend on the process that cholesterol is utilized.For example, as the embodiment of the invention 3 was illustrated, the steroid production of carrying out in the female reproduction gland was not subjected to block fully the influence that from the beginning sterol is produced.Therefore, but same expectability, and blocking-up sterol de novo synthesis path will be improved cholesterol to the contribution of stable state and be taken in (lipoprotein picked-up and free cholesterol discharge) and the interior mobilization of born of the same parents (cholesterol-ester hydrolysis).Yet present embodiment 3 and 6 proof results in the active cell fraction of ovary and epididymis (the storage position of male mature sperm) are the decline of every weight unit or phosphatide unit.Embodiment 1 and this sterol of 9 proofs descend and are accompanied by the improvement of sexual cell growth and/or quality.

On the other hand, even can expect blocking sterol from the beginning biosynthesizing will cause sterol in other Biochemical processes to use descending, cause that the output as steroid descends, but owing to this can intervene the steroid output and depend on the sexual cell ripening process of steroid, so will seriously undermine the potential therepic use of the inventive method aspect the raising fertility.Present method proved pharmacology check sterol from the beginning biosynthesizing can cause the female sex cell quality to improve this surprising discovery.This scheme does not influence steroid negatively and generates, and just depends on the process that cholesterol is used.Thereby sterol uses in two fens modes in female sex cell and/or sexual cell environment, this makes that cell can be by reducing the sterol output in a cell and/or the biological chemistry compartment, and it is unaffected and make should in the use of sterol and/or aspect existing to keep another compartment at the same time.

The present invention relates to utilize known generalization compound to generate and improve the Mammals fertility by suppressing from the beginning sterol.Cause that the potential mechanism that this verified fertility is improved may be the adjustment of sterol/phosphatide ratio.The present invention proof by pharmacological intervention reduce the Mammals sterol from the beginning biosynthesizing can produce the female sex cell of developmental potency with improvement.This sterol reduces mechanism and is being guided out aspect the opposite sex: male sex-cell also will have benefited from being based upon the scheme on the progamous sterol restraining effect.

The sex time of gestation that causes as well known to those skilled in the art is chosen between different plant species and changes.Therefore the present invention relates to Mammals and produce the inconvenient situation of offspring, for example female mammal is in the unshielded sexual intercourse that surpasses a day, for example 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 30 days, January, February, March, April, May, June, July, August, September, October, November, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, 20 years or more than the situation that can not realize gestation after the unshielded sexual intercourse in 60 years.

The selection of time of unshielded sexual intercourse mentioned above is known relevant for the period that the purpose of becoming pregnant has sexual intercourse regularly with Mammals.

Since the invention discloses new and surprising inhibition sterol from the beginning biosynthesizing can improve the sophisticated observations of Mammals sexual cell, thereby the present invention also relates to not have sterile hardship but only wish to obtain the higher chance of becoming pregnant and quicken the Mammals of becoming pregnant, particularly its level of becoming pregnant was with proportional decline of age from 28 to 42 years old human women the age.The present invention also relates to benefit from the human women that this treatment obtains twins or triplets.Generally speaking, thus present method relates to can benefit from domestic animal and other the valuable economically Mammals that number that this each gestation produces fetus improves.

Sterile focus is certainly not only only based on female.In the present invention, sophisticated influence supposition also can be reflected on the male sex-cell to female sex cell.Thereby, in another embodiment, the present invention relates to suppress the sterol de novo synthesis at least one Mammals sexual cell and/or the gamete, improve at least one Mammals gamete thus, zygote, body early embryo, blastocyst of implanting and/or embryo's developmental potency and/or Mammals gamete, zygote, body early embryo, the method of blastocyst of implanting and/or embryo's number, this method is included in ejaculation can suppress the from the beginning biosynthetic compound of sterol to male using in preceding 0 hour-12 days, for example ejaculated preceding 0 hour-11 days, 0 hour-10 days, 0 hour-9 days, 0 hour-8 days, 0 hour-7 days, 0 hour-6 days, 0 hour-5 days, 0 hour-4 days, 0 hour-3 days, 0 hour-2 days and 0 hour-24 hours.The term ejaculation relates in the present context from male deferential any seminal fluid release.

An importance of the present invention relates to number and the developmental potency at least one Mammals sexual cell of external raising and/or gamete, improve the method for the blastocyst of the blastocyst of the fertility of at least one Mammals gamete and/or gained zygote, body early embryo, implantation and/or embryo's developmental potency and/or Mammals gamete, zygote, body early embryo, implantation and/or embryo's number thus, this method comprises adding in substratum and can suppress the from the beginning biosynthetic material of sterol in the cell.

Another aspect of the present invention relates to the sterol outflow that improves at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side, improve the method for the developmental potency of described at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side thus, described method comprises compound or the compound combination of adding the sterol outflow that can promote any above-mentioned cell or tissue in substratum.

The purpose of the preferred embodiment of the invention is to provide various cell culture mediums, its comprise can by improve the sterol outflow and/or suppress sterol from the beginning biosynthesizing reduce the multiple different compounds of phosphatide/sterol ratio.Thereby in a preferred embodiment, the present invention relates to comprise the cell culture medium of cyclodextrin.In another preferred embodiment, the present invention relates to comprise the cell culture medium of statin.In the most preferred embodiment, the present invention relates to comprise the cell culture medium of statin and cyclodextrin.

There is multiple commercially available substratum, it can support gamete to breathe and/or maturation, and also can cultivate the blastocyst and/or the embryo of mammalian zygote, body early embryo, implantation, such as but not limited to Ham ' s F10, Menezo B2, Medi-Cult Universal IVF substratum, Dulbecco ' s improvement eagle substratum (DMEM), McCoyShi 5A, Waymouth ' s substratum, TCM-199, MEM, RPMI-1640 and Leibovitz L-15.

In the present context, in-vitro culture medium is defined as supporting at least one ovocyte individually or in its somatocyte environment and/or at least one sperm is breathed and/or sophisticated medium, and described medium can comprise to be supported (IVF) scheme in vitro fertilization and/or support outer survival of gametophyte and sophisticated cell culture medium.Can be for being used for (IVF) scheme in vitro fertilization and/or supporting gamete survival and sophisticated substratum to set up a plurality of parameters to limit described substratum.The content of the pH when first and foremost, carrying out culture medium culturing, osmolarity and buffer air has high importance.Thereby in the present context, in-vitro culture medium preferably has osmolarity between 240 to 320mOsm/l, 7 to 7.8 pH and at 3-7%CO ₂Atmosphere or 53-7%CO2,1-5%O ₂And 88-96%N ₂Down by CO ₂Buffering.

Osmolarity between 200 to 370mOsm/l can relate more specifically to for example 250 to 310mOsm/l, 260 to 300mOsm/l, 270 to 290mOsm/l, 275 to 285mOsm/l, 276 to 284mOsm/l, 277 to 283mOsm/l, 278 to 282mOsm/l or 279 to 281mOsm/l level.In present embodiment preferred, osmolarity is about 280mOsm/l.

In the present context, the pH between 7 to 7.8 for example can relate more specifically to 7.1 to 7.7,7.2 to 7.6,7.3 to 7.5,7.35 to 7.45,7.36 to 7.44,7.37 to 7.43,7.38 to 7.42 7.39 to 7.41 pH levels for example.In preferred embodiments, pH is about 7.4.

