EP1463804A1 - Mobilisation de gametes et amelioration de la competence de developpement chez les mammiferes au moyen de l'inhibition de la biosynthese des sterols de novo et/ou de l'activation de la sortie des sterols - Google Patents

Mobilisation de gametes et amelioration de la competence de developpement chez les mammiferes au moyen de l'inhibition de la biosynthese des sterols de novo et/ou de l'activation de la sortie des sterols

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Publication number
EP1463804A1
EP1463804A1 EP02792712A EP02792712A EP1463804A1 EP 1463804 A1 EP1463804 A1 EP 1463804A1 EP 02792712 A EP02792712 A EP 02792712A EP 02792712 A EP02792712 A EP 02792712A EP 1463804 A1 EP1463804 A1 EP 1463804A1
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Prior art keywords
mammalian
oocyte
sterol
compound
cell
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EP02792712A
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German (de)
English (en)
Inventor
Mogens Baltsen
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Rigshospitalet
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Rigshospitalet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/225—Polycarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/10—Conditioning of cells for in vitro fecondation or nuclear transfer

Definitions

  • 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 phophohpid/sterol ratio of said cells.
  • infertility is defined differently depending on the woman's age.
  • the standard definition of infertility is the inability to achieve a pregnancy after one year of unprotected intercourse in couples where the woman is under age 35.
  • In parallel to this fertility treatments display a drop that starts around the age of 34 years of age falling to almost zero in women over 42 unless donor eggs are used.
  • improved methods for treating infertility are needed since the average maternal age is increasing.
  • the germ cell progenitor in the female is the so-called primary oocyte.
  • Primary oocytes replicate their DNA and enter the prophase of the first meiotic division. At this time cell degeneration begins and many primary oocytes become atretic and die. All surviving primary oocytes, however, have entered the prophase of the first meiotic division and they are now individually surrounded by a layer of somatic epithelial cells: the primordial follicle.
  • follicles of most mammals including humans develop a fluid filled cavity, the so-called antrum, and these follicles are from then termed antral or Graafian follicles.
  • gonadotropins mainly LH
  • Germ cell progenitors in the male are the spermatogonia. Pre-spermatogonia are surrounded by a somatic cell and enclosed in so-called testis cords prior to or shortly after birth. This represents a major structural similarity of germ cell development in the two sexes. The surrounding cells are called Sertoli-cells and function as nursing cells for the germ cell development in analogy with the granulosa cell nursing of the oocyte.
  • a major difference between female and male germ lines is the timing and progression of the meiotic cycle.
  • the diploid pre-spermatogonia somehow differentiate into a resting pool of stem spermatogonia and a mitotically active pool of spermatogonia.
  • the division of the latter give rise to A-spermatogonia that still able to divide mitotically and B- sermatogonia that are committed to enter meiosis.
  • Male germ line meiosis takes place only after onset of puberty but, unlike the female germ line diplotene arrest, then proceeds with no arrests until four haploid germ cells are produced from a single B-spermatogonia (figure 1).
  • the product of the two meiotic divisions is spermatocytes (1 ' and 2 ' ) and the secondary spermatocytes differentiate further into spermatides and later into spermatozoa that are released into the lumen of the seminiferous tubuli in the testes.
  • capacitation takes place in the uterine tract of the female and involves, amongst other possible factors, a loss of cell membrane cholesterol to the surroundings.
  • the number and quality of the male germ cells is dependent of a number of factors involving hormones, cellular processes, environmental factors and stress. In principle, the number of male germ cells is unlimited because of the existence of stem-spermatogonia that will produce dividing B-spermatogonia and later mature spermatozoa. However, in practice the number of Sertoli-cells in the testes that nurse the maturation of the male germ cells limits the number.
  • Low numbers of mature spermatozoa causes male sub- and infertility, low quality of the ejaculated spermatozoa, or both.
  • Low sperm quality is a broad operational term that may cover genetic, biochemical and morphological components and even environmentally induced factors.
  • Sterols are important constituents of all the membranes of all metazoans. Cholesterol is the most prevalent sterol and the natural biosynthetic end-point of sterol biosynthesis in mammals. Sterols are defined as substances containing the perhydrocyclopentan- ophenanthrene four-ring system with at least a carbonl7 (C17) substituted side-chain and a C3 ⁇ -hydroxyl substitution (figure 4). In the present context the notion of sterols may be limited to structures that concur to these structural requirements and appear as intermediates from lanosterol to cholesterol in a physiological context (see figure 2 for examples).
  • cetic acid is the precursor for cholesterol biosynthesis de novo.
  • this is not synonymous with the contention that acetic acid only will be used for cholesterol biosynthesis, because a number of branches occur on the metabolic pathway of acetic acid, of which the branch that give rise to cholesterol production is one only (see figure 3).
  • the biosynthesis magnitude of any of the products depend on the physiological potentiation of the biosynthetic pathway combined with the regulatory pattern that gives some branches premium as opposed to the other branches.
  • Sterols and sterol dynamics in germ cells Sterols are important constituents of bio-membranes and constitute together with phospholipids the vast lipid material in all membrane structures in cells. Sterols appear as free, i.e. non-covalently modified, sterols in membranes but are stored as sterol-esters in sub-cellular compartments. In mammals, cholesterol is the dominant sterol species and the quantitative appearance of this sterol decides important physical qualities of the membrane. Changes in the cholesterol to phospholipid ratio in the plasma membranes affect membrane permeability, membrane transport properties, cell fusogenicity, enzyme activities and membrane fluidity.
  • the mammalian spermatozoa has a unique composition of sterols as evidenced by the finding of desmosterol in rhesus monkey spermatozoa and desmosterol and cholesta-7,24- dien-3 ⁇ -ol in hamster spermatozoa (Awano et al., 1989) and testicular meiosis activating sterol (T-MAS) in human spermatozoa (Baltsen et al., 1998).
  • T-MAS testicular meiosis activating sterol
  • cholesterol seems to be the major sterol in mammalian spermatozoa.
  • the non-cholesterol sterols may constitute up to 90% of the total amount of sterols in certain spermatozoa stages of some species (Awano et al., 1989).
  • the sperm capacitates during uterine and tubal transport.
  • the acrosome reaction of the spermatozoa is induced by binding to the zona pelucida (eggshell) on the oocyte prior to the cellular fusion but also by progesterone that is also produced by the newly ovulated cumulus cells of the cumulus-oocyte complex in the oviduct.
  • Spermatozoa capacitate in vitro by exposition to agent that confers cholesterol loss from the membrane, and exogenous cholesterol and desmosterol inhibit the acrosomal responsiveness to progesterone (Cross, 1996).
  • the sterol content or the content of a molecule in equilibrium with sterols is therefore a key parameter during male germ cell development.
  • sterol content of spermatozoa membrane determines the extent of expression of certain surface protein important for sperm-egg recognition (Benoff et al. 1993a, b). Cholesterol release is also associated with increasing tyrosine phosphorylation of sperm proteins and rise in the intracellular pH, which both are important for acrosomal release and post-fertilisation events. Cholesterol release from spermatozoa in vitro resulting in sperm capacitation can be achieved by incubation with a number of substances known to bind cholesterol.
  • the total amount of cholesterol per spermatozoa may vary more than ten-fold between subjects, but the fertilisation capacity that relates to cholesterol content seem mostly to be correlated to the ability to loose cholesterol during the post-ejaculatory period (Benhoff et al., 1993b).
  • the absolute sterol content of spermatozoa may not be as important as the ratio between free cholesterol and phospholipids.
  • Unexplained infertile men and oligoasthenospermic men may have an increased free cholesterol/phospholipid (C/PL) ratio as compared to normal fertile men but the clinical importance of the cholesterol level appear to be ambiguous (Sugkraroek et al., 1991; Huacuja et al., 1981).
  • Treatment of human spermatozoa with phospholipid preparations increased their binding to the egg zona pellucida concomitant with a reduction in sperm C/PL ratio (Gamzu et al., 1987).
  • the lipid content of spermatozoa is associated to motility parameters (Connor et al., 1997). Moreover, the C/PL ratio is negatively correlated to motility (Hamamah et al., 1995) and fusogenicity of spermatozoa. Whereas the C/PL ratio may be of importance for the sperm quality, the absolute amount of sterols is still an unresolved matter.
  • statin is the generic term for a compound that competitively and reversible inhibits the HMG-CoA-reductase enzyme. Applied in vivo, statins mediate a decrease in serum low density lipoproteins (LDL) and an increase in serum high density lipoproteins (HDL) by inhibiting the body's own production of cholesterol.
  • statin covers any compound, which inhibits de novo cholesterol biosynthesis by inhibiting the hydroxymethylglutaryl coenzyme A reductase (HMG-CoA-reductase) enzyme.
