Involvement of steroid hormones on in vitro maturation of pig oocytes

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.     

Role of glutathione in the maturation and fertilization of pig oocytes in vitro

The present study examined, by treatment of buthionine sulfoximine (BSO), which is a specific inhibitor of glutathione (GSH) synthesis, the role of GSH in the maturation and fertilization of pig oocytes in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughterhouse were cultured for 36 h in Waymouth MB 752/1 with or without BSO (1 mM), fertilized in vitro, and assessed for GSH concentration (before insemination), maturation, and fertilization. The addition of BSO to maturation medium immediately after culture (Group I), 12 h after culture (Group II), or 24 h after culture (Group III) significantly decreased the GSH concentration in pig oocytes compared with the control (P < 0.01), whereas the rate of cumulus mass expansion at 36 h of culture and the rates of nuclear maturation and sperm penetration following in vitro insemination did not differ. However, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the control and Group III than in oocytes matured in Groups I and II (P < 0.01). In experiment 2, when BSO was added to maturation media 15, 18, 21, or 24 h after culture, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the medium supplemented with BSO at 21 or 24 h of culture than in oocytes matured in other groups (P < 0.05 or P < 0.01). The results indicate that GSH synthesis occurs throughout in vitro maturation of pig oocytes and GSH is an important cytoplasmic factor for regulating sperm nuclear decondensation and male pronucleus formation following sperm penetration in pig oocytes. © 1993 Wiley-Liss, Inc.     

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.     

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Effects of extracellular potassium (K⁺) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whitten's medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K⁺ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K⁺-free medium. Millimole concentrations of K⁺ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K⁺ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K⁺ was higher than in media with 12 and 16 mM K⁺. These results suggest that extracellular K⁺ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K⁺ in maturation media impair cytoplasmic maturation.     

Nuclear and cytoskeletal dynamics during oocyte maturation and development of somatic cell cloned pig embryos injected with membrane disintegrated donor cells

The objectives of this study were to characterize the nuclear and cytoskeletal changes of pig oocytes during in vitro maturation (IVM) and the development of the reconstructed embryos after injection with membrane intact or disintegrated donor cells. Cumulus-oocyte complexes (COCs) were collected from abattoir ovaries by follicle (2-8mm) aspiration. In Experiment 1, COCs were cultured in NCSU-23 medium for 0, 11, 22, 33, and 44 h. Oocytes were fixed at different time points for nuclear and cytoskeletal labeling. Forty-three percent and 75% oocytes progressed to MII stage at 33 and 44 h after IVM culture, respectively. Dynamic shift of spindle and cytoplasmic microtubules was evident. In Experiment 2, matured oocytes were injected with either the whole cumulus cell with or without intact cell membranes after enucleation. The reconstructed oocytes were fixed at 0, 2, or 4 h after cell injection for nuclear and cytoskeletal evaluation. When an intact cumulus cell was injected, the injected cell remained intact within 4h after injection. When a cell with disintegrated membrane was injected, 59-63% (n=146) of the injected cell underwent premature chromosome condensation (PCC). In Experiment 3, the reconstructed pig oocytes received membrane-disintegrated cumulus cells or fetal fibroblasts were cultured in PZM medium. The blastocyst rate of the fibroblast-injected embryos was 10%, which was lower than the non-cloned parthenotes (33%, P<0.05) but higher than the cumulus cell-injected embryos (2.7%). These results suggest that pig oocytes are subjected to nuclear and cytoskeletal reorganization during maturation. Pig oocytes injected with membrane-disintegrated fibroblast cells support better blastocyst development of the cloned embryos.     

Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes

Supplementing in vitro maturation medium with porcine follicular fluid (FF) improves maturation rate, male pronucleus formation, and monospermic fertilization of pig oocytes. This study examined, (1) if there are differences in FF derived from large follicles (LF, 5–6 mm in diameter) and small follicles (SF, 3–4 mm in diameter) on the effect of supplementing the maturation medium with FF on the progression of nuclear maturation, fertilization rate, and developmental competence of porcine oocytes; (2) whether the FF source influences the effect of the FF on the maturation medium on the survival rate and proliferation rate of cumulus cells (CCs) and the expansion of cumulus-oocyte-complexes (COCs); (3) whether the oocyte source (oocytes collected from LFs or SFs) influences the effect of FF on the progression of the nuclear maturation of oocytes; (4) whether the factors in the FF that affect the kinetics of nuclear maturation are proteins, and the range of the molecular weight of the FF factors.

