Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro.

The present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM-199, Waymouth MB 752/l, and mTLP-PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P less than 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/l with or without pFF than in oocytes matured in the other two media (P less than 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP-PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P less than 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/l, can remarkably promote this ability.     

Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection

In vivo and in vitro matured porcine oocytes were fertilized by subzonal sperm injection (SUZI), and their subsequent development in vitro was examined to determine whether ooplasmic incompetence is the major cause of limited developmental ability of in vitro matured/fertilized porcine oocytes (Experiment 1). There was no significant difference in rates of fertilization (61% vs. 70%), monospermy (37% vs. 45%), and male pronuclear formation (77% vs. 61%) between in vivo and in vitro matured oocytes. Blastocyst formation rate was significantly lower for in vitro matured oocytes (11% vs. 42%; P < 0.001). Forty-six percent of in vivo matured oocytes cleaved to the 2-4 cell stage by 24 hr in culture after SUZI, compared with 3% of in vitro matured oocytes (P < 0.01). In experiment 2, in vitro development of in vitro matured oocytes with evenly and unevenly granulated cytoplasm were compared after SUZI to examine whether developmentally competent in vitro matured oocytes can be identified on the basis of morphological appearance. Most of the blastocysts obtained developed from oocytes with unevenly granulated cytoplasm (7/56 vs. 1/45; P > 0.05). Experiment 3 revealed that the proportion of oocytes with evenly granulated cytoplasm was originally low (11%) in the population of oocytes used for in vitro maturation, and it increased approximately 3-fold (36%; P < 0.001) after maturation. These results suggest that ooplasmic incompetence in porcine in vitro matured oocytes is the major cause of their limited developmental competence. Cytoplasmic maturation measured by male pronucleus formation does not directly reflect developmental competence of the oocytes. It was also shown that evenness of granulation of the cytoplasm is not a useful morphological indicator of developmental competence. © 1996 Wiley-Liss, Inc.     

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.     

Morphologic comparison of ovulated and in vitro–matured porcine oocytes,with particular reference to polyspermy after in vitro fertilization

This study was conducted to evaluate morphologic differences in pig oocytes matured in vivo and in vitro, with particular reference to the potential relationship between oocyte morphology and the occurrence of polyspermy after in vitro fertilization (IVF). In vivo–matured oocytes were surgically recovered from the oviducts of gilts with ovulated follicles on day 2 of estrus, and in vitro–matured oocytes were obtained by culturing follicular oocytes in a oocyte maturation system that has resulted previously in production of live offspring following IVF. Comparisons were made of the cytoplasm density, the diameter of oocytes with or without zona pellucida (ZP), the thickness of the ZP, the size of the perivitelline space (PVS), ZP dissolution time, and cortical granule (CG) distribution before IVF, and CG exocytosis and polyspermic penetration after IVF. Oviductal oocytes have clear areas in the cytoplasm cortex, while in vitro–matured oocytes have very dense cortex. The diameter of ovulated oocytes with ZPs was significantly (P < 0.001) greater than that of in vitro–matured oocytes. However, no difference was observed in the diameter of the oocyte proper. Significantly (P < 0.001) thicker ZPs and wider PVSs were observed in the ovulated oocytes. The ZPs of ovulated oocytes were not dissolved by exposure to 0.1% pronase within 2 hr, but the ZPs of in vitro–matured oocytes were dissolved within 131.7 ± 7.6 sec. The ZPs of ovulated oocytes, but not of in vitro–matured oocytes, were strongly labeled by a lectin from archis hypogaea that is specific for β-D-Gal(1–3)-D-GalNAc. Polyspermy rate was significantly (P < 0.01) higher for in vitro–matured oocytes (65%) than for ovulated oocytes (28%). CGs of oviductal oocytes appeared more aggregated than those of in vitro–matured oocytes. Most of CGs were released from both groups of oocytes 6 hr after IVF regardless of whether they were polyspermic or monospermic oocytes. These results indicate that in vitro–matured and in vivo–matured pig oocytes possess equal ability to release CGs on sperm penetration. Unknown changes in the extracellular matrix and/or cytoplasm of the oocytes while in the oviduct may play an important role(s) in the establishment of a functional block to polyspermy in pig oocytes. Mol. Reprod. Dev. 49:308–316, 1998. © 1998 Wiley-Liss, Inc.     