  • the present invention offers a new and simple method to increase the number and the developmental competence of both female and male germ cells and the organisms resulting from the fertilisation of such germ cells.
  • the fertility improvement is obtained without the side effects of the hormone treatment applied in the most of the presently used methods.
  • the present invention can also be applied in combination with the presently used in vitro methods used in the clinic such as In Vitro Maturation (IVM), and In Vitro Fertilisation-Embryo Transfer (IFV-ET) technologies employing varying extents of exogenous gonadotrophin stimulation and germ cell culturing.
  • IVM In Vitro Maturation
  • ISV-ET In Vitro Fertilisation-Embryo Transfer
  • the present invention relates to methods whereby the number and quality of mammalian gametes and embryos can be increased.
  • the methods applied in vivo or in vitro either inhibit the sterol de novo biosynthesis of germ cells of both sexes and their neighbouring cells or promote sterol efflux from female germ cells prior to fertilisation, or both.
  • the processes that are imagined to be manipulated by the substances described in the present invention are described in figure 11.
  • the working mechanism is postulated to involve a lowering of the ratio between free (underivatised) sterol and phospholipid in mammalian germ cells and/or gametes, but other mechanisms that involve the use of mevalonate and post-mevalonate products may likewise be involved.
  • the invention uses a pharmacological regime that inhibits the endogenous de novo biosynthesis of cholesterol in vivo and/or in vitro by administering or adding a compound that antagonises the 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme (HMG-CoA-reductase) in germ cells and germ cell supporting tissues.
  • HMG-CoA-reductase inhibitors are used in a pulsate manner, which contrasts the normal use of these compounds for atheroscleretic prophylactics.
  • the HMG-CoA-reductase inhibitors are used in a concentration that may be considerably above the concentration usually used for atheroscleretic prophylactics.
  • the treatment mediates (an) intracellular effect(s) that rescue otherwise non-viable or non-fertilisable germ cells and renders such germ cells viable and prone to fertilisation.
  • the treatment results in an increased germ cell survival rate and an enhanced germ cell maturation.
  • the treatment leaves the germ cells more prone to maturation and participation in post-maturational events with impact on fertilisation and early embryo development, possibly by affecting the ratio between sterols and phospholipids.
  • the number of germ cells that can be used for later fertilisation is thereby increased, which cause an increasing conception success and an increase in the number of progeny per adult female per reproductive cycle.
  • the present invention relates to a non-hormonal pharmacological treatment during the follicular phase that results in an increased number of retrieved oocytes and an improvement in their quality in terms of morphology and fertilisability.
  • the method of the invention operates without the risks of possible side effects of the conventional hormonal treatment.
  • This new non-hormonal pharmacological method is also useful in combination with conventional IVF treatment protocols for women, as well as with applied techniques for livestock or animal breeding in general and under conditions where in vitro techniques are applied as there is a need for improved methods for providing an increased number of retrievable germ cells and improvements in their quality in terms of viability and fertilisability.
  • the present invention relates to a method for inhibiting the sterol de novo biosynthesis with the effect of decreasing the sterol content of the germ cells and possibly decreasing the ratio between free sterols and phospholipids in at least one mammalian germ cell and/or gamete and thereby increasing the developmental competence of at least one mammalian germ cell, gamete, zygote, early embryo, blastocyst, implanted blastocyst and/or embryo and/or the number of mammalian gametes, zygotes, early embryos, implanted blastocysts, embryos and/or foetus, the method comprising administering a compound which is capable of inhibiting the sterol de novo biosynthesis to a mammal in need thereof.
  • the present invention relates to a method for increasing the sterol efflux from at least one mammalian ovary and/or one mammalian oocyte and/or mamma-lian female gamete and/or ovary derived cell surrounding an oocyte, thereby increasing the developmental competence of said at least one mammalian ovary and/or mammalian oocyte and/or mammalian female gamete and/or ovary derived cell surrounding an oocyte, the method comprising administering a compound or a combination of compounds capable of promoting sterol efflux to a mammal in need thereof.
  • the term "germ cell” relates to any mammalian cell capable of producing a gamete and a "gamete” is defined as a mature male or female germ cell possessing a haploid chromosome set and capable of initiating formation of a new diploid individual by fusion with a gamete of the opposite sex, the term thus include e.g. a secondary spermatocyte, spermatide, a spermatozoon, a cumulus enclosed oocyte (oocyte and cumulus cells) and an isolated oocyte.
  • the term "mammal” relates to the highest class of the subphylum Vertebrata comprising all animals that nourish their young with milk secreted by mammary glands such as human, horse, cow, rat, mouse, pig, sheep, goat, llama, dog, cat and mink.
  • mammary glands such as human, horse, cow, rat, mouse, pig, sheep, goat, llama, dog, cat and mink.
  • the present invention relates to humans.
  • the present invention relates to mammals that are industrially applicable due to human agriculture and pet industry, such as the animals mentioned above.
  • zygote is used in the present context to describe a cell formed by the union of two gametes that develop through pro-nuclei generation and pro-nuclei fusion and initiate the first cellular cleavage, or, in a broader context, as the developing individual produced from gametes.
  • the term "early embryo” is in the present context defined as the two-cell or multicellular organism produced from the zygote until the blastocystic stage, a blastocyst being defined as the stage of the early embryo where a fluid filled cavity, the blastocoel, emerges.
  • the term "implanted blastocyst” relates in the present context to a blastocyst that has initiated hatching from the surrounding eggshell, the zona pellucida, and has just begun the penetration of the uterine lining of the foster mother.
  • An embryo is in the present context defined as the stage of the mammalian development from the termination i of the implantator ⁇ process and the beginning of the early stages of growth and differentiation that are characterised by cellular cleavage, the laying down of fundamental tissues and the formation of primitive organs and organ systems. Especially this term refers to the developing human individual from the time of implantation to the end of the eighth week after conception, which divides embryo from foetus.
  • developmental competence is defined as the ability to develop into a specific unicellular or multicellular stage and includes in the present context any cellular differentiation, development and maturation, fertilisation, as well as post-fertilisation processes covering early embryo development, blastocyst formation, implantation and embryonic and foetal growth.
  • the germ cell is a human germ cell such as an oocyte, an immature oocyte retrieved by aspiration of a pre- antral follicle(s), an immature oocyte retrieved by aspiration of small or medium sized antral follicle(s) and/or an immature oocyte obtained after culture of at least one immature follicle at the primordial to the preantral stage, an ejaculated spermatozoa, an immature spermatozoa retrieved from the male reproductive tract, a spermatid or a spermatocyte.
  • the term "immature” relates to germ cells and gametes not capable of conceiving naturally in their present stage.
  • a compound to be used in the method of the present invention and capable of inhibiting the sterol de novo biosynthesis is preferably selected from the class of compounds that antagonise the 3-hydroxy-3-methylglutaryl coenzyme A reductase enzyme (HMG-CoA- reductase), such as any substance that binds the HMG-CoA-reductase enzyme reversibly or irreversibly, competitively or non-competitively with HMG-CoA, and reduces the amount of molar mevalonate produced per molar HMG-CoA-reductase per unit time.
  • One or more compounds may be used, i.e. the compounds may be used separately or two or more compounds may be used in combination.
  • a HMG-CoA-reductase inhibitor relevant in the present context may be qualified by a test involving the purified solved HMG-CoA-reductase enzyme, freeze-thaw-solved HMG-CoA- reductase enzyme, microsomal fractions obtained from gonads or gonadal cells from a mammal. Such cellular and sub-cellular fractions are referred to as "HMG-CoA-reductase enzyme preparations" below.
  • V c molar mevalonate produced/mg enzyme preparation/second.
  • the amount of molar mevalonate produced per molar HMG-CoA-reductase per unit time during steady-state will be:
  • V i( ioo M M) molar mevalonate produced/mg enzyme preparation/second.
  • the substance then reduced the amount of molar mevalonate produced per molar HMG- CoA-reductase per unit time to 90% or less of control values, a control value being the conversion rate when no inhibitor is applied. If such condition of at least 10% inhibition at 100 ⁇ M substance added is met, the substance qualifies in the present context as an HMG- CoA-reductase inhibitor.
  • said 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%.
  • the compound effectuates an almost complete decrease of the rate of conversion of 3-hydroxy-3-methylglutaryl-coenzyme A into mevalonic acid under the aforementioned conditions
  • enzymes to be inhibited are mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, dimethylallyl transferase, geranyl transferase, squalene synthase, squalene monoxygenase, oxidosqualene lanosterol cyclase and lanosterol synthase.
  • the inhibitory potential of such drugs should be assessed by their ability to decrease the amount of enzyme product produced per molar enzyme per unit time in incubated tissue or tissue fragments from gonads and compared to incubations that differ from this by having no potential inhibitor present.