Be not intended to limit the scope of the invention, other general feature of IVF-substratum can be that described substratum comprises 111 to 171mEq/l Na ⁺, 2.1 to 6.1mEq/l K ⁺, 96 to 146mEq/l Cl ^-, 22.6 to 26.6mEq/l HCO ₃ ^-, 0.3 to 1.2mM Ca ⁺⁺, 29 to 69mg/l phosphorus, 0.56 to 1.56g/l glucose, 0-50000IU/l penicillin, 0-50000IU/l Streptomycin sulphate, 0.1 to 10.0g/l albumin, 0-75IU/l FSH, 0-250IU/l LH and 0-6g/l total protein.Other possible additive comprises for example for example tocopherol and/or xitix, hormone, Mg of maternal serum, antioxidant of foetal calf serum, human serum ⁺⁺, pyruvate salt, lactic acid salt, phenol red, purine, pyrimidine, amino acid and cholesterol.Usually, substratum is cultivated at 37 ℃ (+/-1 ℃).

In the preferred embodiment of the invention, cell culture medium relates to the cell culture medium that chemical ingredients is determined.Term " substratum that chemical ingredients is determined " is used in reference to the substratum of the serum material that does not have the biology extraction, and wherein all components is known and description is arranged with its concentration.In substratum, add hormone or serum source material if desired, preferred recombinant hormone or serum source material.In an alternative embodiment, substratum contains BSA or the HSA that obtains by recombination method, thus eliminated with mammalian blood serum between contact.In highly preferred embodiment, the human gamete of prematurity is cultivated in the substratum that chemical ingredients is determined and is not added the product of serum direct sources or patient's self serum or any other is directed to the serum product of Mammals (for example mankind or ox).

Somatomedin is used for the IVM treatment at present such as but not limited to gonad-stimulating hormone.Thereby, the preferred embodiment of above-described any substratum further relates to the cell culture medium that also comprises at least a somatomedin, wherein said somatomedin be selected from gonad-stimulating hormone (FSH, LH, CG), IGF, EGF, Regular Insulin, tethelin, interleukin or other peptide.

Method of the present invention will preferably originate in prematurity or not full ripe megagamete.In the women, the immature egg parent cell can be used as have tight ovarian cumulus material, the ovocyte of no polar body or visible germinal vesicle and being identified.The personal identification that these ovocytes are participated in conventional IVF-treatment easily is the immature egg parent cell, thereby in embodiment, the present invention relates to make the sophisticated method of immature egg parent cell that obtains from the primordial follicle stage to the hole last stage, be included in and cultivate described immature egg parent cell in the cell culture medium as indicated above.

In preferred embodiments, the amount that applies statin in in-vitro culture medium depends on specific statin, and is 0.01-1000 μ M, 0.05-1000 μ M, 0.1-1000 μ M, 0.5-1000 μ M, 1-1000 μ M, 5-1000 μ M, 10-1000 μ M, 20-1000 μ M, 25-1000 μ M, 30-1000 μ M, 50-1000 μ M, 75-1000 μ M, 100-1000 μ M, 200-1000 μ M, 300-1000 μ M, 400-1000 μ M, 500-1000 μ M, between 750-1000 μ M or the 10-900 μ M, 25-750 μ M, 50-500 μ M, 100-400 μ M, 200-300 μ M, 0.01-20 μ M, 0.01-15 μ M, 0.05-15 μ M, 0.1-10 μ M or 1-10 μ M.

Can optimize the cultivation of at least one ovocyte in in-vitro culture medium of carrying out according to the inventive method in the following way, to suppress from the beginning biosynthesizing and/or promote the sterol outflow and/or reduce ratio between free sterol and the phosphatide of sterol: in in-vitro culture medium, cultivate independent or be at least one ovocyte 1 second to 15 days in its somatocyte environment, for example 1 second to 14 days, 1 second to 13 days, 1 second to 12 days, 1 second to 10 days, 1 second to 9 days, 1 second to 8 days, 1 second to 7 days, 1 second to 6 days, 1 second to 5 days, 1 second to 4 days, 1 second to 3 days, 1 second to 2 days, 1 second to 1 day, 1 second to 23 hours, 1 second to 22 hours, 1 second to 21 hours, 1 second to 21 hours, 1 second to 20 hours, 1 second to 19 hours, 1 second to 18 hours, 1 second to 15 hours, 1 second to 12 hours, 1 second to 11 hours, 1 second to 10 hours, for example 1 second to 5 hours.

The sperm that the method according to this invention is carried out in in-vitro culture medium is cultivated can be by cultivating sperm 1 second to 8 days in in-vitro culture medium, for example 1 second to 7 days, 1 second to 6 days, 1 second to 6 days, 1 second to 4 days, 1 second to 3 days, 1 second to 2 days, 1 second to 1 day, 1 second to 23 hours, 1 second to 22 hours, 1 second to 21 hours, 1 second to 21 hours, 1 second to 20 hours, 1 second to 19 hours, 1 second to 18 hours, 1 second to 15 hours, 1 second to 12 hours, 1 second to 11 hours, 1 second to 10 hours, for example 1 second to 5 hours and optimize, to suppress from the beginning biosynthesizing and/or promote the sterol outflow of sterol.

The IVF treatment generally comprises implements hormonotherapy to the female mammal of needs treatment.Though hormonotherapy has many side effects really, it remains one of current sterile the most effective methods of treatment.The standard I VF treatment of infertile couples generally includes with exogenous hormone treatment back and collects the mature egg sample from the women.This scheme means that usually the women handles with gonadoliberin agonist or antagonist, and the degree that they all cause hypophysis to discharge gonad-stimulating hormone substantially descends.Provide external source gonad-stimulating hormone by intramuscular or peritoneal injection subsequently, it guarantees many ovocyte growths and ripe.Theory of this type of treatment back is that the circulate exact amount of gonad-stimulating hormone of women can regulate and control arbitrarily, the result optimizes to can be used for the ovocyte number of being fertilized subsequently, and under any circumstance this number will be above the ovocyte number of preparing fertilization among treatment (spontaneous circulation) women not (usually only one).In common solution, this treatment is included in the gonad-stimulating hormone initiation ovum of using and obtained ovocyte by suction from the women before ovary is discharged into uterine tube.

Use the compound that can suppress the from the beginning biosynthetic compound of sterol and/or can promote the sterol outflow with other hormons of gonad-stimulating hormone or the IVF of being used for as well known to those skilled in the art treatment, this that add up and collaborative possibly effect is the preferred embodiment of the invention.

In female mammal, can suppress the from the beginning biosynthetic compound of sterol and/or can promote the interior therapeutic of the compound of sterol outflow to unite external source gonad-stimulating hormone, the interior therapeutic of gonadotropin releasing hormone (gnrh) or gnrh agonist and/or antagonist, accumulation/synergistic effect is provided, compare with standard I VF treatment, improved and prepared in the body or the number of mature egg in vitro fertilization, and/or the number of the ovum of being fertilized in the total ovum, and/or the number of blastocyst in the ovum of fertilization, and/or implant the number of blastocyst and/or the number of the filial generation of sending in each reproductive cycle in the growth blastocyst.

And, use the from the beginning amount of the external source gonad-stimulating hormone of ovum, blastocyst, implantation and/or the gestation subsequently of biosynthesis inhibitor and/or the mature oocyte that can promote the compound of sterol outflow may cause saving to be used to obtain certain number, fertilization of sterol to the women in suction last stage of hormonal stimulation.This hormone scheme of saving will become the alternative replacement scheme of utilizing a kind of side effect reduction and the expense reduction of exogenous hormone separately, and this has constituted another essential scope of the present invention.Ovary stimulation oversaturation syndromes (OHSS) and polycystic ovary syndrome (PCO) are arranged in the side effect of not expecting.Utilize statin will cause the minimizing of gonad-stimulating hormone dosage used in the body and the reduction of these side effect incidences subsequently during stimulating ovary in vivo as auxiliary agent.

In the present context, term " gonadotropin releasing hormone " or " gnrh " relate to by the stimulation hypophysis of hypothalamus generation or the endogenous Mammals peptide hormone of other organ or tissue release gonad-stimulating hormone.Other title of this parahormone comprises luteinizing hormone releasing factorl (LRF) and luteotropic hormone releasing hormone.

