Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization

The present study examined the effect of epidermal growth factor (EGF) during in vitro maturation (IVM) and embryo culture on blastocyst development in the pig. In experiment 1, cumulus oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, hormonal supplements, and with or without EGF (0–40 ng/ml) for 20–22 hr. They then were cultured for an additional 20–22 hr without hormones. After maturation, cumulus-free oocytes were co-incubated with frozen-thawed spermatozoa for 5–6 hr. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 hr. In experiment 2, oocytes were matured in medium containing 10 ng/ml EGF, inseminated, and putative embryos were cultured in the presence of 0–40 ng/ml EGF. In experiment 3, oocytes were cultured in the presence of 0, 10 and 40 ng/ml EGF to examine the kinetics of meiotic maturation. In experiment 4, 2- to 4-cell and 8-cell to morula stage embryos derived from oocytes matured with 10 ng/ml EGF were transferred to the oviduct and uterus, respectively, of each of three recipient gilts (3 and 4 days post-estrus, respectively). The presence or absence of EGF during IVM did not affect cumulus expansion, nuclear maturation, fertilization parameters, or cleavage rate. However, compared to no addition (21%), presence of 1 (33%) and 10 ng/ml EGF (42%) during IVM increased (P < 0.01) the rate of blastocyst development in a concentration-dependent manner. Compared to 10 ng/ml EGF, higher concentrations (20 and 40 ng/ml) reduced (P < 0.01) blastocyst development in a concentration-dependent manner (35% and 24%, respectively). No difference was observed between no addition and 40 ng/ml EGF (22%). Compared to no addition and 10 ng/ml EGF, a significantly (P < 0.001) higher proportion (25% vs. 55%) of oocytes reached metaphase II stage 33 hr after IVM with 40 ng/ml EGF. However, no difference was observed at 44 hr. Transfer of embryos to six recipient gilts resulted in three pregnancies and birth of 18 piglets. The results show that EGF at certain concentrations in IVM medium can influence the developmental competence of oocytes. However, addition of EGF during the culture of pig embryos derived from oocytes matured in the presence of EGF is without effect. Birth of piglets provides evidence that embryos derived from oocytes matured in a medium containing EGF are viable. Mol. Reprod. Dev. 51:395–401, 1998. © 1998 Wiley-Liss, Inc.     

Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro

Gonadotropins, steroids and growth factors stimulate or inhibit cumulus expansion, nuclear maturation, or both, of most mammalian oocytes in vitro. The objective was to evaluate the effects of epidermal growth factor (EGF) and various hormone combinations on in vitro granulosa/cumulus (G-C) expansion and nuclear maturation of domestic dog oocytes derived from advanced preantral and early antral follicles. Follicles were collected after enzymatic digestion of ovarian tissue and cultured for 66 h in F-12/DME with 20% fetal bovine serum, 2mM glutamine and 1% antibiotic-antimycotic (Control). Treatments comprised the following groups; each was cultured both with and without EGF (5 ng/mL): Control, FSH (0.5 microg/mL), LH (5 microg/mL), estradiol-17beta (E2, 1 microg/mL), FSH+LH, and FSH+LH+E2. Granulosa/cumulus expansion was scored on a scale of 0 (no expansion) to +3 (maximum expansion). The interaction between EGF and hormone treatment affected (P=0.011) maximum G-C expansion. With the exception of the E2 group, EGF increased (P<0.05) the proportion of oocytes exhibiting +3 expansion. The synergism of E2 with FSH+LH enhanced maximum G-C expansion; compared to all other treatments, the greatest expansion was observed in the FSH+LH+E2+EGF group (83.5+/-3.5%). When cultured in EGF alone, oocytes failed to reach metaphase I-II (MI-MII) stages. The interaction between EGF and hormone treatment tended (P=0.089) to increase the proportion of oocytes resuming or completing nuclear maturation (GVBD-MII). In addition, supplementing culture media with hormones increased (P=0.010) the GVBD-MII rate. Therefore, EGF in combination with FSH and LH enhanced G-C expansion of cultured canine oocytes, with no significant effect on the proportion of oocytes derived from advanced preantral and early antral follicles that reached MI-MII.     

Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor-b?₁ (TGF-b?₁), follicle-stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A₄), and estradiol-17b? (E₂) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte-cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E₂ (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A₄ (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF-b?₁ had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley-Liss, Inc.     

Effect of follicular cells,IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

Several signaling pathways are involved in the control of cattle oocyte maturation

The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment.     

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.     

Insulin-transferrin-selenium (ITS) improves maturation of porcine oocytes in vitro

The objective of this study was to determine if insulin-transferrin-selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.     