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine.

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, beta-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 microM BME, 0.5 microgram/ml LH, 0.5 microgram/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5-6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78-89%). The mean differences in penetration rate (69-77%), polyspermy rate (31-40%), male pronuclear formation rate (93-96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32-39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13-15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.     

Maturation, fertilization and complete development of porcine oocytes matured under different systems

This study was designed 1) to determine the effectiveness of 2 in vitro maturation systems commonly employed to produce nuclear and cytoplasmically mature pig oocytes, 2) to assess the effects of boar, sperm concentration and maturation system on oocyte penetrability and male pronucleus formation and 3) to determine the ability of the in vitro matured oocytes to be fertilized in vivo by artificial insemination (AI) of sows. The differences examined between the 2 maturation systems included the culture medium (Waymouth vs TCM199), hormones, additives, culture conditions (static vs gentle agitation) presence or absence of porcine follicular fluid (PFF) and presence or absence of follicular shells. The results showed that nuclear maturation rate was similar in both systems (83.3 +/- 3.5 vs 86.4 +/- 2.5%), and intracellular content of glutathione was 5.21 +/- 0.73 vs 3.5 +/- 0.39 pmol/oocyte, although no correlation between these parameters was observed. The penetration rate and number of sperm cells per oocyte were dependent on the boar, maturation system and sperm concentration, but the rate of male pronuclear formation seemed to be influenced only by the boar and the maturation system but not by sperm concentration. In vivo fertilization of in vitro matured oocytes showed that both maturation systems could yield viable oocytes since 3 of 4 gilts and 2 of 4 gilts, respectively, became pregnant. Failure to become pregnant was not associated with inadequate oocyte maturation since control gilts, which received their own ovulated oocytes rather than in vitro matured oocytes at transfer, also did not become pregnant. We conclude that polyspermy may be an inherent problem in the IVF but not in the IVM systems.     

Effects of the addition of glutathione during maturation on in vitro fertilisation of prepubertal goat oocytes.

Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is a ubiquitous intracellular free thiol that improves development of the male pronucleus at fertilisation and has also been implicated in promoting the development of preimplantation embryos. The objective of this study was to evaluate the effects of adding GSH or cysteine to the in vitro maturation medium on intracellular GSH amounts after in vitro maturation and fertilisation of prepubertal goat oocytes. Oocytes were matured in TCM199 medium supplemented with 10% bovine fetal serum, 1 mg/ml 17beta-estradiol, 10 microg/ml o-FSH, 10 microg/ml LH and 50 mg/ml gentamicin. In vitro maturation medium was completed with two independent treatments: GSH at different concentrations (0, 0.25, 0.50 and 1.00 mM) and L-cysteine at different concentrations (0, 150, 300, 600 and 900 microM). After 27 h of culture at 38.5 degrees C in 5% CO2 in air, the nuclear stage was evaluated. Simultaneously, another sample of oocytes was frozen and the intracellular GSH level was evaluated with spectrophotometric methodology. Oocytes were inseminated with fresh semen (2-3 x 10(6) sperm/ml) in TALP medium supplemented with 1 mg/ml hypotaurine. Oocytes were fixed at 20 h post-insemination to evaluate the in vitro fertilisation. Oocytes matured in 1.00 mM GSH-supplemented medium exhibited higher amounts of intracellular GSH (3.23 pmol per oocyte). The percentage of normal fertilisation (17-27%) was similar for the treatment groups. In conclusion, the addition of 1.00 mM GSH to the maturation medium could be a useful method for increasing the intracellular GSH levels of prepubertal goat oocytes. However, this increase was not associated with a higher normal fertilisation rate of prepubertal goat oocytes.     

