KR101249268B1 - Composition that blocks and synchronizes the meiotic progression of oocytes comprising 3-methyladenine and Method thereof - Google Patents

Abstract

The present invention relates to meiosis inhibition and synchronization composition and method of oocytes comprising 3-methyl adenine as an active ingredient.

Description

Composition that blocks and synchronizes the meiotic progression of oocytes comprising 3-methyladenine and Method according to oocytes containing 3-methyladenine as an active ingredient

The present invention relates to meiosis inhibition and synchronization composition and method of oocytes comprising 3-methyl adenine as an active ingredient.

Meiosis is the only fundamental event of germ cells on which sexual reproduction is based. Meiosis includes two meiosis. During primary division, exchange between maternal and parental genes occurs before the chromosome pair separates into two daughter cells. They contain only half (1n) of chromosome and 2c DNA. Secondary meiosis proceeds without DNA synthesis. Thus, this division results in the formation of haploid germ cells with only 1c DNA.

The meiosis event is similar in male and female germ cells, but the timing and differentiation processes leading to ovum and sperm are very different. All female germ cells begin the first meiosis early in life, mainly before birth, but are stopped as oocytes in the period from puberty to ovulation (the reticulum). Thus, from the beginning of life, women have stored oocytes that are released until exhausted. The female meiosis is not complete until after fertilization, there is only one egg and two non-polar pole cells per germ cell. On the other hand, only some of the male germ cells begin meiosis at puberty and remain as germ cells in the liver population for life. Once started, meiosis in male cells proceeds with no noticeable delay and produces four sperm.

Asynchronous meiosis progression is commonly observed in mammalian oocytes recovered from overovulated live female or slaughterhouse derived ovaries [Motlik J, Fulka J. J Exp Zool 1976; 198: 155-162; Trounson A, Anderiesz. C, Jones GM, Kausche A, Lolatgis N, Wood C. Hum Reprod 1998; 13 Suppl 3: 52-62; discussion 71-55; Uhm SJ, Gupta MK, Yang JH, Chung HJ, Min TS, Lee HT. Theriogenology 2010; 73: 1024-1036. This asynchronous not only causes serious differences in experimental studies, but also involves differences in developmental function among individual oocytes [Gupta MK, Uhm SJ, Lee HT. Anim Reprod Sci 2008; 108: 107-121; Vanhoutte L, De Sutter P, Nogueira D, Gerris J, Dhont M, Van der Elst J. Hum Reprod 2007; 22: 1239-1246. Thus, some strategies have been reported before recovery from follicles [Bordignon V, Morin N, Durocher J, Bousquet D, Smith LC. Theriogenology 1997; 48: 291-298; Mattioli M, Galeati G, Barboni B, Seren E. J Reprod Fertil 1994; 100: 403-409] and later [Vanhoutte L, De Sutter P, Nogueira D, Gerris J, Dhont M , Van der Elst J. Hum Reprod 2007; 22: 1239-1246, has been proposed for meiotic synchronization of oocytes.

Mitosis synchronization of oocytes can be achieved in vitro by in vitro maturation (IVM) or by chemically induced transient nuclear arrest in premature (Prematuration culture (PMC). Reel cyclic AMP [Funahashi H, Cantley TC, Day BN. Biol Reprod 1997; 57: 49-53; Sun QY, Lu Q, Breitbart H, Chen DY. Reprod Fertil Dev 1999; 11: 81-86], Paul Scorin [ Sun QY, Lu Q, Breitbart H, Chen DY.Reprod Fertil Dev 1999; 11: 81-86; Shimada M, Terada T. Mol Reprod Dev 2002; 62: 124-131], hypoxanthine, xanthosine and purine [Downs] SM, Daniel SA, Bornslaeger EA, Hoppe PC, Eppig JJ.Gamete Res 1989; 23: 323-334] to maintain elevated levels of cAMP maintained by using chemicals such as germinal vesicles (' GV ') may be arrested.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a substance that inhibits meiosis.