In the present context, term " GuRH-A " relates to any material that the gonadotropin-releasing hormone receptor that stimulates Mammals hypophysis or other places produces the second messenger, and described second messenger participates in normally that the gnrh-acceptor combines with gnrh and gonad-stimulating hormone generation and/or gonad-stimulating hormone conduct from the signal between the release in hypophysis or other places.

In the present context, term " antagonists of gonadotropin-releasing hormone " relates to and suppresses gnrh and gnrh-receptors bind and/or suppress normally to participate in the conduction of gnrh-bonded signal, is contacting any material that the back produced and/or discharged gonad-stimulating hormone with gnrh thereby hinder the cell that carries the gnrh-acceptor.

Standard I VF treatment comprises malely sends the seminal fluid sample by masturbation or electro-ejaculation.Before acquisition is used for the seminal fluid sample of IVF treatment, use and to suppress the from the beginning biosynthetic compound of sterol and/or can promote that the synergy of the compound of sterol outflow is the preferred embodiment of the invention equally.In boar, the from the beginning biosynthetic compound of sterol can be suppressed and/or combination therapy in the body of compound of sterol outflow can be promoted, the accumulation synergistic effect is provided, compare with standard I VF treatment, improved and prepared in the body or the number of mature sperm in vitro fertilization, and/or the number of the ovum of being fertilized in the total ovum, and/or the number of blastocyst in the ovum of fertilization, and/or implant the number of blastocyst and/or the number of the filial generation of sending in each reproductive cycle in the growth blastocyst.

The notion of IVF treatment is also included within behind male or female withdrawal sexual cell, cultivates these sexual cell under many sperms and at least one ovocyte blended felicity condition.Another embodiment preferred of the present invention is to utilize from the beginning biosynthesis inhibitor and/or can promote the compound of sterol outflow to add in the substratum of incubation ovum and/or sperm before mixing sperm and ovum and add in the substratum of incubation sperm and ovum of sterol, so that obtain the ovum of fertilization.This type of interpolation will cause comparing with standard I VF treatment, improve and prepare in the body or the number of mature egg in vitro fertilization, and/or the number of the ovum of being fertilized in the total ovum, and/or the number of blastocyst in the ovum of fertilization, and/or implant the number of blastocyst and/or the offspring's who sends in each reproductive cycle number in the growth blastocyst.

Any feature and/or the aspect that are to be understood that relevant the method according to this invention discussed above are applicable to according to similar applications of the present invention.

Description of drawings

Fig. 1

The bipartite graph that Mammals is female and male sex-cell is grown.A key distinction male and that female sex cell is grown was shown by the tranquillization stage.Male sex-cell is tranquillization before the reduction division stage, and ovocyte is at reduction division stage (in earlier stage) tranquillization.The tranquillization stage is fetal development or the birth back life stage between early stage and pubescence, and wherein sexual cell is nondividing.Another key distinction male and that female sex cell is grown shows as the male sex-cell group and upgrades by stem cell mitotic division, and this is opposite with female sex cell group fixed number.

Fig. 2

Mammals is the preceding and sterol biosynthesizing path of sterol from the beginning.Biosynthesizing fundamentally originates in acetate and coenzyme A condensation formation acetyl-coenzyme A before the sterol.Acetyl-coenzyme A is processed in the short step of multienzyme, can cause producing squalene.Squalene becomes lanosterol by two enzymatic conversions, and it is the basis of sterol biosynthetic pathway.7 enzymes shown in sterol biosynthesizing in the Mammals endoplasmic reticulum comprises at least, but may parallelly therewith also comprise many enzymes with similar catalytic characteristics.Enzyme marks in gray background with italic, and the sterol intermediate is with common letter representation, subscript indication two keys of relevant C-C and non-cholesterol methyl substituents.Four-headed arrow represents that enzymatic step is a reversible.Dotted arrow between 24-alkene (left hand figure) and the saturated sterol of side chain (right hand figure) represents that sterol Δ 24 reductase enzymes may have multiple sterol substrate.

Fig. 3

The branch of biological synthesis path before the sterol.Go up sterol metabolism shown in Figure 2 and only be one of many possible result that the mevalonic acid of HMG-CoA-reductase enzyme mediation generates.Two arrows between the metabolite are represented the multistep Enzymatic transformation.

Fig. 4

Structure in the cyclopentanoperhydrophenanthrene ring structure, carbon numbering and ring name.The C-numbering is arranged according to IUPAC.

Fig. 5

The preadolescence female mice that gonad-stimulating hormone stimulates is being used Mevacor ^Handle the 2-cell number in the uterine tube of back.The standard error of mean value is represented by error bars.This figure represents 5 independently experiments.The mean value of all experiments shows on the right side.

Fig. 6

In FSH and statin (mevastatin), cultivate the subtrahend maturing rate of back mouse ovarian cumulus ovocyte complex body (COC).The ovocyte of GVBD representative no visible germinal vesicle under opticmicroscope, this shows that ovocyte restarts reduction division.PB/GVBD represents to restart in the training period the ratio that polar body forms in the maiotic ovocyte.Each experiment is parallel to be carried out in quadruplicate, and 30-40 COC represented in each experiment.Error bars is represented the standard error of mean value.The different letter representation in bar top is at the significant difference of 95% degree of confidence.

Fig. 7

COC shown in Fig. 6 cultivates after 20 hours and enters the free cholesterol from acetate ³The radioactivity of H is followed the tracks of.DPM on the longitudinal axis is illustrated in the radioactivity of measuring by scintillation detector by behind HPLC method rectification counting efficiency and the purification efficiency.After representing to extract, N is used for separating the sum of the COC of cholesterol by HPLC.

Fig. 8 and 9

Compare with FSH with contrast, with the fertility of oocytes rate of statin (Mevacor) maturation in vitro.Compare the ovocyte (PB of fertilization with the CEO that exists 7.5I.U./L FSH to cultivate down with contrast CEO ²-ovocyte to 4 ⁺Cell) percentage ratio improves by add the statin Mevacor in the IVM-of CEO substratum.And the ovocyte that the (not shown) Mevacor is handled shows cumulus expansion in vitro widely with respect to the contrast ovocyte behind the IVM incubation period.The ovocyte that FSH-handles is in this respect between these two groups.Degree of degeneration is indifference between group.And after embryo culture 72 hours, compare with two control groups, the growth in the Mevacor treatment group arrives more progressive state.

Figure 10

Ovocyte rate of fertilization compared with the control with cyclodextrin (hydroxypropyl-beta-cyclodextrin) maturation in vitro.NO compares the ovocyte (PB of fertilization with contrast ²-ovocyte to 4 ⁺Cell) percentage ratio is by adding the sterol combination and transporting material hydroxypropyl-cyclodextrin and improve in the IVM-of NO substratum.Degradation ratio is not subjected to the influence that cyclodextrin exists in the IVM-substratum.

Figure 11

Look at by the comprehensive of developmental process that the present invention handles.The present invention relates to reach in the body external maturation and fertilization process and do not relate to any fetal development.With sterol from the beginning biosynthesizing suppress relevant notion to male and femalely all be suitable for, and with use sterol in conjunction with and to transport the relevant notion of material only suitable at female ripening period.

Figure 12

The comprehensive of ectogenesis process that the present invention describes look at.

Figure 13

The general formula of sulfated cyclodextrin.If R=CH2CH (OH) is CH3, then this structure is represented hereinafter used hydroxypropyl-beta-cyclodextrin among the embodiment 8.