Nuclear and Cytoplasmic Maturation of Bovine Oocytes Cultured with dbc AMP,FSH AND hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation.

The present study was conducted to examine the effect of ascorbic acid 2-O-alpha-glucoside (AA-2G), a stable ascorbate derivative, on the sustenance of cytoplasmic maturation responsible for subsequent developmental competence after in vitro fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured for 44 h in North Carolina State University 37 medium supplemented with cysteine, gonadotropins, 10% (v:v) porcine follicular fluid, and 0-750 microM AA-2G. When oocytes were matured in the presence of 250 microM AA-2G, their ability to promote transformation of the sperm nucleus into the male pronucleus (MPN) was strongly enhanced after in vitro fertilization. Similarly, the presence of 25 microM beta-mercaptoethanol (ME) enhanced the degree of progression to MPN of penetrated sperm by associating with the increase in intracellular glutathione (GSH) content. Although the AA-2G treatment during oocyte maturation showed no influence on the GSH concentration, significantly higher levels of ascorbic acid (AsA) were detected in these oocytes than in those oocytes cultured without AA-2G (P < 0.05). The length of DNA migration encompassed by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase system, was not increased in the oocytes treated with AA-2G, whereas ME treatment could not block the DNA damage by ROS. These findings indicate that AA-2G in maturation medium can potentiate the cellular protection of oocytes against oxidative stress by continuously supplying AsA. The proportion of development to the blastocyst stage after in vitro insemination was significantly increased in oocytes matured with AA-2G (P < 0.05), and this proportion showed no difference in comparison with that of oocytes treated with ME. These findings suggest that a critical concentration of intracellular AsA, supplied by AA-2G during in vitro maturation, plays an important role in supporting the cytoplasmic maturation responsible for developmental competence after fertilization by prevention of oxidative stress against porcine oocytes.     

Effects of glucocorticoids on maturation of pig oocytes and their subsequent fertilizing capacity in vitro

The aim of this study was to assess the possible role of glucocorticoids in the maturation of pig oocytes and their subsequent fertilizing capacity in vitro. Pig cumulus-enclosed oocytes collected from prepubertal gilts were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microg/ml), LH (0.5 microg/ml), FSH (0.5 microg/ml), and estradiol-17beta (1 microg/ml) in the presence or absence of cortisol or dexamethasone (DEX) for 24 h; they then were cultured without hormonal supplements in the presence or absence of cortisol or DEX for an additional 16-24 h. Treatment of cumulus-enclosed or denuded oocytes with increasing concentrations of cortisol or DEX for 48 h resulted in a dose-response inhibition of germinal vesicle breakdown (GVB). Increasing duration (12-48 h) of treatment with DEX (10 microg/ml) led to a time-dependent inhibition of GVB, which achieved statistical significance by 12 h. The addition of DEX (10 microg/ml) to maturation medium immediately after culture or at 12 h, 24 h, or 36 h after culture also decreased the percentage of oocytes with GVB. When oocytes were exposed to DEX for 48 h, the maturation rate was reduced. The degree of this reduction was dependent on DEX, and a concentration of DEX higher than 0.1 microg/ml was needed. The inhibitory effect of DEX on the maturation of oocytes was prevented by the glucocorticoid receptor antagonist RU-486. Exposure of oocytes to DEX for 40 h did not prevent sperm penetration, affect the incidence of polyspermy, or decrease the ability of oocytes to form a male pronucleus. The intracellular concentration of glutathione (GSH) in cumulus-enclosed oocytes was 4.4 mM per oocyte. Exposure of oocytes to DEX (0.01-10 microg/ml) had no effect on GSH concentration. These results demonstrate that glucocorticoids directly inhibit the meiotic but not cytoplasmic maturation of pig oocytes in vitro. This inhibitory effect is not mediated through a decrease in the level of intracellular GSH.     

Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro

The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 (control), 25, 50, or 100 μM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.01) for oocytes matured in medium containing 100 μM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P < 0.05) in oocytes matured with 100 μM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P > 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH. © 1995 wiley-Liss, Inc.     