Another object of the present invention is to provide a method for inhibiting meiosis.

Another object of the present invention is to provide a material for synchronizing meiosis.

Another object of the present invention is to provide a method for synchronizing meiosis.

Another object of the present invention is to provide a contraceptive composition.

To achieve the above object

Provided is a meiosis inhibiting composition of oocytes containing 3-methyladenine as an active ingredient.

In one embodiment of the present invention, the inhibition is preferably performed by being arrested in the GV step.

In one embodiment of the present invention, the oocyte is preferably an immature oocyte, but is not limited thereto.

In another aspect, the present invention provides a mitosis synchronization composition of oocytes containing 3-methyl adenine as an active ingredient.

In one embodiment of the invention, the synchronization is preferably synchronized in the GV step.

In another aspect, the present invention provides a method for inhibiting meiosis of oocytes comprising treating immature pig oocytes with 3-methyladenine to arrest meiosis.

In one embodiment of the present invention, the treatment time is preferably 20 to 42 hours, but is not limited thereto.

In another aspect, the present invention provides a method for synchronizing meiosis of oocytes comprising treating immature porcine oocytes with 3-methyladenine to synchronize meiosis at the GV stage.

In another aspect, the present invention provides a pill composition comprising 3-methyl adenine as an active ingredient.

The compound for inhibiting oocyte meiosis or a salt thereof and a precursor of the present invention can be achieved by preventing mature oocytes from being produced in female contraception.

When a compound of the present invention is administered to a mammal, they are readily provided in the form of a pharmaceutical composition comprising at least one of a compound or a salt, active metabolite and prodrug thereof in combination with a pharmaceutically acceptable carrier. . For oral use, such compositions are preferably in the form of capsules or tablets.

When used as a contraceptive, the compounds of the present invention or salts, active metabolites and prodrugs thereof must be administered continuously or periodically. When used as a pill by women, it is not used continuously, and the timing of administration associated with ovulation is important.

Pharmaceutical compositions comprising the compounds of the present invention or salts, active metabolites and prodrugs thereof include carriers, diluents, absorbents, enhancers, preservatives, buffers, osmotic pressure regulators, tablet disintegrating agents and other conventionally used in the art. It may further comprise a component. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugars, talc, gelatin, pectin, trackercance, methyl cellulose, sodium carboxymethyl cellulose, low melting wax and cocoa buffer.

Liquid compositions include sterilic solutions, suspensions and emulsions. Such liquid compositions may be suitable for use in connection with injection or in vitro and in vitro fertilization. The liquid composition may be any of those mentioned in the above list and may contain other ingredients conventionally used in the art.

In addition, the composition for transdermal administration of a compound of the present invention is provided in the form of a patch, the composition for nasal administration is provided in the form of a nasal spray in liquid or powder form.

The dosage of the compound of the present invention to be used will be determined by the physician, among other things, depending on the compound used, the route of administration and the purpose of use. In general, the compositions of the present invention are prepared by mixing the active compound with a liquid or solid auxiliary component and then forming the product as a preferred preparation if necessary.

Usually, up to 1000 mg, preferably up to 100 mg, and in some preferred embodiments up to 10 mg of the compound of the invention are administered to a mammal per day.

Hereinafter, the present invention will be described.

In the present invention, the present inventors have found that 3-methyladenine (hereinafter referred to as '3MA') is a MII stage, and then to immature oocytes in the GV stage without affecting the post-activation / fertilization development for their reduction in a medium without maturation and inhibitor. It can be reversibly restrained.

However, unlike other inhibitors for GV arrest, 3MA does not require a PMC period and can be added directly to the initial 22 hours of IVM without increasing the overall duration of the IVM.

Treatment of immature porcine oocytes with 3MA 20-42 hours arrests them at the GV stage, regardless of the presence or absence of cumulus cells.