Embodiment

Embodiment 1

To female mice use statin to the after fertilization body in the influence of 2-cell number

Prepuberal C57 black * DBA 2F1 female mice (M ﹠amp; B A/S, Ry, Denmark) by peritoneal injection in 200 μ L H ₂12IU Menogon among the O ^(Ferring, Denmark) is stimulated superovulation (point in 0 day morning 11).These gonad-stimulating hormone goods contain 50% human FSH and 50% human LH with regard to biologic activity.The Mevacor  of half mouse by will containing the 40mg lovastatin (Merck Sharp and Dohme, NL) sheet is dissolved among the 5ml PBS, and from 0 day peritoneal injection gained 200 μ L slurries and additionally accept the injection of 1.6mg lovastatin for three days on end.The PBS of the similar volume of second half (control mice) injection.At point in the 2nd day afternoon 1, to the ovulate 10IU hCG (Ferring, Denmark) of dosage of animal.Mouse is independently raised in cages with same strain system and supplier's ripe male mice (every female 1 male) separately after 1 hour.Female and male mice at 9 in the 3rd day morning separately, the female indwelling separately again 48 hours.After putting to death female mice and dissecting uterine tube, by the glass valinche being placed on isolating uterine tube one end, and blow by strainer with mouth, the 2-cell is flushed in the vinyl disc with substratum (sees below).Count the 2-total cellular score of going out from every mouse afterwards.Described 2-cell subsequently general purpose I VF-substratum (Medicult, Denmark) go up in couveuse (37 ℃, 95% air, 5%CO ₂) in go up to cultivate at 4 orifice plates (Nunclon 176740, Nunc, Denmark).Cultivate after 4 days, utilize microscope to hole scoring and determine the number of morula, blastocyst or degeneration entity.

When mouse when follicular phase, ripening period was handled with 3 * 1.6mg statin, the 2-cell number of every animal significantly raise (Fig. 4).On behalf of cholesterol, this reduce the main result of treatment aspect reproduction: compare ovocyte number rising discharge and fertilization with control animal.The 2-cell is in vitro culture, and to develop into blastocyst with the nondistinctive recovery rate of contrast.

Embodiment 2

Utilize different schemes to female mice use statin to the after fertilization body in the influence of 2-cell number

Prepuberal C57 black * DBA 2F1 female mice (M ﹠amp; B A/S, Ry, Denmark) handle as in Example 1, but a little variation is arranged.In experiment A, statin dosage is reduced to 100 μ g/ animals, and injects at hCG-and to give as single injection in preceding 15 minutes.In experiment B, statin dosage is reduced to 3 * 100 μ g/ animals, and uses as in Example 1.In experiment C, statin dosage 9 the monthly age mouse hCG-inject and gave as single injection in preceding 15 minutes.In experiment D, gonad-stimulating hormone dosage reduces gradually, and statin dosage is used as among the embodiment.

Generally speaking, this experiment shows that statin dosage can reduce to 1/10th of used dosage among the embodiment 1.And different with the application in the time window of stage follicular phase broad, it be that single injection gives that the statin of suitable concn can stimulate previous crops in the ovulation of hCG.Experiment D has confirmed an additional concepts, that is, the gonad-stimulating hormone of reduction is used can be partly by using the statin compensation altogether.But, before drawing a conclusion aspect these, all experiments in the present embodiment are still needed and will be specialized quantitatively.

Table 2: use external source gonad-stimulating hormone and statin (Mevacor) C57 black * DBA 2 F1 afterwards

2-cell number in the mouse

Mating mating in the laboratory animal stage Gnrh hCG statin treatment application program mating animal

Numbering (I.U.) is 2-cell number number frequency (I.U.)

-?????????????????????????22.0±8.9????????3????????3/4

A preadolescence 12 10 100 μ g Mevacor singles 27.5 ± 3.5 2 2/4

The onset of ovulation

1000 μ g Mevacor singles 15.3 ± 7.3 4 4/4

The onset of ovulation

-?????????????????????????9.5±2.2?????????4????????4/4

B preadolescence 12 10 100 μ g Mevacor singles 19.3 ± 4.1 4 4/6

The onset of ovulation

3 * 100 μ g Mevacor many times 6.5 ± 1.0 4 4/5

Follicular phase

-?????????????????????????25.2±7.1????????5????????5/8

The ripe cycles 15 10 1.0mg Mevacor single 16.8 ± 3.4 5 5/8 of C

The onset of ovulation

12????????????-

12 3 * 1.0mg Mevacor many times 11.4 ± 2.9 6 6/8

Follicular phase

4?????????????-?????????????????????????7.2±1.4?????????6????????6/8

D preadolescence 453 * 1.0mg Mevacor many times 51 1/4

Follicular phase

1.2???????????-?????????????????????????1????????????????1????????1/4

1.2 3 * 1.0mg Mevacor many times 0 0/4

Follicular phase

0????????0/4

Embodiment 3

Use the decline that statin strengthens free cholesterol tissue density in the LH/hCG inductive ovary after stimulating preadolescence mouse propagation gland

Prepuberal C57 black * DBA 2F1 female mice (M ﹠amp; B A/S, Ry, Denmark) by peritoneal injection in 200 μ L H ₂12IU Menogon  (Ferring, Denmark) among the O is stimulated superovulation (point in 0 day morning 11).Mevacor  (the Merck Sharp and Dohme of half mouse by containing the 40mg lovastatin, NL) sheet is dissolved among the 5ml PBS, and from 0 day peritoneal injection gained 200 μ L slurries and additionally be given 1.6mg lovastatin injection (as in Example 1) for three days on end.The PBS of the similar volume of second half (control mice) injection.At point in the 2nd day afternoon 1, to the ovulate 10IU hCG (Ferring, Denmark) of dosage and putting to death after 6 hours of animal, preparation ovary extract also carries out HPLC-and analyzes.Ovary separates in pairs, weighs, and freeze-drying is weighed once more, and subsequently at 1.0ml (v/v) 75% normal heptane: extract in 25% Virahol.Organic extraction is reconstruct in moving phase, carry out straight phase (straight-phase, SP) HPLC separates (ChromSpherSi, 5 μ m, 250 * 4.6mm HPLC post is at (v/v) 99.5% normal heptane (Fischer, Leicestershire, U.K.): 0.5% Virahol (Baker, NL) in divide to run sample) with 1.00ml/.Eluate detects by the ultraviolet absorption between the 200-300nm.Before HPLC analyzes, contain cholesterol, FF-MAS and progesterone (P ₄) standard mixture run sample three times, with the stability of the product response factor that settles the standard.Cholesterol (Steraloids C6760) and P ₄(Sigma P-0130) standard substance are by commercial acquisition, and used FF-MAS standard substance as mentioned previously (Baltsen etc., 1999) in the laboratory, prepare.All typical curves are linear in whole sample value scopes.

Table 3: the free cholesterol (C) in the preadolescence mouse ovarian of the gonad-stimulating hormone sensitization that usefulness hCG and statin stimulate, liquor folliculi reduction division activate sterol (FF-MAS) and progesterone (P ₄).Different suffix letters (runic) are represented the significant difference of t-check.

Stimulating group hCG+ statin hCG-

N?????????????????????13?????????????????6????????????????6

Mouse (g) 14.6 ± 0.2A 15.3 ± 0.3A 14.6 ± 0.3A that weighs

N?????????????????????5??????????????????5????????????????3

Ovary (mg) 2.3 ± 0.1A 2.2 ± 0.2AB 1.9 ± 0.1B that weighs

C, ovary (mg/g weight in wet base) 2.2 ± 0.1A, 2.7 ± 0.1B 3.0 ± 0.2B

C, ovary (mg/g dry weight) 10.5 ± 0.6A ^§11.0 ± 0.4A ^§12.1 ± 0.5A

C, liver (mg/g weight in wet base) 1.7 ± 0.1A ^#(not surveying) 2.9 ± 0.3B

C, liver (mg/g dry weight) 6.5 ± 0.5A ^#(not surveying) 7.1 ± 0.4A

FF-MAS (μ g/g weight in wet base) 0.13 ± 0.05A, 3.0 ± 0.5B, 0.76 ± 0.66A

P ₄(μ g/g weight in wet base) 14.9 ± 0.9A, 13.0 ± 0.5A, 1.3 ± 0.7B

#)N＝3

§) significance boundary line (P=0.055, alternative hypothesis " hCG+ statin "＜" contrast ").Significance boundary line (P=0.061, alternative hypothesis " hCG "＜" contrast ").