In experiment 1, adding FF from LFs (LFF) significantly accelerated nuclear maturation and improved the fertilization rate; the developmental ratio was comparable with those of adding FF from SFs (SFF). In experiment 2, adding LFF, but not SFF, improved the CC survival rate, although the FF source did not affect the proliferation rate. Expansion of COCs was greater with SFF than LFF. In experiment 3, LFF promoted nuclear maturation of oocytes collected from only LFs. There was a significant interaction between the FF source and the oocyte source in the effect on nuclear maturation stages at 36 h of maturation. In experiment 4, treatment of FF with heat or trypsin diminished the difference between the effect of LFF and SFF on the progression of nuclear maturation. In addition, the predominant effect of LFF compared to that of SFF on nuclear maturation was not affected by ultrafiltration of the FF with a 30-kDa filter, but was diminished by ultrafiltration with a 100-kDa filter. The present study suggests that some proteins present in LFF that range in molecular weight from 30 to 100 kDa improve the developmental competence of oocytes probably via progression of nuclear maturation and cumulus cells viability.     

Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte-cumulus complexes of cattle

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte-cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100mM) for 24h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO(3)(-)/NO(2)(-)) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose-response inhibitory effect on cumulus expansion (P<0.05). Addition of 1 and 10mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates (P>0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes (P<0.05), and increased NO(3)(-)/NO(2)(-) concentration when compared to controls (P<0.05). To evaluate if this effect was reversible, 10(-5)M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100mM AG that inhibited the nuclear maturation. However, association of 10(-5)M SNP to 100mM AG did not reverse the effects of AG, but increased NO(3)(-)/NO(2)(-)concentration (P<0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1mM AG (83.9+/-6.2%) did not differ from control oocytes (83.6+/-8.2%; P>0.05), but 10mM partially inhibited migration (3.8+/-6.4%) and 100mM totally inhibited migration (P<0.05). SNP (10(-5)M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10mM AG to IVM medium reduced (73.0+/-8.1%, 15.0+/-8.9%; P<0.05) cleavage and blastocyst development, respectively when compared with controls (89.1+/-3.4%, 37.6+/-7.3%, respectively), but did not differ, (P>0.05), from the group treated with 1mM AG (80.9+/-8.4%, 41.5+/-10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.     

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.     

Steroidogenesis in cumulus cells of bovine cumulus-oocyte-complexes matured in vitro with BSA and different concentrations of steroids.

The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs) to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P<0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P<0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 microg/ml, but these increases were less (P<0.05) than those observed in the control medium. However, in the presence of 1.0 microg/ml progesterone, estradiol accumulation was equal to that of the control medium (P>0.05). Progesterone accumulation (P<0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P<0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 microg/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes

The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.     

Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes

Nitric oxide (NO) is a highly reactive free radical involved in intra- and intercellular signaling in various stages of reproduction. The objective of the present study was to evaluate the effect of the addition of sodium nitroprusside (SNP), a NO donor, on nuclear and cytoplasmic in vitro maturation of bovine oocytes. Analysis of variance was conducted and the means were compared by t test at a level of 5%. Low (10(-7) and 10(-9)M) and intermediate (10(-5)M) concentrations of SNP had no significant effect on nuclear maturation, however, when a greater concentration of SNP (10(-3)M) was added, oocytes remained in metaphase I (MI) after 24 h culture (P<0.05) and did not show cumulus expansion. To evaluate if this effect was reversible and if a retardation or inhibition had occurred in the progression from MI to MII, oocytes were cultured in presence of 10(-3)M of SNP for 24 h followed by culture for an additional 24 h in medium with or without SNP. After 48 h, the oocytes remained in MI even when the medium was changed at 24 h with or without SNP. The kinetics of nuclear maturation was assessed to evaluate if there had been or not a retardation in the progression of meiosis with the concentration of 10(-3)M SNP. This concentration delayed germinal vesicle breakdown (VGBD) at 8 h of culture (P<0.05), and at 12 h there was no significant difference between the control and the treated group. The concentrations that did not induce alterations in nuclear maturation were evaluated for cytoplasmic maturation. The concentration of 10(-5)M improved the percentage of peripheral cortical granules (P<0.05), and significantly increased the percentage of blastocysts. These results demonstrate that SNP at greater concentrations (10(-3)M) has a cytotoxic effect, but at intermediate (10(-5)M) concentrations it increases blastocyst rates. NO exhibits a dual effect on bovine oocytes, inhibits (10(-3)M of SNP) nuclear and cytoplasmic maturation or stimulates (10(-5)M of SNP) cytoplasmic maturation, depending on concentration in the culture medium.     