In addition, significantly higher proportions (60.9 ± 13.8%) of 3MA-treated oocytes require nucleolus completely surrounded by highly aggregated chromatin rims (GV-II stage). However, the GV-arresting effect of 3MA is completely reversible for their further culture in the absence of 3MA. Cumuls-oophorus-complexes (COCs) arrested at the GV stage for 22 hours by 3MA were further incubated for 22 hours in the absence of 3MA, resulting in 96.1 ± 1.5% of oocytes at 42 hours of IVM. Reached and did not differ from untreated control oocytes in their ability to fertilize, eggplant and blastocyst formation (P> 0.05) for in vitro fertilization (IVF) or unit reproduction activation (PA).

These data indicate that 3MA effectively blocks and synchronizes meiotic progression of porcine oocytes at the GV stage without affecting their ooplasmic maturation in terms of post fertilization / activation in in vitro culture development.

Hereinafter, the present invention will be described in detail.

COCs 3 about their meiosis progression MA Effect

We investigated the effect of 3MA on meiotic progression of oocyte-enclosed porcine oocytes. Porcine COCs recovered from slaughterhouse-derived ovaries were subsequently stained with Hoechst 33342 for 22 hours incubation in the absence (control) and presence of 3MA and to assess their chromatin morphology. The results showed that 99.2 ± 0.8% of 3MA-treated COCs were arrested in the GV stage while 46.0 ± 10.1% of the untreated control oocytes progressed to the GVBD stage (Table 1).

Table 1 is a table in which pig COCs were incubated in the absence (control) and presence of 3MA for 22 hours and showed chromatin morphology. The numbers in parentheses in the table indicate the number of oocytes, and the various letters (a, b) in the column indicate the statistical difference (P <0.05).

In addition, a significantly higher percentage of oocytes (60.9 ± 13.8 vs. 33.8 ± 9.8%) showed the nuclear morphology of the GV-II stage with phosphorus completely surrounded by highly condensed chromatin te (FIG. 1). .

We then conducted a time dependent study to evaluate the effect of treating pig COCs with 3MA for different times. Porcine COCs were incubated for 20, 28, 36 or 42 hours in the presence or absence of 3MA (control) and then stained with Hoechst 33342 to analyze their chromatin morphology.

The results indicated that oocytes from untreated controls of 51.7 ± 4.4, 51.2 ± 7.7, 86.2 ± 5.2 and 95.5 ± 2.8% were in GVBD, MI, MII and MII stages at 20, 28, 36 and 42 hours of incubation. (FIG. 2).

However, in the presence of 3MA, 96.1 ± 3.9, 97.6 ± 2.4, 80.0 ± 8.4 and 70.1 ± 8.4% of oocytes remained arrested at the GV stage.

COCs treated with 3MA for 42 hours also reduced the degree of egg swelling with 84.2 ± 8.5% COC, indicating no egg swelling for 7.2 ± 2.0% in untreated control COC (P <0.05) (FIG. 3A).

However, 3MA-treatment for 42 hours did not affect oocyte survival as assessed by plasma esterase activity and plasma membrane properties in the FDA assay (93.4 ± 6.9 vs. 93.5 ± 4.7%).

DOs Meiosis Progress  About 3 MA effect

In order to investigate whether 3MA acts on the surrounding cumulus cells through their action or directly on the cumulus itself, we investigated the effect of 3MA on cumulative-free DOs.

Porcine COCs were infiltrated completely without cumulus cells and incubated for 22 hours or 42 hours in the presence or absence of 3MA (control) and stained with Hoechst 33342 to examine their chromatin morphology. At 42 hours of IVM, untreated DO oocytes showed lower MII-plate formation (68.8 ± 0.6%) compared to those obtained with COCs.

Nevertheless, 22 or 42 hours treatment with 3MA arrested 96.5 ± 1.8 and 86.6 ± 8.1% DOs at the GV stage, respectively (Table 2).

Table 2 shows the chromatin morphology of swine DO aged in the absence (control) and presence of 3MA for 22 or 42 hours. The numbers in parentheses in the table represent the number of oocytes, and the various letters (a, b) in the column represent statistical differences (P <0.05) between the same treatment duration groups.