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.     

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.     

Effect of 2-mercaptoethanol and cysteine supplementation during in vitro maturation on the developmental competence of oocytes from hormone-stimulated lambs

The objective was to determine the effect of 2-mercaptoethanol and cysteine on in vitro developmental competence of oocytes from lambs (4-8-week old) stimulated with eCG and pFSH. Oocytes were matured in medium (TCM199) with no supplement (Control group) or with 100muM 2-mercaptoethanol and 600muM cysteine (GSH group). Oocytes from adult sheep were also included (Adult group). The addition of 2-mercaptoethanol and cysteine did not improve nuclear maturation or microtubule configuration 12, 15, 18, or 24h after placement in maturation medium. Sperm head decondensation and male pronucleus formation were evaluated at 6, 12, and 18h after commencement of IVF; sperm decondensation appeared earlier in the GSH group (6h after the start of IVF). There were differences (P<0.05) between the Control group and the GSH and Adult groups for: fertilization rate at both 12h (55.4, 77.0, and 80.6%, respectively) and 18h (67.9, 86.9, and 88.7%); parthenogenesis rate at both 12h (25.0, 10.8, and 5.6%) and 18h (28.3, 9.8, and 4.5%); and polyspermy rate at 18h (26.4, 4.9, and 5.7%). Blastocyst rate at 7d was higher in the GSH group than the Control group (23.9% vs. 14.9%, P<0.05), but both were lower (P<0.05) than the Adult group (38.3%). The addition of 2-mercaptoethanol and cysteine improved sperm decondensation and rates of fertilization and the blastocyst development to 7d, with no effect on blastocyst rate at 9d.     

Effects of LH and FSH on the maturation of pig oocytes in vitro

This research was designed to investigate the effects of LH and FSH (50 ng/ml) on pig oocyte maturation in vitro. The following parameters were studied: a) the degree of heterologous coupling between cumulus cells and oocytes, evaluated by measuring the (3)H-uridine and (3)H-choline uptake in cumulus enclosed oocytes; b) meiotic maturation; c) cytoplasmatic maturation, evaluated by analyzing the ability of the oocytes to promote male pronucleus formation after in vitro fertilization. Despite the marked cumuli expansion induced by gonadotropins, uridine uptake was not influenced by LH or FSH. By contrast, choline uptake in LH-treated oocytes was significantly higher than in FSH-treated or control oocytes (3199 cpm +/- 251 vs 1686 cpm +/- 142, P<0.01). Gonadotropins accelerated meiotic progression, and after 30 hours of culture the percentage of oocytes at the germinal vesicle stage was significantly lower (P<0.01) in LH-(24%, 24 102 ) and FSH-(20%, 18 90 ) treated oocytes than in control oocytes (76%, 64 84 ). After 44 hours of culture, the percentage of oocytes reaching the MII stage was significantly higher (P<0.01) in the presence of LH (76%, 92 120 ) and FSH (86%, 92 108 ) than in the controls (35%, 40 116 ). The percentage of oocytes capable of sustaining male pronucleus formation was similar in the control (48.4%, 63 132 ) and FSH-treated oocytes (44.3%, 51 116 ), while it was markedly increased (P<0.01) by the addition of LH (72.7%, 143 197 ). The data reported indicate that in vitro pig oocytes tend to undergo meiotic maturation even in the absence of hormones. However, in our in vitro system, LH and FSH accelerated and facilitated meiotic progression, and LH selectively improved cytoplasmic maturation which is required to promote the formation of a male pronucleus.     