  • the experimental set-up should therefore be similar to those above-mentioned conditions for test of HMG-CoA-reductase inhibitory substances, apart from involving the natural substrate of the enzyme in question instead of HMG-CoA, and this substrate should be used in free concentrations equal to the Km (the Michaehs-Menten constant) of the enzyme-substrate reaction.
  • the inhibitory effect of the substance under conditions similar to the above-mentioned conditions for HMG-CoA-reductase inhibition, i.e. when the substance is applied in concentrations of 100 ⁇ M, should be at least 10% in order to be qualified as a sterol synthesis inhibitor.
  • Such list will include, but not be limited to, zaragozic acid, azasqualene, bisphosphonates (e.g. cycloheptylam ⁇ nomethylene-l,l-b ⁇ sphosphon ⁇ c acid, (3-(l-pyrol ⁇ d ⁇ no)-l- hydroxypropyl ⁇ dene-l,l-b ⁇ sphosphon ⁇ c acid) and PHPBP (3-(l-p ⁇ pe ⁇ d ⁇ no)-l- hydroxypropyl ⁇ dene-l,l-b ⁇ sphosphon ⁇ c acid)) and other squalestatms (inhibitors of squalene synthase), imidazoles, pamidronate, alendronate and oxysterols.
  • bisphosphonates e.g. cycloheptylam ⁇ nomethylene-l,l-b ⁇ sphosphon ⁇ c acid, (3-(l-pyrol ⁇ d ⁇ no)-l- hydroxypropyl ⁇ dene-l,l-
  • the present invention relates to the use of any compound which, during its interaction with the gonadal tissue, will inhibit the de novo sterol biosynthesis and thereby lead to a decreased amount of free sterols in epididymal spermatozoa or oocytes and cumulus-oocyte complexes of mammals possibly resulting ultimately in a lowered sterol/phospholipid ratio in the germ cell supporting cells.
  • the invention relates to any combinations of compounds and administration that through an inhibitory interaction with enzymes involved in the pre-sterol pathway, i.e.
  • the invention relates to any of the above mentioned regimes that result in a decrease of at least 10%, such as at least 5% of free sterols in the epididymal spermatozoa or oocytes and cumulus-oocyte complexes of mammals as compared to a situation where the mammal receives no such treatment.
  • the invention also relates to any combination of a de novo sterol biosynthesis inhibitory regime and known techniques that are applied in order to propagate germ cells in mammals in the need thereof, such as IVM and IVF-ET techniques, stimulation with exogeneous gonadotrophins and/or other techniques.
  • gonadotrophin relates to a mammalian peptide hormone produced by the pituitary or the placenta that contains two peptide subunits, ⁇ and ⁇ , of which the ⁇ -subunit is shared by them and the ⁇ -subunit distinguishes their chemical nature and biological characteristics.
  • Known gonadotrophins so far include follicle stimulating hormone (FSH), lutemizing hormone (LH) and cho ⁇ on gonadotrophin (CG).
  • the compound used for inhibition of sterol biosynthesis de novo is a statin.
  • statin is selected from the group consisting of atorvastatin, ce ⁇ vastatin, dalvastatin (RG 12561), fluvastatm, BAY W 62, pravastatin, lovastatin (formerly called mevinolin or monacolin K), 28, HR 780, pravastatin, simvastatin, compatin, methyl-compactin, mevastatin (formerly called campactm or ML-236B), ML- 236A, ML-236C, dihydrocompactin, monacolin ., monacolin L, monacolin M, monacolin X, dihydromonacohn L, mevinolin, 3 ⁇ -hydroxycompact ⁇ n acid monosodium salt (pravastatin)
  • a compound capable of promoting sterol efflux is any substance that during incubation of tissue or cells will lead to an increase in the diffusion of sterol(s) into the medium and therefore efflux of said sterol(s) from the biological material to a sterol acceptor that is initially unsaturated by sterol(s), e.g. serum albumin or another protein, carbohydrate or lipid material.
  • Sterol efflux can be assayed by incubating conditioned and radio labelled Fu5AH rat hepatoma cells in Eagle's minimum essential medium supplemented with human serum albumin at 3 mg/mL (3 %o w/v), as well as the putative sterol binding or transporting substance in question. Conditioning and labelling of the cells as well as cell density during culture must conform to literature descriptions in Moya et al. (Moya et al., 1994, Arte ⁇ oscler. Thromb., 14(7): 1056-1065).
  • Fu5AH rat hepatoma cells are incubated under these conditions for 4 hours at 37°C and, subsequently, after incubation spent media are separated by centrifugation and washing of the cells in new medium (Eagle 's minimum essential medium supplemented with human serum albumin at 3 mg/mL) and radioactivity measured in the two fractions, then:
  • RA ce n s represents the total radioactivity in the cell fraction and RA medlum represents the total radioactivity in the fraction representing medium including the medium from three consecutive washings. If no other substance than serum albumin at 3 mg/mL is present in the medium, then:
  • the fractional release of 3 H-Cholesterol from the cells is increased 50 % or more after 4 hours of incubation at 37°C, i.e. :
  • the amount of SBTS added to the medium results in an increase in the medium osmolarity of 0.001 - 10%. In another preferred embodiment the amount of SBTS added to the medium is in the range of 0.001 - 10 mM. In yet another preferred embodiment the amount of SBTS added to the medium is in the range of 0.01 - 50%o (w/w), such as but not limited to 0.5-4%o.
  • cyclodextrins capable of promoting the sterol efflux could be cyclodextrins, chemically modified cyclodextrins, such as but not limited to sulphated cyclodextrins, high density lipoproteins (HDL), an apoprotein derived from HDL, sterol carrier protein I and II or any other protein capable of solubilizing sterols.
  • cyclodextrins chemically modified cyclodextrins, such as but not limited to sulphated cyclodextrins, high density lipoproteins (HDL), an apoprotein derived from HDL, sterol carrier protein I and II or any other protein capable of solubilizing sterols.
  • HDL high density lipoproteins
  • sterol carrier protein I and II any other protein capable of solubilizing sterols.
  • the compound or a combination of compounds capable of promoting the sterol efflux is selected from the group consisting of cyclodextrin, chemically modified cyclodextrins, such as but not limited to sulphated cyclodextrins, high density lipoproteins (HDL), an apoprotein derived from HDL, and sterol carrier protein I and II.
  • cyclodextrin chemically modified cyclodextrins, such as but not limited to sulphated cyclodextrins, high density lipoproteins (HDL), an apoprotein derived from HDL, and sterol carrier protein I and II.
  • the compound capable of promoting the sterol efflux is a cyclodextrin.
  • cyclodextrin relates to a homologous group of cyclic glucans consisting of alpha-1,4 bound glucose units obtained by the action of cyclodextrin glucanotransferase on starch or similar substrates. Cyclodextrins form inclusion complexes with a wide variety of substances and thereby co-solubilize substances that are otherwise insoluble or low-soluble in water or buffers. Cyclodextrins differ from one another by the number of glucopyranose units in the structure.
  • the parent cyclodextrins contain 6, 7 and 8 glucopyranose units and are referred to as alpha (a-), beta ( ⁇ -), and gamma (g-) cyclodextrin respectively.
  • statin or compound capable ofinhibiting sterol biosynthesis de novo that should be administered to the mammal in the need thereof depends on the particular statin and/or compound chosen and is between 0.01-100 mg per kg body weight per day, such as 0.05-90 mg per kg body weight per day, 0.1-80 mg per kg body weight per day, 1-50 mg per kg body weight per day, 1-25 mg per kg body weight per day, 1-10 mg per kg body weight per day, 1.0-10 mg per kg body weight per day, 2.0-10 mg per kg body weight per day, 2.5-10 mg per kg body weight per day, 3.0-10 mg per kg body weight per day, 4.0-10 mg per kg body weight per day, 5.0-10 mg per kg body weight per day, 6.0- 10 mg per kg body weight per day, 7.0-10 mg per kg body weight per day, 8.0-10 mg per kg body weight per day, 9.0-10 mg per kg body weight per day or between 1.0-9.0 mg per kg body weight per day, 2.0-8.0 mg per kg body weight per day, 2.5-7.5 mg per kg
  • the amount of compound capable of promoting sterol efflux that should be administered to the mammal in the need thereof depends on the particular compound chosen but will likely be between 0. 01-1000 mg per kg body weight per day, such as 0.5-900 mg per kg body weight per day, 1-800 mg per kg body weight per day, 10-500 mg per kg body weight per day, 10-250 mg per kg body weight per day, 10-100 mg per kg body weight per day, 20- 100 mg per kg body weight per day, 25-100 mg per kg body weight per day, 30-100 mg per kg body weight per day, 40-100 mg per kg body weight per day, 50-100 mg per kg body weight per day, 600-100 mg per kg body weight per day, 70-100 mg per kg body weight per day, 80-100 mg per kg body weight per day, 90-100 mg per kg body weight per day or between 10-90 mg per kg body weight per day, 20-80 mg per kg body weight per day, 25-75 mg per kg body weight per day, 40-60 mg per kg body weight per day or 45-55 mg per kg body weight per day
  • the reproductive state of the female mammal is cyclic with a complex interaction between the hypothalamus, anterior pituitary and the ovaries leading to the process of ovulation.