3 MA Reversibility

We investigated whether the effect of 3MA in meiosis arrest was reversible for removing 3MA from the culture medium.

To test this possibility, pig COCs and DOs were cultured separately in the presence or absence (control) of 3MA for 22 hours and an additional 22 hours without 3MA.

The results showed that both COCs and DOs arrested in the GV stage by 22 hours 3MA could recover to reach the MII stage perfectly if they were further incubated without 22 hours 3MA (Table 3).

Table 3 is a table showing the reversibility of meiosis progression for 22 hours further cultured in the 3V absence of porcine oocytes arrested at the GV stage for 22 hours with 3MA. COCs: cumulus-oophorus-complexs; DOs: cumulus-free denuded oocytes, respectively, and the COCs and DOs were further cultured for 22 hours at 3MA in the absence of 3MA of porcine oocytes arrested at the GV stage for 22 hours. The numbers in parentheses in the table indicate the number of oocytes, and the same superscript (a) in the column indicates no significant difference (P> 0.05) within the COCs or DOs group.

The percentage of oocytes that reached the MII stage was not significantly different for both COCs (96.8 ± 1.5 vs. 96.1 ± 1.5%) and DOs (80.8 ± 8.3 vs. 83.4 ± 5.5%) between the control and 3MA treated groups. (P> 0.05).

However, as compared to the untreated control (0.0 ± 0.0%), 29.0 ± 4.2% of 3MA-treated COCs did not show egg swelling (FIG. 3B).

Importantly, 22 hours cultured 3MA-treated GV-arrested oocytes in IVM permeable medium are sufficient for them to obtain MII stage chromatin form, so the duration of total IVM is 42-fold in both control and 3MA treated groups. 44 hours was maintained.

In another set of experiments, we further tested meiosis resumption in pig COCs arrested in the GV step for 42 hours of 3MA treatment. Porcine COCs were incubated in the presence or absence of 3MA (control) for 42 hours and further incubation for 22 hours in the absence of 3MA. 96.6 ± 0.4 and 95.7 ± 0.1% COCs progressed to the MII stage, while 3.4 ± 0.4 vs. 3.2 ± 1.0% of COCs maintained the GV stage in the control and 3MA-treated groups, respectively.

3 MA Temporarily for 22 hours by GV  In step Arrested COCs of In vitro  develop

We assessed whether transient arrest of porcine oocytes at the GV stage by 3MA treatment affected their in vitro developmental function.

First we evaluated the in vitro developmental function of 3MA treated oocytes for unit reproduction.

Porcine COCs cultured in the presence or absence of 3MA for 22 hours (control) and further cultured for 22 hours in the absence of 3MA were subjected to unit reproduction and in vitro development and embryo quality were examined.

The untreated control and 3MA treated oocytes showed no difference (P> 0.05) with respect to egg yield (89.8 ± 1.7 vs. 84.6 ± 5.1%) and blastocyst formation rate (44.3 ± 12.4 vs. 45.1 ± 7.6%).

The blastocysts from both groups also had similar (P> 0.05) swelling and hatching capacity (80.7 ± 9.5 vs. 76.6 ± 8.9%) and the number of cells per blastocyst (57.4 ± 2.8 vs. 54.3 ±) of similar (P> 0.05). 3.2) shows that 3MA induced transient GV arrest has no detrimental effect on oocyte maturation of oocytes (Table 4; FIG. 4).

Table 4 shows the post-activation (mean ± standard deviation) in in vitro development of porcine COCs with nuclear arrest in the GV phase by 3MA for 22 hours. COCs in the table were incubated in the presence or absence of 3MA (control) for 22 hours and further mature for 22 hours in the absence of 3MA. The values in parentheses represent the number of embryos, and the values with the same superscript (a) in the column are not significantly different (P> 0.05).

3 MA Temporarily for 22 hours by GV  In step Arrested COCs Fertility and development after fertilization

We assessed the fertility and post-fertilization development of porcine oocytes arrested at the GV stage temporarily for 22 hours by 3MA.