The tissue density of free cholesterol decline in the mouse ovarian before the ovulation.HCG stimulates the downtrending of free cholesterol tissue density in the ovary of back to handle obviously because of statin.Described decline can not be explained by the swelling effect of the hCG hormone that produces steroid fully, because cholesterol tissue density also descends when estimating based on stem organization's weight.Apply HCG to simulate natural preovulatory LH peak.Emphasis is pointed out that the influence that the degree of progesterone (steroid) generation is not handled by statin.And, should be noted that statin blocks the accumulation of LH/hCG inductive cholesterol premetabolite FF-MAS fully, and make that the level of FF-MAS is lower than the mouse that does not stimulate with hCG in the ovary.And, the relatively announcement of the mouse ovarian that stimulates with contrast and hCG with regard to tissue density, the absolute decline of cholesterol be FF-MAS raise more than 100 times.

Embodiment 4

The maturation of the ovocyte that the HMG-CoA-reductase inhibitor surrounds at the stimulated in vitro ovarian cumulus ripe and the ovocyte that surrounds at the ovarian cumulus that external enhancing gonad-stimulating hormone stimulates

Prepuberal C57 black * DBA 2F1 female mice (M ﹠amp; B A/S, Ry, Denmark) as in embodiment 1 and 2, by peritoneal injection in 200 μ L H ₂12IU Menogon  (Ferring, Denmark) among the O is stimulated superovulation (point in 0 day morning 11).After 46 hours, ovary is immersed in is supplemented with in the hypoxanthic substratum of 4mM (α-MEM 22571-020, Gibco BRL, Scotland) (seeing below), by with preovulatory ovarian follicle in the needle penetration ovary of 0.20mm, get ovocyte back from preovulatory follicle from ovary.By the microscopic examination of preparation property and draw with the opening valinche that film is housed subsequently and collect ovarian cumulus-ovocyte complex body (COC), and in α-MEM in couveuse (37 ℃, 95% air: 5%CO ₂) in go up to cultivate at 4 orifice plates (Nunc, Denmark).Culture medium supplemented has 4mM xanthoglobulin (HX) (Sigma H-9377, Sigma-Aldrich, Denmark), 200mM L-glutaminate (Gibco BRL, Scotland), 20000IU/L penicillin (Gibco BRL, Scotland), 20000IU/L Streptomycin sulphate (Gibco BRL, Scotland) and 3mg/ml BSA (SigmaA-7030, Sigma-Aldrich, Denmark).All incubations add 25mCi ³(NEN is U.S.A.) so that measure the de novo synthesis of cholesterol by flicker behind incubation for the H-acetate.Interpolation 50mM is in the statin mevastatin (Sigma among the DMSO (substratum DMSO final concentration is 1%v/v in all settings (comprising contrast)), Sigma-Aldrich, Denmark), and the recombinant human follicle-stimulating hormone (Gonal-F of interpolation 7.5IU/L final concentration, Serono, DK).Cultivate after 22 hours, estimate the breaking (GVB) and the existence of polar body (PB) of existence, germinal vesicle of germinal vesicle (GV) in the ovocyte.These 3 states are represented the process of oocyte maturation.

As if cholesterol reduces scheme itself and has the ripe effect of reduction division, and this effect can be accumulated on the ripe effect of known FSH (Fig. 4).The cell of four parts of parallel laboratory tests is converged, and measure from acetate metabolism ³The amount of H, the from the beginning biosynthesizing that confirms cholesterol are really by adding statin descend (Fig. 5).The effect of mevastatin is remarkable, and from the beginning the effect aspect the biosynthesizing is not too effective and FSH is suppressing cholesterol.This experimental results show that utilize statin suppress cholesterol from the beginning biosynthesizing strengthen the external natural ripe incident that causes by gonad-stimulating hormone, and strengthen blocking-up simultaneously to the cholesterol de novo synthesis.

Embodiment 5

Cholesterol is from the beginning biosynthetic in external mediation mouse ovarian stops for hCG

Prepuberal C57 black * DBA 2F1 female mice (M ﹠amp; B A/S, Ry, Denmark) as among the embodiment 1-4, by peritoneal injection in 200 μ L H ₂12IU Menogon  (Ferring, Denmark) among the O is stimulated superovulation (0 day).Separate ovary after 46 hours as in Example 4 and at 0.25mCi ³(NEN cultivates in α-MEM (Gibco BRL, Scotland) under existence U.S.A.) H-acetate.Add the combination of 0 (contrast) or 5IU/ml hCG and 0 (contrast) or 20%FCS in the culture.2 ovaries common incubation in the 1.0ml substratum.After 22 hours, stop cultivating, the flushing ovary, and as embodiment 3 explains, extract.The sterol that extracts separates with the HPLC post: ChromSpherSi 250 * 4.6mm, 5 μ m; Moving phase: 99.65% normal heptane (Fisher, Leicester, U.K.): 0.35% Virahol (Baker, Deventer, NL) (vol.: vol.)), regeneration: 90% normal heptane: 10% Virahol.Before straight phase sample analysis, to containing squalene, 4,4-dimethyl sterol, 7-dehydrocholesterol, desmosterol, cholesterol and P ₄Standard substance carry out continuous 3 times and run samples so that the time window of collecting is tabulated.Collection contain squalene window 1 (2 ' 50 " 4 ' 00 "), contain 4, the window 2 of 4-dimethyl sterol (10 ' 30 " 14 ' 00 "), contain cholesterol and desmosterol window 3 (25 ' 00 " 29 ' 00 "), contain 5, window 4 of 7-alkene (29 ' 30 " 33 ' 00 ") and window 5 (contain P ₄), dry also place anti-phase: LiChrospher RP-8.5 μ m, 250 * 4.6mm HPLC post are at (v/v) 92.5% acetonitrile: divide in 40 ℃ with 1.00ml/ in 7.5% water and run samples.Analyte is identified by the UV photoabsorption between the 200-300nm.Squalene (Merck S21362), lanosterol (Sigma L5768), zymosterol (Steraloids C3200), lathosterol (Steraloids C7400), 7-dehydrocholesterol (Steraloids C3000), lanosterol (steraloids C7400), 7-dehydrocholesterol (steraloids C3000), desmosterol (Steraloids C3150), cholesterol (Steraloids C6760) and P ₄(Sigma P-0130) is by commercial acquisition, and FF-MAS and T-MAS standard substance are in prepared in laboratory.All hereinafter listed analytes in reverse phase separation as the unimodal wash-out of homogeneous.Collect the peak, (Packard NL) mixes with the 3.0ml scintillation solution in scintillation vial.In Beckman LS 1801 liquid flashing counting devices, count.Proofread and correct the CPM value at counting efficiency and sample injection.

Table 4: exist ³Pass through the flicker data of the isolating analyte of HPLC during H acetate behind the external incubation mouse ovarian

Analyte contrast/hCG P contrast/FCS P

Squalene 80 ± 9% 0.099 136 ± 20% 0.139n.s.

Lanosterol 22 ± 7% 0.000074 331 ± 98% 0.065n.s.

FF-MAS??????????72±16％???????0.133???????????63±11％?????????0.021 ^**

T-MAS???????????43±5％????????0.000059????????96±13％?????????0.76n.s.