Effect of growth factors, EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes

The objective of these experiments was to determine the effect of exogenous addition of insulin-like growth factor-I (IGF-I, 100 ng/mL), epidermal growth factor (EGF, 10 ng/mL) and estradiol (E2, 100 ng/mL) to the maturation medium of sheep oocytes on their subsequent development in vitro. Addition of IGF-I to the maturation medium did not improve nuclear or cytoplasmic maturation of sheep oocytes at the concentration tested. However, EGF improved significantly the resumption of meiosis (84% oocytes in metaphase II stage after IVM vs. 59% in medium alone). Cleavage rate and blastocyst development rates were improved (P<0.01) after addition of EGF (60% and 29%, respectively), as compared with maturation in TCM 199 alone (39% and 19%, respectively), but remained lower than rates observed after maturation in complete medium containing follicular fluid (FF, 10%) and FSH (81% and 35%, respectively). No additive effect of EGF over FSH was observed during these experiments. Addition of FF to FSH containing maturation medium improved significantly both cleavage (P<0.001) and blastocyst rates (P<0.05). Addition of E2 to the IVM medium is not required when medium already contains FF. However, in defined conditions supplementation of maturation medium with E2 had a positive effect. These results suggest that EGF, FSH and E2 may play an important role in the nuclear and cytoplasmic maturation of sheep oocytes in vitro.     

The effects of 17beta-estradiol and protein supplement on the response to purified and recombinant follicle stimulating hormone in bovine oocytes

During in vitro maturation of bovine oocytes, effects of gonadotropins (FSH, LH) and growth factors such as epidermal growth factor (EGF) vary among studies. Now that we can use defined or semi-defined medium, it becomes possible to evaluate recombinant products to assess their roles. Therefore, this study was designed to evaluate the effect of purified porcine (pFSH) or recombinant human (r-hFSH; 5, 50, or 500 ng/ml) follicle stimulating hormone, luteinising hormone (LH; 50,500 or 5000 ng/ml) and epidermal growth factor (EGF; 5, 10, 30 or 50 ng/ml) on subsequent embryonic development of in vitro matured bovine oocytes. In addition, the presence of bovine serum albumin (BSA; 8 mg/ml) as a protein supplement during in vitro maturation (IVM) was studied. For all treatments, cumulus-oocyte complexes were matured in defined maturation medium consisting of synthetic oviduct fluid. Addition of LH to the maturation medium at all concentrations studied did not increase the proportion of oocytes developing to the morula and blastocyst stages. However, morula and blastocyst yield were improved (p < 0.05) after addition of EGF (30 ng/ml) as compared with maturation medium alone (29.3% vs 18.0%, respectively). Addition of r-hFSH to the maturation medium in the presence of 17beta-estradiol (E2) significantly (p < 0.0001) increased the morula and blastocyst rate compared with maturation medium alone (40.3% vs 19.3%, respectively). The presence of BSA alone during IVM significantly reduced the developmental competence of oocytes as reflected by the morula and blastocyst yield. These results demonstrate the essential role of FSH, EGF and E2 on the kinetics of nuclear and cytoplasmic maturation that are essential for the formation of an egg capable of fertilisation and development. Also, supplementation of r-hFSH and E2 during IVM under our conditions increases morula and blastocyst yield following in vitro fertilisation and in vitro culture in defined medium. Finally, the presence of BSA as the only protein supplement during IVM may be detrimental to oocyte maturation.     

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17beta-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 microg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.     

Effect of EGF on in vitro maturation of domestic cat oocytes

The objective of this study was to evaluate the influence of different concentrations of epidermal growth factor (EGF) on in vitro maturation of domestic cat oocytes. A total of 444 cat oocytes were matured in MSOF (maturation synthetic oviductal fluid) in the presence of varying EGF concentrations: (1) MSOF (control); (2) MSOF+10 ng/mL EGF (EGF10); (3) MSOF+25 ng/mL EGF (EGF25); and (4) MSOF+50 ng/mL EGF (EGF50). After IVM, oocytes were in vitro fertilized to verify the effect of adding EGF on cytoplasmic maturation. Cleavage rate was recorded and noncleaving oocytes were stained with Hoechst 33258 and examined to determine nuclear maturation rate. Cleaved zygotes were cultured in vitro and embryo stages were evaluated on days 6 and 7. There was no difference among groups in the total number of oocytes reaching the metaphase II (MII) stage (P>0.05). The EGF25 group had the highest (P<0.01) blastocyst yield (37.5%) and developmental competence (60.9%). Cleavage rate and resulting morulae and blastocysts on day 6 for EGF25 group were higher (P<0.01) than control and EGF50 groups. Although EGF did not significantly enhance nuclear maturation rate, it had a dose-related positive effect on cytoplasmic maturation, since the oocyte's ability to cleave and reach the blastocyst stage was improved at 25 ng/mL, with intermediate improvement at 10 ng/mL, but 50 ng/mL had no significant benefit. In conclusion, the addition of EGF to the maturation medium enhanced cytoplasmic maturation of cat oocytes in vitro.     