Consistent with PA embryos, untreated controls and 3MA-treated oocytes did not differ in maintaining embryo development from fertilization to blastocyst stage (P> 0.05) (Table 5).

The blastocysts generated from 3MA treated oocytes had similar (P> 0.05) swelling and hatching capacity (74.2 ± 8.8 vs. 80.5 ± 8.2%) as obtained from untreated control oocytes and similar (P> 0.05) Number (69.7 ± 7.5 vs. 59.8 ± 6.5).

3MA-treated oocytes differed for overall fertilization rate (62.3 ± 9.9 vs. 60.3 ± 6.3%) and monosperm fertilization rate (47.3 ± 5.2 vs. 48.4 ± 1.9%) compared to untreated control oocytes None (P> 0.05), thus further confirming that 3MA induced transient GV arrest had no deleterious effect on oocyte maturation of oocytes (FIG. 5).

Table 5 shows the post-fertilization (mean ± standard deviation) in in vitro development of pig COCs with nuclear arrest in the GV phase by 3MA for 22 hours. COCs in the table were incubated in the presence or absence of 3MA (control) for 22 hours and further mature for 22 hours in the absence of 3MA. The values in parentheses represent the number of embryos, and the values with the same superscript (a) in the column are not significantly different (P> 0.05).

As can be seen from the present invention, it can be seen that 3MA effectively blocks and synchronizes meiosis progression of porcine oocytes at the GV stage without affecting their egg maturation in terms of in vitro embryonic fertilization / activation. In addition, it can be seen that the meiosis inhibitory effect of 3MA is observed in both COCs and DOs.

FIG. 1 shows the distribution of several GV steps in swine COCs matured in the absence (open box) or presence (black box) of 3MA for 22 hours. The values at the top of the bar represent the average percentage of total COCs at each nuclear step. The various letters (a, b) at the top of the bar represent the statistical difference (P <0.05) between the control and 3MA groups for each nuclear step.
2 shows the chromatin form of porcine COCs matured with or without 3MA for 20h, 28h, 36h or 42h. The values at the top of the bar represent the average percentage of COCs in the open box (GV), closed box (GVBD), crossed box (MI) and dotted box (MII) steps. The various letters (a, b) at the top of the bar represent the statistical difference (P <0.05) between the control and 3MA groups for each nuclear step at each time point.
3 shows cumulus expansion in swine COCs matured in the absence (open box) or presence (black box) of 3MA for 42 hours.
A. Porcine COCs incubated in the absence or presence of 3MA for 42 hours
A. Porcine COCs were cultured in the absence or presence of 3MA for 22 hours and further cultured in the absence of 3MA for another 22 hours.
Cumulus Cell Dilation: Degree 2 (Complete Cumulus Cell Expansion): Homogeneous expansion of all cumulus cells; Degree 1 (suitable cumulus expansion): 70 percent of the cumulus cells homogeneously expand; Degree 0 (no minor or swelling): Oocytes strongly adhere to Jonas Felucida. Oocytes with ovarian dilatation of degrees 1 and 2 are considered mature. Values above the bar indicate the average percentage. Several letters (a, b) at the top of the bar indicate statistical differences (P <0.05) between the control and 3MA groups for each degree of ovarian dilatation.
4 shows blastocysts generated from parthenogenetic activation (PA) of porcine COCs temporarily arrested at the GV stage by 3MA.
COCs were incubated in the absence (A) or presence of 3MA for 22 hours and further in the absence of 3MA for another 22 hours. Panel 1: bright field; Panel 2: Hoechst 33342 stained.
The values above the bar expand and represent the percentage of hatched blastocysts (A) or the total number of nuclei (B) per blastocyst. The same superscript (a) between groups is not significantly different (P> 0.05).
5 shows the in vitro fertilization (IVF) ability of swine COCs with nuclear arrest temporarily for 22 hours by 3MA. Prior to IVF, COCs were incubated for 22 hours in the absence (open box) or presence of 3MA (black box) and further incubation in the absence of 3MA for 22 hours.
The values at the top of the bar represent the average percentage calculated from the total number of COCs initially used for the IVM-IVF. Several letters (a, b) at the top of the bar represent statistical differences (P <0.05) between the control and 3MA groups. .