Pronuclear synchronization and nuclear morphology of mature and in vitro matured oocytes in the rat: An ultrastructural study

Summary The objective of this study was to evaluate synchronous and asynchronous pronucleus (PN) formation and the related patterns of juxtapositional nucleolus (n) formation in immature (prophase I [PI] and metaphase I [MI]) and mature (metaphase II [MII]) oocytes after fertilization, both ultrastructurally and at the level of light microscope. A single dose of 15 IU gonadotrophin was injected subcutaneously to twenty four 26-wk-old, female Wistar rats to induce ovulation. Human chorionic gonadotrophin (4 IU) was administered 40 h later, and after 4–6 h the ovaries were dissected, and the oocytes were aspirated. A total of 214 rat oocytes were classified according to a maturation index as follows: group I, 80 PI oocytes; group II, 50 MI oocytes; and group III, 84 MII oocytes. Immature oocytes were in vitro matured for 18–36 h. Spermatozoa were acquired by microepididymal sperm aspiration and processed using swim-up technique. Intracytoplasmic sperm injection was performed on mature oocytes after 2 h of incubation and on in vitro matured (IVM) oocytes 4 h after maturation. Pronuclear synchronization [both pronucleases (PNs) centrally located, equal sized, with equal numbers and sizes of juxtapositional nucleoli (Nn)] was observed in fertilized oocytes. Asynchronous PN formation (diversity between male and female PNs, related to dimensions, localization, and the number of Nn) in groups I, II, and III was found in 75, 86, and 47% of preembryos, respectively. There was a significant difference of synchronous pronuclear formation between mature and IVM oocytes (P<0.05). In IVM oocytes, asynchronous PN formation is high, and juxtapositional pronucleolar patterns are observed to be low by transmission electron microscope (TEM).     

Low oxygen tension during in vitro maturation of porcine follicular oocytes improves parthenogenetic activation and subsequent development to the blastocyst stage

To establish a reliable in vitro maturation system for activation and subsequent development as nuclear recipients for the effective production of pig clones, we assessed maturation, activation and parthenogenetic development in response to the following: (1) type of immature oocytes (cumulus-oocyte complexes (COCs) or parietal granulosa plus cumulus-oocyte complexes (GCOCs)); (2) oxygen (O(2)) tension (5 or 20%); and (3) maturation period (36-60 h). The rate of nuclear maturation to metaphase-II (M-II) in the GCOC group (73.0 +/- 3.1%) was higher than that in the COC group (P < 0.05, 60.6 +/- 3.5%), but the rates did not differ between the 5 and 20% O(2) tension groups. M-II rate increased (P < 0.05) to about 70% after 42 h and then remained constant until 60 h of culture. When oocytes were matured under 5% O(2) tension and stimulated, the rate of normal oocyte activation (a female pronucleus formation and emission of the second polar body) was higher (P < 0.05, 38.5 +/- 3.9%) than when oocytes were matured under 20% O(2) tension (24.5 +/- 3.9%). On the other hand, the rate of normal activation was not significantly different between the COC and GCOC groups, and the highest (P < 0.05) normal activation rate was obtained in oocytes cultured for 48 and 54 h (48.4 +/- 5.5% and 47.9 +/- 8.2%, respectively). When COC and GCOC matured for 48 h under 5 and 20% O(2) tension were stimulated and subsequently cultured in vitro for 6 days, the rate of blastocyst formation did not differ between the oocyte types nor between the O(2) tension groups. However, blastocyst quality, as measured by mean total cell number, was significantly higher in the 5% O(2) group (P < 0.05, 34.6 +/- 2.0 for COC; 33.8 +/- 1.8 for GCOC) compared with the 20% O(2) group (25.9 +/- 1.8 for COC; 27.0 +/- 2.0 for GCOC). In conclusion, low O(2) tension (5%) during in vitro maturation of porcine oocytes promoted their ability to be activated normally and improved the quality of parthenogenetic blastocysts developed in vitro in modified NCSU-37 solutions. This knowledge may be applicable for preparation of in vitro matured oocytes with good quality as recipient oocytes for generating pig clones.     