  • the cycle is repeated with an average period around 28 days (range 21-35 days).
  • the first phase, menstruation, lasts 3-5 days.
  • the first day of a cycle in a human is the first day of the first phase, that is the first day of menstrual bleeding.
  • the second follicular phase of the human ovary corresponds to the proliferative phase of the endometrium and is least 5-16 days (i.e. highly variable).
  • a luteal phase which corresponds to the secretory phase of the endometrium and is usually more constant at around 14 days.
  • the moment of ovulation is the timing of the discharge of an ovum from the ovary.
  • the period for which it is necessary to use treatment regimen of the present invention is calculated within each ovulatory cycle.
  • the ovulatory cycle differs between the individual species among the mammals, therefore the optimum period where the administration is needed should preferably be calculated in regards to each individual species based on their ovulatory cycle.
  • the duration of the ovulatory cycle differs highly from the human cycle. The average duration of the ovulatory cycle in various mammals are given in table 1 below:
  • a female mammal in need thereof is to be administered a compound which is capable of inhibiting the sterol de novo biosynthesis and/or promoting sterol efflux during a period of 0 hour-12 days before ovulation, such as 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 before ovulation.
  • a compound which is capable of inhibiting the sterol de novo biosynthesis and/or promoting sterol efflux during a period of 0 hour-12 days before ovulation such as 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 before ovulation.
  • the administration of a compound which is capable of inhibiting the sterol de novo biosynthesis and/or promoting sterol efflux could be extended up til 3 days after ovulation, such as 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours or 72 hours after ovulation.
  • One important aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a combination of a compound capable of inhibiting the sterol de novo biosynthesis and a compound capable of increasing the sterol efflux, optinonally in a combination with a pharmaceutically acceptable carrier.
  • a synergistic effect between the statin and the cyclodextrin is obtained due to the temporal coincidence of the two different processes that have an effect on the sterol content of the cell thereby creating an effect that is above the summation of the two regimes used separately, preferably by at least a factor 3.
  • a preferred composition according to the invention is the combination comprising a statin and a cyclodextrin.
  • the invention relates to the use of a statin and a cyclodextrin together with a pharmaceutically acceptable carrier for the preparation of a medicament for increasing the fertility of a mammal in the need thereof.
  • growth factors have long been used in the clinic, thus in a preferred embodiment the present invention relates to compositions containing a statin and a cyclodextrin further comprising a growth factor.
  • growth factors could be gonadotropins (FSH, LH, CG), insulin-like growth factors (IGFs), epidermis growth factors (EGFs), Insulin, growth hormone, interleukines or other peptide hormones.
  • the growth factors is selected from the group consisting of gonadotropins (FSH, LH, CG), insulin-like growth factors (IGFs), epidermis growth factors (EGFs), Insulin, growth hormone, interleukines and other peptide hormones.
  • FSH gonadotropins
  • IGFs insulin-like growth factors
  • EGFs epidermis growth factors
  • Insulin growth hormone
  • interleukines interleukines
  • compositions comprising a combination of a compound capable of inhibiting the sterol de novo biosynthesis and a growth factor.
  • said compound is a statin.
  • compositions comprising a combination of a compound capable of promoting the sterol efflux and a growth factor.
  • said compound is a cyclodextrin.
  • the compound and/or any of the compositions mentioned above are administrated to the mammal 1-10 times a day, such as 1 time a day, 2 times a day, 3 times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day or 10 times a day
  • the compound and/or compositions may also be administered one to several times or on a continuous basis via a tissue-pump device during this period.
  • the compound and/or compositions may be administered one to several times on a continuous basis via a tissue- pump device during this period.
  • the compound(s) and/or compositions may be administered in a formulation that retards its release in the body.
  • Such formulation will provide for the possible administration of the compound(s) and/or compositions in a period of say 0-30 days prior to the aforementioned period, but effecting a tissue exposition in the very same period by a slow release in vivo. Such administration will thereby provide the similar desired effect on the germ cell development as an administrative regime resulting in an immediate body release.
  • the present invention also relates to the use of any of the above mentioned compositions for the preparation of a pharmaceutical composition for use in any of the methods of the invention.
  • an egg is an ovum that has undergone chromosomal reduction, and therefore may be ready for fertilisation, and takes the form of a relatively large non-motile gamete which contributes to most of the cytoplasm of the zygote.
  • These eggs even though released as mature and ready to be fertilised, somehow could lack the ability to complete the fertilisation process, which is helped by adding a compound capable of inhibiting the sterol de novo biosynthesis.
  • Mevacor ® a pharmaceutical product of the statin group that works by inhibiting HMG-CoA-reductase, the key regulatory step of cholesterol biosynthesis de novo, causes a decrease in serum cholesterol, and, as shown here, influences the amount of free cholesterol in organs like the liver and the ovary (example 3).
  • the ovaries respond by producing a larger number of fertihsable oocytes and/or increase the developmental competence of the prevalent ovulated eggs, which is evidenced by the increased number of 2-cells in tuba following mating with normal (untreated) male mice (example 1).
  • statins with respect to meiotic maturation is demonstrated by example 4, which shows that applying a statin (Compactin, Sigma) to the culture medium mediates oocyte meiotic maturation in vivo. Moreover, the effect appears to be additive to the well-known meiosis maturation effect of gonadotrophins (here FSH).
  • statin Compactin, Sigma
  • Metabolic data are included that show correlation between oocyte maturation and de novo biosynthesis of cholesterol.
  • the replacement of Mevacor ® with Compactin in vitro was necessary because the active component in Mevacor ® , lovastatin, is only active after bioconversion in the liver.
  • the cholesterol-lowering enhancement by statins in comparison to hCG as the more natural ovulatory trigger is evidenced by example 3.
  • the overall decrease of free cholesterol during hormone induced oocyte development and ovulation in the mouse is evidenced by data represented in example 5.
  • statin Compactin, Sigma
  • example 7 shows that applying a statin (Compactin, Sigma) to the IVM culture medium mediates improved oocyte fertilisation by sperm using IVF. It is demonstrated that the presence of compactin during IVM mediated an increased number of fertilised oocytes after IVF as compared to a control incubation and an incubation with FSH. The rate of generated oocytes remains the same (fig 8). It is surprisingly that the statin has an enhanced effect as compared to the addition of FSH. In figure 9 the results show that cells treated with Mevinolin has reached a more progressed state after 72 hours of culture following IVF, as compared to the two aforementioned groups for comparison.
  • the rate of degeneration after 72 hours of culture is lower in the Mevinolin treated group as compared to the two aforementioned groups for comparison, which may correspond to the fact that the oocytes cultured in the presence of Mevinolin displayed a high degree of cumulus expansion after IVM which was not visible during the microscope examination. Cumulus expansion is pre-requisite for proper oocyte maturation and fertilisation preparation.
  • a background for the present invention is the prior knowledge that loss of free sterol from the plasma membrane is a pre-requisite for proper terminal maturation of the mammalian spermatozoa.
  • the present invention is based on three new major ideas.
  • the membrane sterol decrease is a biochemical process that also takes place during final pre-fertilisation maturation of the oocyte. No knowledge on ovarian or oocyte loss of sterol prior to fertilisation has hitherto been presented in the scientific literature or elsewhere.
  • the sterol loss and/or processes related to a lowered intracellular sterol content can be enhanced in germ cells of both sexes by applying inhibitors of the de novo sterol biosynthesis pathway. The inhibition will not lead to full sterol compensation by the germ cell or germ cell environment by for instance increasing poprotein intake or increasing sterolester hydrolysis. This is probably because the vast majority of the sterols of the germ cell environment closely prior to fertilisation are produced in situ under natural conditions.
  • the sterol biosynthesis inhibitory action will lead to a significant decrease in the membrane sterol content that may substitute the natural sterol loss stimulated by hormones. Moreover, the natural sterol loss will be enhanced by the inhibition of the sterol biosynthesis de novo.
  • the de novo sterol biosynthesis inhibition mediates important maturational processes per se, e.g. meiotic maturation, and constitutes a mechanism that mediates pivotal processes for germ cells development apart from capacitation in spermatozoa Also, the membrane sterol loss mediates important maturational processes.
  • the present application provides experiments wherein statin is used in order to manipulate the level of free sterol negatively in the oocyte and the oocyte environment.