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

All chemicals used in the present invention are Sigma-Aldrich Co. (St. Louis, MO, USA). Each experiment was performed at least three times and randomly assigned oocytes recovered from the same batch of slaughterhouse-derived ovaries to each group.

Example  1: oocyte harvesting In vitro  Maturity( IVM )

Extraction of oocytes and IVM from porcine ovary was performed by Gupta MK, Uhm SJ, Lee HT. Some modifications were made to the content of Theriogenology 2007; 67: 238-248.

In summary, Prepubertal pig ovaries were collected at local slaughterhouses and maintained in saline at 30-37 ° C. The oocyte complex (CUC) was transferred to a laboratory using a 10 ml syringe with 18G needles. Extracted from follicles (3-6 mm diameter). The oocyte cumulus complexes were washed three times in TL-HEPES medium containing 1 mg / ml bovine serum albumin (BSA) (low carbonate TALP; Parrish et al., 1988), followed by 10% 25 mM NaHCO ₃ (v / v). Porcine follicular fluid, 0.57mMCystein, 0.22 μg / ml sodium pyruvate, 25 μm / ml gentamicin sulfate, 0.5 μg / ml p-FSH (Follitropin V; Vereprepharm, Canada), 1 μg / ml estradiol-17B, and 10 ng / ml epidermis 500 μl of tissue culture medium 199 (TCM-199; Gibco BRL, Grand Island, NY) with Earle's salt supplemented with cell growth factor (EGF; E-4127, Sigma) was divided into 50 groups of 5% CO 2 air. Mature at 42 ° C. under mineral oil at 39 ° C. in a humid atmosphere.

To determine the effect of 3MA, 10 mM 3MA was added or not added to the IVM medium for each experimental design. The concentration of 3MA (10 mM) was determined based on preliminary experiments that did not affect oocyte viability but inhibit meiosis progression.

In experiments to determine the effect of 3MA on denuded oocytes (DO), the oocytes were removed from the COC prior to IVM by repeated pipetting in 0.1% hyaluronidase.

Example  2: oocyte maturation, Survivability  And assessment of meiosis progression

Oocyte maturation was determined by Gupta MK, Uhm SJ, Lee HT. Evaluation was based on cumulus expansion and MII-plate formation as described in Anim Reprod Sci 2008; 108: 107-121.

The meiosis progression of oocytes was Uhm SJ, Gupta MK, Yang JH, Chung HJ, Min TS, Lee HT. Measurements were made after fluorescence Hoechst 33342 staining as described in Theriogenology 2010; 73: 1024-1036.

In summary, the derivatized oocytes of the oocytes were fixed for 5 minutes in a fixed solution containing 2% formalin and 0.25% gluteraldehyde using 0.1% hyaluronidase, and placed on a glass slide and glycerol-based Hoechst 33342 (12.5 μg / mL). Stain for 10 minutes in solution. The stained nuclei show blue (excitation: 330-385 nm; emission: 420 nm; dichromatic: 400 nm) when visualized under UV irradiation with an epifluorescent microscope equipped with a blue filter, based on GV, Classified as GVBD, MI, or MII [Motlik J, Fulka J. J Exp Zool 1976; 198: 155-162; Uhm SJ, Gupta MK, Yang JH, Chung HJ, Min TS, Lee HT. Theriogenology 2010; 73: 1024-1036.

The viability of oocytes was evaluated based on plasma membrane morphology and their esterase enzyme activity by FDA (3 ′ 6 ′ diacetyl fluorescein) analysis [Gupta MK, Uhm SJ, Lee HT. Fertil Steril 2010; 93: 2602-260740].

Example  3: Activation of oocyte regeneration

Presumed diphroid unit reproduction is Gupta MK, Uhm SJ, Han DW, Lee HT. Produced by electro-activity as described in Mol Reprod Dev 2007; 74: 435-444.