Effect on fertilization and development by re-culture after freezing and thawing of bovine oocytes matured in vitro

This study evaluated the effect of 6-h repeat culture before insemination of frozen-thawed, in vitro-matured oocytes on their fertilizability and developmental capacity. Immature oocytes were cultured for 18 h or 24 h and were frozen incrementally. Post-thaw oocytes were repeat cultured for 0 h (control) or 6 h before insemination. With fertilizability, the proportion of enlarged sperm head was significantly (P<0.05) higher in oocytes cultured for 24 h without repeat culture (24:0 h; 51.8%) than those cultured for 24 h with repeat culture (24:6 h; 26.1%) and nonfrozen oocytes (25.9%). However, the proportion of male pronucleus (MPN) in the group of 24:0 h (32.4%) was significantly (P<0.05) lower than that of nonfrozen oocytes (64.3%); the formation of the female pronucleus and MPN were also significantly (P<0.01) lower (17.2%) than that of nonfrozen oocytes (56.1%). Polyspermic oocytes cultured for 18 h with repeat culture for 6 h (18:6 h) were significantly (P<0.05) higher (47.5%) than for nonfrozen oocytes (25.6%). Development to 8-cell stage in the group of 18:6 h was significantly (P<0.05) lower (1.6%) than that of nonfrozen oocytes (18.5%). The cleavage rates in the groups of 24:0 h (16.3%) and 24:6 h (23.4%) were also significantly (P<0.05) lower than for nonfrozen oocytes (63.3%). Development to blastocysts was low (3.9%), but hatched blastocysts were observed in frozen-thawed oocytes cultured for 18:0 h. These results indicate the post-thaw 6-h repeat culture did not greatly improve the fertilizability and embryonic development of oocytes cultured for 18 h or 24 h before freezing. Frozen-thawed oocytes after 24 h in vitro maturation without repeat culture especially had impaired capacities for fertilizability and development.     

Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.     

Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor

This study examined the ability of epidermal growth factor (EGF) to improve the developmental competence of pig oocytes matured in a protein-free (PF) in vitro maturation (IVM) system. Oocyte maturation was done in one of three media: 1. PF-TCM: tissue culture medium (TCM) 199 + 0.1% polyvinylalcohol (PVA); 2. PF-TCM+EGF: PF-TCM + 10 ng/ml EGF; and 3. +ve CONT: North Carolina State University (NCSU) 23 medium + 10% porcine follicular fluid. All media contained 0.57 mM cysteine. Hormonal supplements, 0.5 microg/mL LH and 0.5 microg/mL FSH, were present only for the first half (20 to 22 h) of the culture period. After maturation, oocytes were co-incubated with frozen-thawed spermatozoa for 5 to 6 h and transferred to embryo culture medium, NCSU 23 containing 0.4% BSA, for 144 h. In Experiment 1, differences in cumulus expansion were observed for oocytes matured in +ve CONT (Category 4), PF-TCM (Category 2) and PF-TCM+EGF (Category 3). However, no significant differences in nuclear maturation to metaphase II stage were observed. In Experiment 2, no differences in fertilization parameters were observed. Significant (P < 0.01) differences in cleavage rates were observed among the three media for a proportion of the oocytes matured (52, 60 and 69% in PF-TCM, PF-TCM+EGF, and +ve CONT, respectively). Oocytes matured in PF-TCM showed the lowest (P < 0.01) blastocyst development (22%). However, the same rate of blastocyst development was obtained for +ve CONT (37%) and PF-TCM+EGF (37%). Blastocyst cell numbers were significantly higher when oocytes were matured in the presence of EGF (26 vs. 37 to 41). In Experiment 3, oocytes matured in PF-TCM+EGF had a significantly (P < 0.05) higher intracellular glutathione (GSH) concentration (5.9 vs. 11.4 pmol/oocyte) compared with PF-TCM. Twenty-two of 25 embryo transfer recipients became pregnant (Experiment 4). Four animals returned to estrus in within 60 days. Six pregnant animals slaughtered at 26 to 45 days had 43 fetuses (range: 4 to 12) and the remaining 12 animals farrowed 82 piglets (range: 3 to 12). These results indicate that EGF enhances the developmental competence of pig oocytes matured in a protein-free culture medium which is correlated with higher GSH level in oocytes. Birth of piglets indicate that embryos derived from oocytes matured in the presence of EGF are viable.