  • the experiments relating to the present invention show that enhancement of the natural sterol loss during final oocyte maturation results in increased viability and fertilisability of oocytes, probably by rescuing a number of "borderline" oocytes that would otherwise have become atretic and thereby lost during the hormone dependent growth phase.
  • giving statin to a male mouse in vivo results in a decreased sterol de novo biosynthesis and/or decrease of phospholipid/sterol ratio in the motile cells (normal spermatozoa) of the epididymis.
  • the present invention demonstrates (figure 5) that hormone stimulated and statin treated prepubertal female mice produce on average about 70% more 2-cells as compared to the same mice stimulated with hormone alone. It is contemplated that even higher percentages may be obtained by tuning the statin dose and period of treatment.
  • the example demonstrates that applying a statin to a female mammal increases the developmental competence of its gametes.
  • the present invention demonstrates that a statin can be administered during the follicular phase maturation of follicles in vivo with the effect on enhancing the maturational events.
  • the effect possibly relies on a decrease in the amount of total free sterols in the germ cell as compared to animals where de novo sterol biosynthesis is taking place, which leads to a decreasing sterol to phospho pid ratio in the plasma membrane resulting in a germ cell more prone to participation in the process of fertilisation.
  • in vitro oocyte maturation and fertilisation will benefit from a treatment that inhibits sterol de novo biosynthesis, resulting in increased rates of meiosis and enhanced post-meiotic maturation.
  • Example 1 and 9 shows that this sterol decrease is accompanied by an improved germ cell development and/or quality.
  • a sterol usage dichotomy is therefore operating in the female germ cell and/or germ cell environment that enables the cells to respond by decreasing the sterol output in one cellular and/or biochemical compartment and at the same time leaving another compartment unaffected with respect to sterol usage and/or presence.
  • the present invention relates to the use of known generic compound for improving the fertility of a mammal by inhibiting sterologenesis de novo.
  • the underlying mechanism in the demonstrated fertility improvement may be a modulation of the sterol/phospholipid ratio.
  • the present invention shows that decreasing the sterol biosynthesis de novo in mammals by pharmacological intervention produces female germ cells with an improved developmental competence.
  • the cross-sexual aspects of the sterol decreasing mechanism has guided the belief that also male germ cells will benefit from a regime built on sterol inhibition prior to fertilisation.
  • the present invention relates to situations where mammals have difficulties in producing offspring, such as in situations where the female mammal is unable to achieve a pregnancy after more than one day of unprotected intercourse, such as 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15, years, 20 years or more than 60 years of unprotected intercourse.
  • timing of unprotected intercourse described above is related to the period in which the mammals regularly have intercourse with the purpose of conceiving.
  • the present invention also relates to situations in which the mammal does not suffer from infertility, but merely wishes to obtain a higher conception chance and thereby faster conception, especially in respect to female humans in the age range from 28 to 42 in which the conception level decreases proportionally with age.
  • the present invention also relates to human females that may benefit from the treatment by obtaining twin-carriage or tri- carriage.
  • the present method relates to livestock and other economically valuable mammals that could benefit from it by producing an increased number of foetuses per pregnancy.
  • the present invention relates to a method for inhibiting the sterol de novo biosynthesis in at least one mammalian germ cell and/or gamete and thereby increasing the developmental competence of at least one mammalian gamete, zygote, early embryo, implanted blastocyst and/or embryo and/or the number of mammalian gametes, zygotes, early embryos, implanted blastocysts and/or embryos, the method comprising administering to a male a compound which is capable of inhibiting the sterol de novo biosynthesis during a period of 0 hour- 12 days before ejaculation, such as 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
  • One important aspect of the present invention relates to a method for improving the number and developmental competence of at least one mammalian germ cell and/or gamete in vitro and thereby increasing the fertilising ability of at least one mammalian gamete and/or the developmental competence of the resulting zygote, early embryo, implanted blastocyst and/or embryo and/or the number of mammalian gametes, zygotes, early embryos, implanted blastocysts and/or embryos, the method comprises adding to a medium a substance which is capable inhibiting the sterol de novo biosynthesis in cells.
  • Another aspect of the present invention relates to a method for increasing the sterol efflux from at least one mammalian ovary and/or mammalian oocyte and/or mammalian female gamete and/or ovary derived cell surrounding an oocyte, thereby increasing the developmental competence of said at least one mammalian ovary and/or mammalian oocyte and/or mammalian female gamete and/or ovary derived cell surrounding an oocyte, the method comprising adding to a medium a compound or a combination of compounds capable of promoting the sterol efflux in any of the above mentioned cells or tissues.
  • the present invention thus relates to a cell culture medium comprising a cyclodextrin.
  • the present invention relates to a cell culture medium comprising a statin.
  • the present invention relates to a cell culture medium comprising a statin and a cyclodextrin.
  • an in vitro medium is defined as a medium which supports the respiration and/or maturation of at least one oocyte in its somatic cellular context or alone and/or at least one spermatozoo, said medium may comprise cell culture media which 5 support in vitro fertilisation (IVF) protocols and/or support survival and maturation of gametes in vitro.
  • IVF in vitro fertilisation
  • Several parameters for a medium that is likely to be useful for in vitro fertilisation (IVF) protocols and/or support survival and maturation of gametes can be established to define said medium. Foremost the concern to pH, osmolarity and the content of the buffered air under which the media is cultured is of high importance.
  • an in vitro medium has preferably an osmolarity between 240 to 320 mOsm/l, a pH of 7 to 7.8, and is C0 2 -buffered under 3-7% C0 2 in atmospheric air or 53-7% C0 2 , 1-5% 0 2 and 88-96% N 2 .
  • An osmolarity between 200 to 370 mOsm/l may more specifically relate to levels such as 15 250 to 310 mOsm/l, 260 to 300 mOsm/l, 270 to 290 mOsm/l, 275 to 285 mOsm/l, 276 to 284 mOsm/l, 277 to 283 mOsm/l, 278 to 282 mOsm/l or 279 to 281 mOsm/l.
  • the osmolarity is about 280 mOsm/l.
  • a pH between 7 to 7.8 pH may more specifically relate to levels such as 7.1 20 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 e.g. 7.39 to 7.41 pH.
  • the pH is about 7.4.
  • an IVF-medium could be that the medium contains 111 to 171 mEq/l
  • the medium is to be cultured at 37°C (+/- 1°C).
  • the cell culture medium relates to a chemically defined cell culture medium.
  • the term "chemically defined medium” is used to 35 denote a medium without biologically extracted serum substances, and where all components and their concentrations are known and described. If hormones or serum derived substances are to be added to the medium, recombinant hormones or serum derived substances are preferred.
  • the culture medium contains BSA or HSA obtained by recombinant methods, thereby eliminating the inter- mammal serum contact.
  • the immature human gametes are cultured in a chemically defined medium without addition of directly serum-derived products or the patients' own serum or any other serum product derived directly from a mammal, such as a human or cattle.
  • growth factors such as, but not limited to, gonadotropins, are presently used in IVM treatment.
  • a preferred embodiment of any of the media described above further relates to a cell culture medium further comprising at least one growth factor, wherein said growth factor is selected from the group comprising gonadotropin (FSH, LH, CG), IGF, EGF, Insulin, growth hormone, interleukines or other peptides.
  • the method of the present invention will preferably start with immature or not fully matured female gamete.
  • an immature oocyte will be recognised as an oocyte with tight cumulus masses, no polar bodies or germinal vesicles visible.
  • These oocytes are readily recognised by a person involved in routine IVF-treatments as being immature oocytes, and thus one embodiment of the present invention relates to a method for maturating an immature oocyte obtained from a stage between the primordial follicle and the pre-antral stage comprising culturing said immature oocyte in a cell culture medium as described above.
  • the amount of statin administered to an in vitro medium depends on the particular statin and is 0.01-1000 ⁇ M, 0.05-1000 ⁇ M, 0.1-1000 ⁇ M, 0.5- 1000 ⁇ M, 1-1000 ⁇ M, 5-1000 ⁇ M, 10-1000 ⁇ M, 20-1000 ⁇ M, 25-1000 ⁇ M, 30-1000 ⁇ M, 50-1000 ⁇ M, 75-1000 ⁇ M, 100-1000 ⁇ M, 200-1000 ⁇ M, 300-1000 ⁇ M, 400-1000 ⁇ M, 500- 1000 ⁇ M, 750-1000 ⁇ M or between 10-900 ⁇ M, 25-750 ⁇ M, 50-500 ⁇ M, 100-400 ⁇ M, 200- 300 ⁇ M, 0.01-20 ⁇ M, 0.01-15 ⁇ M, 0.05-15 ⁇ M, 0.1-10 ⁇ M or 1 -10 ⁇ M.