In summary, the stripped oocytes were washed three times with TL-HEPES-BSA medium and then electrically activated with an active medium containing 0.3 M mannitol, 0.1 mM MgSO₄ and 1.0 mM CaCl2. Activation delivered the oocytes that were stripped between the 1 mm wide and 0.2 mm diameter platinum electrodes covered with activation medium in a chamber to which an electric pulsing machine (BTX Electo Cell Manipulator 200; BTX, San Diego, Calif.) Was connected. Activation was induced for 30 m seconds with a single direct current pulse of 1.0 kv / cm. After activation, the Northern California State University Medium 23 (NCSU23) supplemented with 7.5 mg / ml Cytochalasin B (Sigma, St. Louis, MO, USA) for 4 hours before transferring the activated oocytes to the culture medium in vitro for further culture. Cultured in medium.

Example  4: in vitro fertilization ( IVF )

The IVF of mature porcine oocytes was Gupta MK, Jang JM, Jung JW, Uhm SJ, Kim KP, Lee HT. Proteomics 2009; 9: 2846-2860, as described.

In summary, all oocytes matured in vitro were inoculated with oocytes three times with modified medium (modified Tris-buffer medium) containing 0.1 mM hyaluronidase to escape the oocytes and containing 1 mM caffeine sodium benzoate and 0.1% BSA. Washed and placed in groups of 10 to 15 oocytes per 50 μl of fertilization medium. Spermatozoa were recovered from the myoblastomas in TL-HEPES and centrifuged at 800 rpm for 5 minutes to pelletize. The soft pellets were then subjected to a swim-up in Sp-TALP medium for 10 minutes. The swarmed sperm cells were collected and used for fertilization of oocytes at a final sperm concentration of ⁵ × 10 ⁵ sperm / mL. Sperm and oocytes were incubated for 6 hours at 39 ° C., 5% CO _2, and then transferred to in IVC medium for further culture.

Example  5: in vitro culture of embryos ( IVC )

Putative conjugates produced by PA or IVF were incubated for 7 days in NCSU23 medium supplemented with 0.4% fatty acid free bovine serum albumin (BSA; Sigma; A6033). At 2 and 7 days of IVC, egg shedding rate and blastocyst formation rate were recorded based on the total number of oocytes used for IVM, respectively.

Example  6: Measurement of fertility and embryo quality

Fertilization ability of oocytes matured in vitro was measured 18 hours after fertilization, while embryo quality of PA and IVF blastocysts was evaluated on day 7 of IVC [Uhm SJ, Gupta MK, Yang JH, Chung HJ, Min TS, Lee HT. Theriogenology 2010; 73: 1024-1036; Gupta MK, Uhm SJ, Lee HT. Fertil Steril 2010; 93: 2602-2607.

The nuclei of fertilized embryos and blastocysts were visualized by fluorescence Hoechst 33342 staining as described above. Fertilized oocytes were classified as either unfertilized (oocytes with no penetrating sperm or with pronucleus) or fertilization (oocytes with two or more germ cells but without MII plates). Fertilized oocytes with two or more germ cells were considered polyspermic.

Statistical analysis used in the present invention was performed using SPSS software (SPSS Inc, Chicago, IL, USA) for Chi-square test or ANOVA. Percent data were subjected to arc sine transformation before statistical analysis. Data are presented as mean ± standard deviation. The difference was considered significant at P = 0.05.

Claims (9)

delete delete delete delete delete Method of inhibiting meiosis of oocytes comprising treating immature pig oocytes with 3-methyladenine to arrest meiosis. 7. The method for inhibiting meiosis of oocytes according to claim 6, wherein the treatment time is 20 to 42 hours. A method of synchronizing meiosis of oocytes comprising treating immature porcine oocytes with 3-methyladenine to synchronize meiosis at the GV stage. Contraceptive composition comprising 3-methyl adenine as an active ingredient.

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