5,7-alkene 35 ± 4% 0.000071 161 ± 33% 0.142n.s.

Desmosterol 58 ± 7% 0.0041 91 ± 13% 0.55n.s.

Cholesterol 226 ± 39% 0.032 ^*111 ± 32% 0.75n.s.

Progesterone 13 ± 5% 0.000085-n.c. ^{* *}

The P value is obtained from the paired check of 6+6 sample, supposes that control value is not equal to test value. ^*) descend after adding hCG. ^*) along with FCS adds and increases. ^{* *}) do not compare owing to hang down flicker numerical value.N.s. representative is not remarkable.

This experiment confirm after hCG handles, in the mouse ovarian cholesterol from the beginning biosynthesizing descend and cumulative rises of sterol before the cholesterol that from the beginning produces.And the radioactivity amount relevant with progesterone (hCG/LH handles the main steroid that produces in the ovary of back) descends.This discloses when ovary during by induction of ovulation, originates from the substrate total amount decline of the cholesterol that from the beginning produces.In fact, the radioactivity amount that is incorporated in the various sterol fractions is independent of replenishing of lipoprotein (FCS), and this shows that ovulation stimulates the biological mechanism of cholesterol reduction in the ovary of back not to be subjected to the influence of common cholesterol homeostatic mechanism.

Embodiment 6

Change C/PL in the mouse epididymis swarm cell fraction than (C/PL-than) by using statin in advance

Use postpubertal C57 black * DBA 2F1 male mice (M ﹠amp; B A/S, Ry, Denmark).Give 4 mouse 3 peritoneal injections by every 30g body weight 1.0mg lovastatin, be specially: Mevacor  (the Merck Sharp and Dohme that will contain the 40mg lovastatin, NL) sheet is dissolved among the 8ml PBS, and epididymal sperm is collected diary do 0 day, in 1 peritoneal injection gained, 200 μ L slurries in-6 ,-3 and-1 day afternoon.The PBS of the similar volume of 4 other mouse (control mice) injection.All mouse of 10 execution in 0 day morning, and in physiological saline, prepare epididymis.Afterwards, epididymis transferred to contain MEM (Gibco BRL, Scotland) in the 1-mL culture hole, wherein be supplemented with 2.3mM pyruvic acid, 200mM L-glutaminate (Gibco BRL, Scotland), 20000IU/L penicillin (Gibco BRL among the MEM, Scotland), 20000IU/L Streptomycin sulphate (Gibco BRL, Scotland) and 0.2mg/ml BSA (Sigma A-7030, Sigma-Aldrich, Denmark), then epididymis is placed couveuse (37 ℃, 95% air: 5%CO ₂) in.

Behind the incubation 1.5 hours, the upper phase sucking-off, and transfer in the new vial also centrifugal.After centrifugal, abandon supernatant, will precipitate freeze-drying, afterwards by 50% methyl alcohol: 50% chloroform (vol./vol.) extracts.Subsequently extract is divided into two equal portions.Half is pointed among the embodiment as mentioned carries out the sterol analysis, and calculates by cholesterol, desmosterol, courage steroid-7 total sterol content of 24-diene-3 β-alcohol, T-MAS and lanosterol composition.Second half method according to Bartlett (Bartlett, 1958) is carried out phospholipid determination.Calculate the C/PL-ratio of each individual animals.

Think that the C/PL-ratio in the statin treatment group is lower than the C/PL-ratio in the control group.

Embodiment 7

Apply the rate of fertilization that statin improves the ovocyte (CEO) that the mouse ovarian cumulus surrounds behind the IVF during the IVM

Oocyte of mouse separates according to the description among the embodiment 3 and cultivates, but has a place to change, promptly the IVM substratum also be supplemented with the 1mg/mL Pp63 glycophosphoproteins (Sigma F-3385, Sigma, U.S.A.).CEO is divided into 3 groups, and in the plate that contains the substratum that has added following composition, cultivate: the recombinant human follicle-stimulating hormone (Gonal-F of 1% (v/v) ethanol (contrast), concentration 7.5IU/L, Serono, DK) together with 1% (v/v) ethanol (FSH) or 10 μ M mevastatin (Mevacor M2147, Sigma-Aldrich, Denmark) together with 1% (v/v) ethanol (Mevacor).In order to dissolve Mevacor in substratum, ethanol is essential.At maturation in vitro after 22 hours, ovocyte is transferred to by minimum essential medium (MEM) (21090-022, Gibco BRL, Scotland), 200mM L-glutaminate (Gibco BRL, Scotland), 20000IU/L penicillin (Gibco BRL, Scotland), 20000IU/L Streptomycin sulphate (Gibco BRL, Scotland), 3mg/ml BSA (Sigma A-7030, Sigma-Aldrich, Denmark), 2.3mM Sodium.alpha.-ketopropionate (S-8636, Sigma), 10mM ethylenediamine tetraacetic acid (EDTA) (EDTA, Merck 1.08418.0250) and the 1mg/mL Pp63 glycophosphoproteins (Sigma F-3385, Sigma is U.S.A.) in the fertilization substratum of Zu Chenging, before ovocyte being transferred to the fertilization substratum, obtain to be used for the mouse sperm of IVF in the following manner: C57 black * DBA 2F1 male mice (the M ﹠amp that puts to death the 6-9 monthly age; B A/S, Ry, Denmark), and in physiological saline, prepare epididymis.Afterwards, epididymis is cut into small pieces, and caked material transfer to 4 is contained in the 1-mL culture hole of 800uL IVF-substratum mentioned above, be placed on couveuse (37 ℃, 95% air: 5%CO ₂) in.Behind the incubation 1.5 hours, 300 μ L upper phase of each of 4 holes are sucked out, and transfer in the new vial.The zoidiogamy word bank of gained is counted in nucleonics, and adds 50000 motile sperm cells in each ovocyte culture.The IVF culture in couveuse in 37 ℃, 95% air: 5%CO ₂Incubation 20 hours is transferred to ovocyte under the same conditions in the fresh IVF substratum of equilibrated afterwards.These ovocytes were kept somewhere other 48 hours, and according to the maturity state of ovocyte or the process counting of zygote, described process is GV-ovocyte, GVBD-ovocyte, PB-ovocyte, PB just subsequently ²-ovocyte (ovocyte), 2-cell, 3-4 cell, 4+ cell (having zygote) and degenerated cell (normally granular, as perhaps to have the cell of plasma membrane fold widely) more than 4 blastomeres with visible second polar body.

Embodiment 8

Apply the rate of fertilization that cyclodextrin improves nude mice ovocyte (NO) behind the IVF during the IVM

Is oocyte of mouse according to embodiment? described separation and cultivation are divided into three groups with NO, and contain added 0 ‰, 0.5 ‰, 2.0 ‰ or the plate of the substratum of 5 ‰ hydroxypropyls-cyclodextrin (ICN 153540, ICNOhio, the U.S.) in cultivate.Identical among IVM and IVF condition and the embodiment 6.

Embodiment 9

Improve the mouse sperm quality by using statin in advance

Experiment be provided with comprise with embodiment 6 in identical animal and statin use.By for estimating the methods analyst animal that fertilization potentiality and sperm quality are set up.Think and compare that the animal that statin is handled has the fertilization advantage with control animal.

Reference

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Claims

1. suppress Mammals sexual gland cell and/or at least one Mammals sexual cell and/or gamete and/or support from the beginning biosynthesizing of sterol in the sexual gland derived cell of sexual cell with sterol, improve at least one Mammals gamete thus, zygote, body early embryo, blastocyst of implanting and/or embryo's developmental potency, and/or raising has the number of the Mammals gamete of fertility, cause post-coitum zygote in female each reproductive cycle, body early embryo, the method that blastocyst of implanting and/or embryo's number increase, described method comprise to the administration that needs this method can suppress from the beginning biosynthetic compound of sterol or compound combination.