  • the culturing of at least one oocyte in an in vitro medium according to the method of the present invention is optimised to inhibit the sterol de novo biosynthesis and/or promoting sterol efflux and/or decreasing the ratio between free sterol and phospholipid by culturing the at least one oocyte in its somatic cellular context or alone in an in vitro medium between 1 second and 15 days such as between 1 second and 14 days, between 1 second and 13 days, between 1 second and 12 days, between 1 second and 10 days, between 1 second and 9 days, between 1 second and 8 days, between 1 second and 7 days, between 1 second and 6 days, between 1 second and 5 days, between 1 second and 4 days, between 1 second and 3 days, between 1 second and 2 days, between 1 second and 1 day, between 1 second and 23 hours, between 1 second and 22 hours, between 1 second and 21 hours, between 1 second and 21 hours, between 1 second and 20 hours, between 1 second and 19 hours, between 1 second and 18 hours, between 1 second and 15 hours, between 1 second and 12 hours,
  • the culturing of spermatozoa in an in vitro medium by the method according to the present invention is optimised to inhibit the sterol de novo biosynthesis and/or promoting sterol efflux by culturing the spermatozoa in a in vitro medium between 1 second and 8 days such as between 1 second and 7 days, between 1 second and 6 days, between 1 second and 6 days, between 1 second and 4 days, between 1 second and 3 days, between 1 second and 2 days, between 1 second and 1 days, between 1 second and 23 hours, between 1 second and 22 hours, between 1 second and 21 hours, between 1 second and 21 hours, between 1 second and 20 hours, between 1 second and 19 hours, between 1 second and 18 hours, between 1 second and 15 hours, between 1 second and 12 hours, between 1 second and 11 hours, between 1 second and 10 hours, e.g. between 1 second and 5 hours.
  • 1 second and 8 days such as between 1 second and 7 days, between 1 second and 6 days, between 1 second and 6 days, between 1 second and 4 days, between 1 second and
  • IVF treatments generally involve hormone treatment of the female mammal in the need thereof. Even though hormone treatment does have several side effects, it is still one of the most effective treatments of infertility today.
  • a standard IVF treatment of an infertile couple usually involves a sample of mature egg collected from the woman after treatment with exogeneous hormones. The regime usually implies that the woman is treated with gonadotrophin releasing hormone agonists or antagonists, which both result in a substantially decreased extent of gonadotrophin release from the pituitary. Exogeneous gonadotrophins are subsequently provided by intramuscular or intraperitoneal injections, which secure that a number of oocytes will grow and mature.
  • the additive and possibly synergistic effect of administrating a compound capable of inhibiting the sterol de novo biosynthesis and/or a compound capable of promoting sterol efflux in combination with an administration of gonadotrophins or other hormone(s) known to the person skilled in the art to be used for IVF treatment is a preferred embodiment of the present invention.
  • the combination of treatment in vivo by a compound capable of inhibiting the de novo sterol biosynthesis and/or a compound capable of promoting sterol efflux with the treatment in vivo of exogeneous gonadotrophins, a gonadotrophin-releasing hormone (gnrh) or a gnrh agonist and/or antagonist provides a additive/synergistic effect that increases the number of mature ova ready for fertilisation in vivo or in vitro and/or the number of fertilised ova per total number of ova and/or the number of blastocysts per fertilised ova, and/or the number of implanted blastocysts per developed blastocyst and/or the number of delivered offspring per reproductive cycle as compared to a standard IVF treatment.
  • gnrh gonadotrophin-releasing hormone
  • the administration of de novo sterol biosynthesis inhibitors and/or compounds capable of promoting sterol efflux to women during the hormone stimulation pre-aspiration period will possibly lead to the saving of exogeneous gonadotrophins to be used for obtaining a certain number of mature oocytes, fertilised ova, blastocysts, implants and/or subsequent pregnancy.
  • Such hormone saving regime will represent a side-effect reducing and cost-reducing alternative to the use of exogeneous hormones alone, and this constitutes yet another important scope of the present invention.
  • the unwanted side effects are the development of the ovarian hyperstimulatory syndrome (OHSS) and the polycystic ovarian syndrome (PCO).
  • OHSS ovarian hyperstimulatory syndrome
  • PCO polycystic ovarian syndrome
  • statins as an adjuvant during the stimulation of the ovaries in vivo will result in a reduction in the gonadotrophin dose used in vivo and a subsequent reduction in the incidence of these side effects.
  • hormone-releasing hormone relates to an endogenous mammalian peptide hormone produced by the hypothalamus that stimulates the pituitary or other organs or tissues to release gonadotrophins.
  • Other designations of the hormone class include luteinizing hormone-releasing factor and luteinizing hormone- releasing hormone.
  • the term "gonadotrophin-releasing hormone agonist” relates to any substance that will stimulate the mammalian gonadotrophin-releasing hormone receptor in the pituitary or elsewhere to produce second messengers normally involved in the signal- transduction between binding of the gnrh-receptor and gnrh and gonadotrophin-production and/or gonadotrophin-release from the pituitary or elsewhere.
  • the term "gonadotrophin-releasing hormone antagonist” relates to any substance that will inhibit gnrh to bind gnrh-receptors and/or inhibit the signal- transduction normally involved in gnrh-binding, thereby hindering the cell bearing the gnrh-receptor to produce and/or release gonadotrophins upon stimulation with gnrh.
  • Standard IVF treatment involves that the male deliver a semen sample obtained by masturbation or electro-stimulated ejaculation.
  • the synergistic effect of administrating a compound capable of inhibiting the sterol de novo biosynthesis and/or a compound capable of promoting sterol efflux prior to obtaining the semen sample to be used for IVF treatment is likewise a preferred embodiment of the present invention.
  • the combination of treatment in vivo by a compound capable of inhibiting the de novo sterol biosynthesis and/or a compound capable of promoting sterol efflux provides a additive synergistic effect that increases the number of mature spermatozoa ready for fertilisation in vivo or in vitro and/or the number of fertilised ova per total number of ova and/or the number of blastocysts per fertilised ova, and/or the number of implanted blastocysts per developed blastocyst and/or the number of delivered offspring per reproductive cycle as compared to a standard IVF treatment.
  • IVF treatment also involves that the germ cells after retrieval from the male and female are cultured under appropriate conditions where a number of spermatozoa are mixed with at least one oocyte.
  • Yet another preferred embodiment of the present invention is the use of de novo sterol biosynthesis inhibitors and/or compounds capable of promoting sterol efflux added to the medium in which the ova and/or spermatozoa are incubated prior to mixture as well as to the medium in which the spermatozoa and ova are incubated in order to obtain a fertilised ovum.
  • Such addition will result in an increased number of mature ova ready for fertilisation in vivo or in vitro and/or the number of fertilised ova per total number of ova and/or the number of blastocysts per fertilised ova, and/or the number of implanted blastocysts per developed blastocyst and/or the number of delivered offspring per reproductive cycle as compared to a standard IVF treatment.
  • the mammalian de novo pre-sterol and sterol biosynthesis pathway The mammalian de novo pre-sterol and sterol biosynthesis pathway.
  • Pre-sterol biosynthesis ultimately starts with a condensation between acetate and coenzymeA to form acetyl-coenzymeA.
  • Acetyl-coenzymeA is processed in a multienzymatic step, which may lead to the production of squalene.
  • Squalene is converted by two enzymes into lanosterol, which is the root of the sterol biosynthesis pathway.
  • the mammalian sterol biosynthesis in the endoplasmic reticulum involves at least the indicated 7 enzymes, but may in parallel to these involve a number of enzymes with similar catalytic characteristics.
  • Enzymes are designated by italics on a grey background whereas sterol intermediates are indicated by ordinary letters with superscribed designations of the relevant C-C double bindings and non-cholesterol methyl substitutions.
  • the bi-directional arrow indicates that the enzymatic step is reversible.
  • the broken arrow between 24-enes (left-hand panel) and side-chain saturated sterols (right hand panel) indicates that the sterol delta24 reductase enzyme may have multiple sterol substrates.
  • Fig. 4 Structure, numbering of carbons and ring designation in the perhydrocyclopentanophenanthrene ring structure. C-numbering is in accordance with IUPAC convention. Fig. 5
  • COC mouse cumulus oocytes complexes
  • Compactin statin
  • GVBD represent oocytes in which no germinal vesicle was visible under a light microscope, which indicates that the oocyte has resumed meiosis.
  • PB/GVBD indicates the ratio of polar body formation amongst the oocytes that resumed meiosis during the culture period.
  • Each experiment was performed in quadruplicates and each experiment represents 30-40 COCs. Error bars indicate the standard error of the mean. Different letters above bars indicate a statistical difference at the 95 % confidence level.
  • DPM on the ordinate axis indicate the radioactivity measured by the scintillation detector after correction for counting efficiency and purification efficiencyby the HPLC method.
  • N denotes the total number of COCs extracted for cholesterol isolation by HPLC.