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.     

The presence of cumulus cells on nuclear maturation of sheep oocytes during in vitro maturation

This study was carried out to investigate the role of maturation by cumulus cells and the initial bond between the cumulus cells and the oocyte on nuclear maturation of sheep oocytes. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30–35 °C within 1–3 h after collection. The oocytes of follicles, 2–6 mm in diameter, were recovered by aspiration and collected in a pre-incubated (at 38.6 °C, 5% CO₂, and 100% humidity) Hepes-modified TCM 199 medium. After preliminary evaluation, the oocytes with evenly granulated cytoplasm and which were surrounded with at least two layers of cumulus cells (good quality oocytes) were selected and subjected to culture in pre-incubated bicarbonate-buffered TCM 199 supplemented with 0.05 IU/ml recombinant human follicle stimulating hormone (rhFSH), 1 IU/ml human chorionic gonadotropin (hCG), and 1 μg/ml estradiol (OCM: oocyte culture medium). Before culturing, the selected oocytes were randomly divided into four treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM; Group 2, denuded oocytes cultured in OCM; Group 3, denuded oocytes co-cultured with a cumulus cell monolayer in OCM; Group 4, denuded oocytes cultured in OCM in the presence of cumulus cells-conditioned medium. After an incubation period (26–27 h), the nuclear status of the oocytes in each treatment group was assessed using a 2% orcein stain. The rate of oocytes reaching the metaphase II (MII) stage (metaphase of second stage of meiosis division) was 82%, 5%, 11%, and 47% for Groups 1, 2, 3, and 4, respectively. The differences between groups were significantly (P < 0.05) different. The percentage of MII oocytes in Group 4 (47%) was higher than that obtained in Group 3 (11%), indicating a higher efficiency in a cumulus cell-conditioned medium, compared to the cumulus cells monolayer in providing the proper condition for sheep oocyte nuclear maturation. The results suggest the ability of sheep oocytes to resume meiosis in the absence of gap junctional communication (GJC) between the cumulus cells and oocyte being drastically interrupted while for optimum oocyte nuclear maturation, the intact physical contact between the oocyte and cumulus cells is essential.     

Completion of first meiosis by sperm penetration in vitro of bovine oocytes inhibited at metaphase-I with dimethylsulphoxide

The effects of dimethylsulphoxide (DMSO) on immature oocytes during maturation in culture and following penetration by spermatozoa were examined. Germinal vesicle breakdown (GVBD) was observed in all oocytes cultured in the maturation medium supplemented with 2, 4 and 8% DMSO. When the oocytes were cultured in medium with 8% DMSO, 95% (57 60 ) of them were inhibited at prometaphase-I. Cumulus cells were significantly (P<0.05) beneficial for resumption of oocyte nuclear maturation during further culture in the maturation medium for 4, 8 and 24 h after DMSO treatment. When the oocytes were additionally cultured for 4 and 8 h in the maturation medium after DMSO treatment, the proportions of oocytes reaching metaphase-II were significantly (P<0.05) higher in those cultured with spermatozoa than without (68 vs 49% and 84 vs 56%, respectively). These results indicate that 8% DMSO does not affect GVBD of oocytes, but conversely it inhibits oocytes at prometaphase-I, and that cumulus cells are important for recovery from DMSO inhibition and for the resumption of nuclear maturation of oocytes. Sperm penetration was also found to stimulate the completion of meiotic maturation of oocytes inhibited at metaphase-I with 8% DMSO.     

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.     

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

In the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.     

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.