2. improve the method for the sterol outflow of at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side, improve the method for the developmental potency of described at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side thus, described method comprises the compound or the compound combination that can promote the sterol outflow to the administration that needs this method.

3. according to the method for claim 1 or 2, the Mammals that wherein needs described method is female, and restraining effect and/or outflow occur in before the ovulation 0-12 days and/or ovulation back in one period of 0-3 days.

4. according to the process of claim 1 wherein that the Mammals of the described method of needs is male, and suppress to occur in before the ejaculation in one period of 0-12 days.

5. improve at least one Mammals gamete, zygote, body early embryo, blastocyst and/or embryo's the developmental potency and/or the number of the Mammals gamete that raising has fertility implanted, thereby cause zygote in female each reproductive cycle, body early embryo, the method that blastocyst of implanting and/or embryo's number increase, described method comprise that interpolation can suppress described at least one Mammals gamete in supporting breathing of at least one ovocyte and/or at least one sperm and/or sophisticated in-vitro culture medium, zygote, body early embryo, from the beginning biosynthetic compound of sterol or compound combination among blastocyst of implanting and/or the embryo.

6. improve the sterol outflow of at least one Mammalian Ovary, ovocyte, megagamete and/or ovocyte ovary derived cell on every side, improve the method for the developmental potency of described at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side thus, described method comprises that adding the compound or the compound that can promote the sterol outflow in supporting at least one ovocyte breathing and/or sophisticated in-vitro culture medium makes up.

7. according to each method among claim 1 and the 3-5, compound wherein is the material or the combinations of substances of the mammalian enzyme in the biosynthesizing path between one or more participation acetyl-CoAs of antagonism and the lanosterol, and described enzyme is HMG-CoA reductase enzyme, Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium mevalonic acid decarboxylase, isopentenylpyrophosphate isomerase, Dimethylallyltransferase, geranyl transferring enzyme, squalene synthetase, squalene monooxygenase, oxidation shark alkene lanosterol cyclase and lanosterol synthetic enzyme.

8. according to each method among claim 1, the 3-5 and 7, compound wherein is the material or the combinations of substances of antagonism 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (HMG-CoA reductase enzyme).

9. according to each method among claim 1,3-5 and the 7-8, compound wherein is the combination of a kind of statin or multiple statin.

10. according to claim 1, each method among 3-5 and the 7-9, statin wherein is selected from holder and cuts down his spit of fland, simvastatin, Fluvastatin, BAY W 62, Pravastatin, lovastatin (in the past being referred to as Mevacor or lovastatin K), 28, HR 780, Pravastatin, simvastatin, mevastatin, methyl-mevastatin, Mevastatin (in the past being referred to as mevastatin or ML-236B), ML-236A, ML-236C, the dihydro mevastatin, lovastatin J, lovastatin L, lovastatin M, lovastatin X, dihydro lovastatin L, Mevacor, the single sodium salt of 3 beta-hydroxy mevastatins acid (Pravastatin, CS-514), Simvastatin, simvastatin and nystatin/Triamcinolone Acetonide.

11. according to each method in claim 2-3 and 6, wherein can promote the compound of sterol outflow or cyclodextrin that the compound combination is selected from cyclodextrin, chemically modified, high-density lipoprotein (HDL) (HDL), from apoprotein and sterol carrier protein I and the II of HDL.

12. according to each method among the claim 2-3,6 and 11, compound wherein is a cyclodextrin.

13. according to each method in the aforementioned claim, statin and/or can promote that the amount of application of the compound of sterol outflow is between every kg body weight 0.01-100mg every day wherein.

14. according to each method in the aforementioned claim, the statin of wherein in in-vitro culture medium, using and/or can promote that the amount of the compound of sterol outflow is 0.01-1000 μ M.

15. according to each method in the aforementioned claim, wherein at least one ovocyte was cultivated in in-vitro culture medium 1 second to 15 days.

16. according to each method among claim 1,3-5,7-10 and the 13-15, wherein sperm was cultivated in in-vitro culture medium 1 second to 8 days.

17. according to each method in the aforementioned claim, wherein said using and/or the using and/or add associating of interpolation and gonad-stimulating hormone.

18. according to each method in the aforementioned claim, wherein said using and/or the using and/or add associating of interpolation and Mammals somatomedin.

19. according to the method for claim 17, wherein gonad-stimulating hormone is between the 0-1000 IU/L at external amount of application.

20. according to the method for claim 17, wherein gonad-stimulating hormone amount of application in vivo be every day every kg body weight 0-5 IU FSH and every day every kg body weight 0-200IU LH.

21. according to each method in the aforementioned claim, wherein Mammals is selected from people, horse, ox, rat, mouse, pig, sheep, goat, camel, dog, cat and ermine.

22. according to each method in the aforementioned claim, wherein Mammals sexual gland cell is human cell or an isolating sexual cell of supporting sexual cell with sterol.

23. according to each method in the aforementioned claim, wherein Mammals sexual gland cell is ovocyte or the isolating ovocyte that ovarian cumulus surrounds.

24. according to each method among claim 1,3-5,7-10 and the 13-23, wherein sexual cell is the sperm that penetrates.

25. according to each method in the aforementioned claim, wherein Mammals sexual gland cell is the immature egg parent cell with adherent cumulus cell and/or isolating immature egg parent cell or ovarian follicle, ovocyte with adherent cumulus cell and/or the immature egg parent cell little or that medium sized hole vesicular ovarian follicles is regained before hole that arrogant hole vesicular ovarian follicles is regained, little human hole vesicular ovarian follicles diameter 0.4-＜5mm, medium sized human hole vesicular ovarian follicles is 5-＜15mm, and big human hole vesicular ovarian follicles diameter 15mm or more than.

26. according to each method in the aforementioned claim, wherein the sexual gland cell is the prematurity or the sophisticated ovocyte of acquisition after cultivating from least one prematurity ovarian follicle in stage during the primordial follicle phase is to hole.

27. cell culture medium, it comprises statin or statin combination.

28. cell culture medium, it comprises compound or compound combination, for example cyclodextrin that can promote the sterol outflow.

29. cell culture medium, it comprises statin and cyclodextrin.

30. according to each cell culture medium among the claim 27-29, wherein said substratum is the cell culture medium that chemical ingredients is determined.

31. according to each cell culture medium among the claim 27-30, it further comprises at least a somatomedin.

32. according to each cell culture medium among the claim 27-31, wherein said somatomedin is selected from gonad-stimulating hormone (FSH, LH, CG), IGF, EGF, Regular Insulin, tethelin, interleukin and other peptide factor.

33. make by primordial follicle phase and the hole sophisticated method of immature egg parent cell that the stage obtains between early stage, be included in according to cultivating described immature egg parent cell in each the cell culture medium among the claim 27-32.

34. pharmaceutical composition, it comprises can suppress the from the beginning biosynthetic compound of sterol and the combination of compounds and the pharmaceutically acceptable carrier that can improve the sterol outflow.

35. according to the pharmaceutical composition of claim 34, wherein said compound is statin and cyclodextrin.

36. according to each pharmaceutical composition among the claim 34-35, it further comprises somatomedin.

37. according to the pharmaceutical composition of claim 36, wherein said somatomedin is selected from gonad-stimulating hormone (FSH, LH, CG), IGF, EGF, Regular Insulin, tethelin, interleukin and other peptide factor.

38. pharmaceutical composition, it comprises the combination that can suppress from the beginning biosynthetic compound of sterol and somatomedin.

39. according to the pharmaceutical composition of claim 38, wherein said compound is a statin.

40. pharmaceutical composition, it comprises the compound that can promote sterol outflow and the combination of somatomedin.