  • Fertilisation rate of oocyte matured in vitro with a statin (Mevinolin) compared to control and FSH.
  • the fraction of fertilised oocytes (PB 2 -oocytes to 4+ cells) increased by the addition of the statin Mevinolin to the IVM-medium of CEOs as compared to CEOs cultured in the presence of 7,5 I.UJL FSH and control CEOs.
  • the Mevinolin treated oocytes displayed extensive cumulus expansion after the IVM culture period as opposed to the control oocytes.
  • the FSH-treated oocytes were intermediate between these two groups in this respect.
  • the degree of degeneration was not different between the groups.
  • the development has reached a more progressed state in the Mevinolin treated group as compared to the two control groups after 72 hours of embryo culture.
  • Fig. 10 Fertilisation rate of oocyte matured in vitro with a cyclodextrin (hydroxypropyl-beta- cyclodextrin) compared to control.
  • the fraction of fertilised oocytes (PB 2 -oocytes to 4+ cells) increased by the addition of the sterol binding and transporting substance hydroxypropyl-cyclodextrin to the IVM-medium of NOs as compared to control NOs.
  • the rate of degeneration was not affected by the presence of the cyclodextrin in the IVM- medium.
  • Fig. 11 Overview of the developmental processes that are be manipulated by the present invention.
  • the present invention relates to the process of maturation and fertilisation both in vivo and in vitro and not to any embryonic development.
  • the concept relating to inhibition of sterol biosynthesis de novo applies both in the male and in the female, whereas the concept relating to the use of sterol binding and transporting substances applies during maturation in the female only.
  • mice Prepubertal C57 black x DBA 2 FI female mice (M&B A/S, Ry, Denmark) were stimulated to super-ovulation ( 11 a.m. at day 0) by i.p. injections of 12 IU Menogon® (Ferring, Denmark) in 200 ⁇ L H 2 0.
  • This gonadotrophin preparation contains 50% human FSH and 50% human LH in terms of biological activity.
  • Half of the mice were given additional injections of 1,6 mg lovastatin, by dissolving a tablet of Mevacor® (Merck Sharp and
  • mice Dohme, IML containing 40 mg lovastatin in 5 ml PBS and injecting 200 ⁇ L of the resulting slurry i.p. at three consecutive days, beginning at day 0. The other half (control mice) were injected a similar volume of PBS. At 1 p.m. at day 2 the animals were given an ovulatory dose of 10 IU hCG (Ferring, Denmark). The mice were caged individually one hour later with a mature male mouse of the same strain and supplier (1 male per female). Female and male mice were separated at 9 a.m. at day 3, females were left alone for another 48 h.
  • 2-cells were flushed into a plastic dish with culture medium (see later) by mounting a glass pipette in one end of the isolated tuba and blowing by mouth through a filter. The total number of 2- cells flushed per mouse were hereafter counted.
  • the 2-cells were subsequently cultured in Universal IVF-medium (Medicult, Denmark) in an incubator (37°C, 95% atmospheric air, 5% C0 2 ) in 4-well dishes (Nunclon 176740, Nunc, Denmark). After 4 days of culture, wells were scored and numbers of morula, blastocyst or degenerated entities were determined by use of a microscope:
  • the number of 2-cells per animal increased significantly when the mice were treated with 3 x 1.6 mg statin during the follicular phase maturation (figure 4). This represents the principal outcome with respect to reproduction of the cholesterol lowering treatment: the increase in the number of ovulated and fertilised oocytes as compared to control animals.
  • the 2-cells were cultured in vitro and developed into blast-oocytes with recovery rates indiscriminate to controls.
  • statins Effect of a statin administration in female mice on the number of 2-cells in vivo after fertilisation using various regimes
  • Prepubertal C57 black x DBA 2 FI female mice (M&B A/S, Ry, Denmark) were treated as in example 1 but for a few deviations.
  • the dose of statins was decreased to 100 ⁇ g/animal and given as a single injection 15 minutes prior to the hCG-injection.
  • statins was decreased to 3 x 100 ⁇ g/animal and administered as in example 1.
  • statins was given as a single injection 15 minutes prior to the hCG-injectionin 9 month old mice.
  • the dose of gonadotropins were decreased gradually whereas the dose of statins was administered as in example 1.
  • statins may be decreased to one tenth of the dose applied in example 1.
  • statin may be given as a single injection prior to the ovulatory stimulation by hCG as opposed to the application during a broader time window in the follicular phase.
  • the add-on concept is demonstrated in experiment D where a lowered gonadotropin administration may be compensated for partly by the co-administration of a statin. All the experiments in the present example, however, need quantitative substantiation before conclusions are to made in these respects
  • Table 2 Number of 2-cells in C57 black x DBA 2 Fl mice after administration of exogeneous gonadotropins and a statin (Mevacor).
  • statin augments the LH/hCG induced decrease in the tissue density of free cholesterol in ovaries after gonadal stimulation of pre-pubertal mice
  • mice Prepubertal C57 black x DBA 2 Fl female mice (M&B A/S, Denmark) were stimulated to super-ovulation (11 a.m. at day 0) by i.p. injections of 12 IU Menogon® (Ferring, Denmark) in 200 ⁇ L H z O (as in example 1). Half of the mice were given additional injections of 1.6 mg lovastatin, by dissolving a tablet of Mevacor® (Merck Sharp and Dohme, NL) containing 40 mg lovastatin in 5 ml PBS and injecting 200 ⁇ L of the resulting slurry i.p. at three consecutive days, beginning at day 0 (as in example 1).
  • Mevacor® Merck Sharp and Dohme, NL
  • mice were injected a similar volume of PBS.
  • the animals were given an ovulatory dose of 10 IU hCG (Ferring, Denmark) and killed 6 h after for preparation and HPLC-analysis of ovarian extracts: Ovaries were isolated pair-wise, weighed, freeze-dried, weighed again and subsequently extracted in 1.0 ml (v/v) 75 % n- heptane: 25 % isopropanol.
  • the organic extract was re-constituted in mobilphase for HPLC straight-phase (SP) separation (ChromSpherSi, 5 ⁇ m, 250x4.6 mm HPLC column, running in (v/v) 99.5 % n-heptane (Fischer, Leicestershire, U.K.) : 0.5 % isopropanol (Baker, NL) at 1.00 ml/min.).
  • SP straight-phase
  • Eluted material was detected by ultraviolet light absorption between
  • Table 3 Free cholesterol (C), follicular fluid meiosis-activating sterol (FF-MAS) and progesterone (P 4 ) in gonadotrophin primed prepubertal mouse ovaries stimulated with hCG and statin. Different post-designation letters (boldfaced) denote statistical difference by a t-test.
  • N 13 6 6 mouse weigh (g) 14.6 ⁇ 0.2 A 15.3 ⁇ 0.3 A 14.6 ⁇ 0.3 A
  • liver (mg/g wet w.) 1.7 ⁇ 0.1 A * (not assayed) 2.9 ⁇ 0.3 B
  • liver (mg/g dry w.) 6.5 ⁇ 0.5 A * (not assayed) 7.1 ⁇ 0.4 A
  • the tissue density of free cholesterol decreases in the mouse ovary prior to ovulation.
  • the tendency of decreasing tissue density in the ovaries of free cholesterol after hCG stimulation was pronounced by the statin treatment. The decrease can not be completely explained by the oedematic effect of the steroid generating hCG hormone in that the cholesterol tissue density also decreased when evaluated on the basis of dry tissue weights. HCG was applied in order to mimic the natural pre-ovulatory LH-surge.
  • statin completely blocks the LH/hCG induced accumulation of the pre-cholesterol metabolite FF-MAS and leaves the ovary with a level of FF-MAS below the mice that was not challenged with hCG.
  • statin completely blocks the LH/hCG induced accumulation of the pre-cholesterol metabolite FF-MAS and leaves the ovary with a level of FF-MAS below the mice that was not challenged with hCG.
  • comparing control and hCG-stimulated mouse ovaries reveal that the absolute decrease of cholesterol is > 100 times the increase in FF-MAS with respect to the tissue density.
  • a HMG-CoA-reductase inhibitor stimulates maturation of cumulus enclosed oocytes in vitro and augments gonadotrophin stimulated maturation of cumulus enclosed oocytes in vitro
  • Oocytes from preovulatory follicles were retrieved from the ovaries 46 hours after by puncturing the pre-ovulatory follicles of the ovaries with 0.20 mm needles when ovaries were immersed in a culture medium ( ⁇ -MEM 22571-020, Gibco BRL, Scotland) supplemented with 4 mM hypoxanthine (see below).
  • Cumulus oocyte complexes (COC) were collected by identification through a preparation microscope and subsequent sucking through a membrane mounted mouth pipette and cultured in ⁇ -MEM in an incubator (37°C, 95% atmospheric air: 5% C0 2 ) in 4-well dishes (Nunc, Denmark).
  • the culture medium was supplemented with 4 mM hypoxanthine (HX) (Sigma H-9377, Sigma-Ald ⁇ ch, Denmark), 200 mM L-glutamme (Gibco BRL, Scotland), 20000 IU/L penicillin (Gibco BRL, Scotland), 20000 IU/L streptomycin (Gibco BRL, Scotland) and 3 mg/ml BSA (Sigma A-7030, Sigma-Ald ⁇ ch, Denmark). All incubations were also added 25 mCi 3 H-acetat (NEN, U.S.A.) in order to measure de novo synthesis of cholesterol after incubation by scintillation.
  • HX hypoxanthine
  • NNN 25 mCi 3 H-acetat
  • statin Compactin (Sigma, Sigma-Ald ⁇ ch, Denmark,) was added in 50 mM in DMSO (final concentration of DMSO in cultures were 1% v/v in all set-ups including controls) and recombinant human follicular stimulating hormone (Gonal-F, Serono, DK) was added to a final concentration of 7,5 IU/L.
  • GV germinal vesicles
  • GVB germinal vesicle breakdown
  • PB polar bodies
  • Prepubertal C57 black x DBA 2 Fl female mice (M&B A/S, Ry, Denmark) were stimulated to super-ovulation (day 0) by i.p. injections of 12 IU Menogon® (Ferring, Vanl ⁇ se, Denmark) in 200 ⁇ L H 2 0 as in examples 1-4. Ovaries were isolated 46 hours after as in
  • Window 1 (2'50"-4'00") containing squalene, window 2 (10'30"-14'00") containing 4,4- dimethylsterols, window 3 (25'00"-29'00") containing cholesterol and desmosterol, window 4 (29'30"-33'00") containing 5,7-enes and window 5 (containing P 4 ) were collected, dried and subjected to reversed phase: LiChrospher RP-8, 5 ⁇ m, 250x4.6 mm HPLC column running in (v/v) 92.5% acetonitrile: 7.5% water at 1.00 ml/min, 40°C. Analytes were
  • C/PL-ratio ratio of cholesterol to phospholipids in the motile cell fraction of epididymis in mice by prior administration of a statin
  • Post-pubertal C57 black x DBA 2 Fl male mice M&B A/S, Ry, Denmark
  • mice are given 3 i.p. injections of 1.0 mg lovastatin per 30 g body weight by dissolving a tablet of Mevacor® (Merck Sharp and Dohme, NL) containing 40 mg lovastatin in 8 ml PBS and injecting 200 ⁇ L of the resulting slurry i.p. at 1 p.m. at day -6, -3 and -1, counting the day of epididymal sperm harvest as day 0.
  • Mevacor® Merck Sharp and Dohme, NL
  • mice Four other mice (control mice) are injected a similar volume of PBS. At 10 a.m on day 0 all mice are killed and the epididymis are prepared in physiological saline. Thereafter, the epididymis are transferred to a 1-mL culture well containing MEM (Gibco BRL, Scotland) supplemented with 2.3 mM pyruvate, 200 mM L-glutamine (Gibco BRL, Scotland), 20000 IU/L penicillin (Gibco BRL, Scotland), 20000 IU/L streptomycin (Gibco BRL, Scotland) and 0.2 mg/ml BSA (Sigma A-7030, Sigma-Aldrich, Denmark) and placed in an incubator (37°C, 95% atmospheric air: 5% co 2 ).
  • the upper liquid phase is aspirated and transferred to a new glass-vial and centrifuged. After centrifugation, the supernatant is discarded and the pellet is freeze-dried and thereafter extracted in 50%methanol : 50%chloroform (volJvol.).
  • the extract is subsequently divided in two equal parts. One half is subjected to sterol analysis as designated in the examples above and the total sterol content is calculated to consist of cholesterol, desmosterol, cholesta-7,24-dien-3 ⁇ -ol, T-MAS and lanosterol. The other half is subjected to phospholipid assay according to the method of Bartlett (Bartlett, 1958). The C/PL-ratio is calculated for each individual animal.
  • the C/PL-ratios in the statin treated group is lower the C/PL-ratios in the control group.
  • Statins applied during IVM increase the fertilisation rate in mouse cumulus enclosed oocytes (CEO) following IVF
  • Mouse oocytes were isolated and cultured according to descriptions in example 3 but for a single deviation in that the IVM culture medium was further supplemented with 1 mg/mL Fetuin (Sigma F-3385, Sigma, U.S.A.).
  • the CEOs were divided into three groups and cultured in dishes containing medium added either 1% (v/v) ethanol (control), recombinant human follicular stimulating hormone (Gonal-F, Serono, DK) at a concentration of 7,5 IU/L together with 1% (v/v) ethanol (FSH), or 10 ⁇ M Compactin (Mevinolin M2147, Sigma-Aldrich, Denmark) with 1% (v/v) ethanol (Mevinolin).
  • oocytes were transferred to a fertilisationn medium consisting of minimal essential medium (MEM) (21090-022, Gibco BRL, Scotland), 200 mM L- glutamine (Gibco BRL, Scotland), 20000 IU/L penicillin (Gibco BRL, Scotland), 20000 IU/L streptomycin (Gibco BRL, Scotland), 3 mg/ml BSA (Sigma A-7030, Sigma- Aldrich, Denmark), 2.3 mM sodium pyruvate (S-8636, Sigma), 10 mM ethylen diamine tetra acetic acid (EDTA, Merck 1.08418.0250) and 1 mg/mL Fetuin (Sigma F- 3385, Sigma, U.S.A.).
  • MEM minimal essential medium
  • EDTA ethylen diamine tetra acetic acid
  • Fetuin Sigma F- 3385, Sigma, U.S.A.
  • mouse sperm for IVF was obtained the following way: A 6-9 month old C57 black x DBA 2 Fl male mouse (M&B A/S, Ry, Denmark) was killed and the epididymis prepared in physiological saline. Thereafter, the epididymis were cut in small pieces and the bulk material was transferred to four 1-mL culture well containing the 800 ⁇ L IVF-medium described above and subsequently placed in an incubator (37°C, 95% atmospheric air: 5% C0 2 ). After 1,5 hours of incubation, 300 ⁇ L of the upper liquid phase of each of the four wells was aspirated and transferred to a new glass-vial.
  • the resulting pool of motile sperm was counted in a counting chamber and 50000 motile sperm cells was added each oocyte culture.
  • the IVF cultures were incubated at incubator 37°C, 95% atmospheric air: 5% C0 2 for 20 hours after which the oocytes were transferred to a fresh IVF medium equilibrated under the same conditions. These oocytes were left for another 48 hours and subsequently counted according to the maturational status of the oocyte or the progression of the fertilised zygote, i.e.
  • GV-oocytes GVBD-oocytes
  • PB-oocytes PB 2 -oocytes (oocytes with a visible second polar body), 2-cells, 3-4-cells, 4+ cells (zygotes with more than 4 blastomeres) and degenerated cells (usually granulated or cells with extensive plasma-membrane ondulation).
  • Cyclodextrin applied during IVM increase the fertilisation rate in naked mouse oocytes (NO) following IVF
  • Mouse oocytes were isolated and cultured according to descriptions in example??. The NOs were divided into three groups and cultured in dishes containing medium added either 0 %o, 0.5 %o, 2.0 %o or 5 %o hydroxypropyl-cyclodextrin (ICN 153540, ICN Ohio, U.S.A.). IVM and IVF conditions were the same as in example 6.
  • Example 9
  • the set-up involves the same animals and statin administration as in example 6.
  • the animals are analysed by methods that are developed in order to evaluate the fertilising potential and sperm quality. It is contemplated that statin treated animals will have a fertility premium as compared to the control animals.

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Abstract

La présente invention concerne des méthodes qui permettent d'accroître la compétence de développement d'au moins une cellule germinale, un gamète, un zygote, un embryon précoce, un blastocyste implanté et/ou un embryon de mammifère, par administration d'un composé qui est capable d'inhiber la biosynthèse de novo des stérols et d'établir ainsi les conditions cellulaires qui améliorent leur développement et leur survie. Cette invention se rapporte également à des méthodes qui permettent d'accroître la sortie des stérols avant la fertilisation d'au moins un ovaire, ovocyte, gamète femelle ou cellule dérivée d'ovaire entourant un ovocyte de mammifère, au moyen de l'administration d'un composé qui est capable d'activer la sortie des stérols et de réduire ainsi le rapport entre les phospholipides et les stérols desdites cellules.
EP02792712A 2001-12-21 2002-12-20 Mobilisation de gametes et amelioration de la competence de developpement chez les mammiferes au moyen de l'inhibition de la biosynthese des sterols de novo et/ou de l'activation de la sortie des sterols Withdrawn EP1463804A1 (fr)

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