41. according to the pharmaceutical composition of claim 40, wherein said compound is a cyclodextrin.

42. according to the pharmaceutical composition of claim 38 or 40, wherein said somatomedin is selected from gonad-stimulating hormone (FSH, LH, CG), IGF, EGF, Regular Insulin, tethelin, interleukin and other peptide factor.

43. the from the beginning biosynthetic compound of sterol or the compound that can suppress in Mammals sexual gland and/or at least one Mammals sexual cell and/or gamete and/or the sexual gland derived cell are combined in the purposes for preparing in the medicine, the from the beginning biosynthesizing of sterol that described medicine is used for suppressing at least one Mammals sexual cell and/or gamete and/or supports the sexual gland derived cell of sexual cell with sterol, improve at least one Mammals gamete thus, zygote, body early embryo, blastocyst of implanting and/or embryo's developmental potency, and/or be used to improve the number of Mammals gamete with fertility, cause post-coitum zygote in female each reproductive cycle, body early embryo, blastocyst of implanting and/or embryo's number increase.

44. can promote compound or the purposes of compound combination in the preparation medicine of the sterol outflow of the ovary derived cell around at least one Mammalian Ovary and/or mammal ovocyte and/or mammal oogamete and/or the egg mother cell; Described medicine is used for promoting the sterol outflow of at least one Mammalian Ovary and/or mammal ovocyte and/or mammal oogamete and/or egg mother cell ovary derived cell on every side, improves thus the developmental potency of described at least one Mammalian Ovary and/or mammal ovocyte and/or mammal oogamete and/or egg mother cell ovary derived cell on every side.

45. according to the purposes of claim 43 or 44, wherein Mammals is female, and restraining effect occurs in preceding 0-12 days of ovulation and/or ovulates the back in one period of 0-3 days.

46. according to the purposes of claim 43, wherein Mammals is male, and restraining effect occurred in before the ejaculation in one period of 0-12 days.

Be combined in the purposes for preparing at least one ovocyte of support and/or breathing of at least one sperm and/or the sophisticated in-vitro culture medium 47. can suppress from the beginning biosynthetic compound of sterol or compound, described substratum is used to improve the blastocyst of at least one Mammals gamete, zygote, body early embryo, implantation and/or embryo's developmental potency, and/or be used to improve the number of Mammals gamete with fertility, cause the blastocyst of zygote, body early embryo, implantation in female each reproductive cycle and/or embryo's number to increase.

48. can promote the compound of sterol outflow or compound to be combined in the purposes for preparing at least one ovocyte breathing of support and/or the sophisticated in-vitro culture medium, described substratum is used to improve the developmental potency of at least one Mammalian Ovary and/or mammal ovocyte and/or Mammals megagamete and/or ovocyte ovary derived cell on every side.

49. according to each purposes among claim 43 and the 45-47, compound wherein is the material or the combinations of substances of the mammalian enzyme in the biosynthesizing path between one or more participation acetyl-CoAs of antagonism and the lanosterol, and described enzyme is 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (a HMG-CoA reductase enzyme), Mevalonic kinase, Phosphomevalonic kinase, tetra-sodium mevalonic acid decarboxylase, the isopentenylpyrophosphate isomerase, Dimethylallyltransferase, the geranyl transferring enzyme, squalene synthetase, squalene monooxygenase, oxidation shark alkene lanosterol cyclase and lanosterol synthetic enzyme.

50. according to each purposes among claim 43, the 45-47 and 49, compound wherein is the material or the combinations of substances of antagonism 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (HMG-CoA reductase enzyme).

51. according to each purposes among claim 43,45-47 and the 49-50, compound wherein is statin or statin combination.

52. according to claim 43, each purposes among 45-47 and the 49-51, statin wherein is selected from holder and cuts down his spit of fland, simvastatin, Fluvastatin, BAY W 62, Pravastatin, lovastatin (in the past being referred to as Mevacor or lovastatin K), 28, HR 780, Pravastatin, simvastatin, mevastatin, methyl-mevastatin, Mevastatin (in the past being referred to as mevastatin or ML-236B), ML-236A, ML-236C, the dihydro mevastatin, lovastatin J, lovastatin L, lovastatin M, lovastatin X, dihydro lovastatin L, Mevacor, the single sodium salt of 3 beta-hydroxy mevastatins acid (Pravastatin, CS-514), Simvastatin, simvastatin and nystatin/Triamcinolone Acetonide.

53. according to each purposes in claim 44-45 and 48, wherein can promote the compound of sterol outflow or cyclodextrin that the compound combination is selected from cyclodextrin, chemically modified, high-density lipoprotein (HDL) (HDL), from apoprotein and sterol carrier protein I and the II of HDL.

54. according to each purposes among the claim 44-45,48 and 53, compound wherein is a cyclodextrin.

55. according to each purposes in the aforementioned claim, wherein the amount of application of statin is between every kg body weight 0.01-100mg every day.

56. according to each purposes in the aforementioned claim, the statin of wherein in in-vitro culture medium, using and/or can promote that the amount of the compound of sterol outflow is 0.01-1000 μ M.

57. according to each purposes in the aforementioned claim, wherein at least one ovocyte was cultivated in in-vitro culture medium 1 second to 15 days.

58. according to each purposes among claim 43,45-47,49-52 and the 55-57, wherein sperm was cultivated in in-vitro culture medium 1 second to 8 days.

59. according to each purposes in the aforementioned claim, wherein said using and/or the using and/or add associating of interpolation and gonad-stimulating hormone.

60. according to each purposes in the aforementioned claim, wherein said using and/or the using and/or add associating of interpolation and Mammals somatomedin.

61. according to the purposes of claim 59, wherein the external amount of application of gonad-stimulating hormone is 0-1000IU/L.

62. according to the purposes of claim 59, wherein in the body of gonad-stimulating hormone amount of application be every day every kg body weight 0-5 IU FSH and every day every kg body weight 0-200IU LH.

63. according to each purposes in the aforementioned claim, wherein Mammals is selected from people, horse, ox, rat, mouse, pig, sheep, goat, camel, dog, cat and ermine.

64. according to each purposes in the aforementioned claim, wherein Mammals sexual gland cell is human cell or an isolating sexual cell of supporting sexual cell with sterol.

65. according to each purposes in the aforementioned claim, wherein Mammals sexual gland cell is ovocyte or the isolating ovocyte that ovarian cumulus surrounds.

66. according to each purposes among claim 43,4547,49-52 and the 55-65, wherein sexual cell is the sperm that penetrates.

67. according to each purposes in the aforementioned claim, wherein sexual cell is an ovocyte.

68. according to each purposes in the aforementioned claim, wherein sexual cell is the immature egg parent cell regained by suction or by suction immature egg parent cell little or that medium sized hole vesicular ovarian follicles is regained.

69. according to each purposes in the aforementioned claim, wherein sexual cell is the immature egg parent cell that is in the acquisition to hole at least one prematurity ovarian follicle in early stage of primordial follicle phase in cultivation.

70. according to each purposes in the aforementioned claim, wherein Mammals sexual gland cell is immature egg parent cell with adherent cumulus cell and/or the isolating immature egg parent cell that arrogant hole vesicular ovarian follicles is regained, perhaps ovocyte with adherent cumulus cell and/or the immature egg parent cell that ovarian follicle, little or medium sized hole vesicular ovarian follicles are regained before hole, little human hole vesicular ovarian follicles diameter 0.4-＜5mm, medium sized human hole vesicular ovarian follicles is 5-＜15mm, and big human hole vesicular ovarian follicles diameter 15mm or more than.

71. according to each purposes in the aforementioned claim, wherein the sexual gland cell is the prematurity or the sophisticated ovocyte of acquisition after cultivating from least one prematurity ovarian follicle in stage during the primordial follicle phase is to hole.