Effects of epidermal growth factor on maturation, fertilization and development of bovine follicular oocytes

When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 0 to 50 ng/ml EGF, the rate of metaphase II oocytes of 30 ng/ml EGF (97%) was significantly (P < 0.05) higher than that of the control (77%), 10 (85%), and 50 ng/ml EGF (82%). After in vitro fertilization, the rate of monospermic oocytes of 30 ng/ml (75%) and 50 ng/ml EGF (77%) was significantly (P < 0.05) higher than that of the control (56 %). When bovine follicular oocytes were cultured for 24 h in TCM 199 containing 30 ng/ml EGF and/or 10% FCS and fertilized with frozen-thawed spermatozoa, the rate of monospermic oocytes was significantly (P < 0.05) higher in EGF + FCS (82%) than in EGF (61%) and FCS (67%). The rate of oocytes with 2 pronuclei was significantly (P < 0.05) higher in EGF + FCS (54%) than in EGF (27%). When in vitro-fertilized bovine embryos were cultured for 8 d with granulosa cells in TCM 199 containing 0, 10, 30 and 50 ng/ml EGF, the rate of embryos developing to the blastocyst stage was not significantly different among the control (22%), 10 ng/ml (20%), 30 ng/ml (18%), and 50 ng/ml (20%) EGF groups. These results indicate that EGF has a beneficial effect on in vitro maturation and fertilization of oocytes and that EGF plus FCS also have a beneficial effect on normal fertilization of oocytes. However, EGF had no beneficial effect on in vitro development of embryos when they were co-cultured with granulosa cells in medium with FCS.     

Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes

The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 μg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6–8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[³⁵S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes. Mol. Reprod. Dev. 46:401–407, 1997. © 1997 Wiley-Liss, Inc.     

Transforming growth factor-alpha augments meiotic maturation of cumulus cell-enclosed mouse oocytes

Growth factors have been shown to play an important role in the regulation of ovarian function. In this study, we examined the effects of transforming growth factor-alpha (TGF-alpha) on the meiotic maturation of immature mouse oocytes in vitro. Cumulus cell-enclosed oocytes were exposed to TGF-alpha with or without the meiotic inhibitor hypoxanthine (HX), and oocyte maturation was assessed by germinal vesicle breakdown (GVBD). Likewise, mechanically denuded oocytes were examined for GVBD following exposure to HX and TGF-alpha. When cumulus cell-enclosed oocytes were exposed to TGF-alpha (1 microgram/ml) in the presence of HX (4 mM), an increase in GVBD was observed first after 5 hours of culture. Maximal stimulation was reached at 24 hours when 70% of the oocytes underwent maturation in the presence of TGF-alpha and HX as compared to 33% with HX only. Concentrations of TGF-alpha as low as 0.1 ng/ml produced a similar stimulatory response after 24 hours of culture. Spontaneous maturation in the presence of TGF-alpha, but without HX, was also enhanced. The stimulation of GVBD by TGF-alpha showed an increase over time both with and without HX. When denuded oocytes were exposed to TGF-alpha in the presence of HX, no effect was observed. Our results suggest that TGF-alpha is a potent stimulator of mouse oocyte maturation in vitro and that its effect is mediated by the surrounding cumulus cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro maturation of pig oocytes with different media, hormone and meiosis inhibitors

This study evaluated in vitro maturation of pig oocytes in two maturation media (TCM199 and NCSU23) supplemented with 10% porcine follicular fluid (pFF) or 0.1% polyvinyl alcohol (PVA) and four hormonal treatments. The best media was then used to evaluate the effect of reversible meiosis inhibitors cycloheximide (5 microgram/ml) [DOSAGE ERROR CORRECTED]and butyrolactone I (12.5M) on the maturation of pig oocytes was evaluated. After maturation for 44 h, the oocytes were fixed, stained, and examined under epifluorescence microscopy. The comparison of the proportion of oocytes in metaphase II revealed that hormonal treatment 2(incubation for 22 h - 10 ng EGF/ml, 10 IU hCG/ml and 10 IU eCG/ml, followed by incubation for 22 h - 10 ng EGF/ml) presented higher repeatability percentages: TCM+ PVA (54.5% - 61/112); TCM+ pFF (65.0% - 63/97);NCSU23 + PVA (54.6% - 65/119), and NCSU23 + pFF (58.1% - 61/105). The comparison of maturation media showed that TCM199 presented more constant results than NCSU23. Regarding supplementation with pFF or PVA, TCM199 with pFF presented better results. The comparison between butyrolactone I and cycloheximide demonstrated that both drugs effectively inhibited meiosis; however, only cycloheximide presented metaphase II percentages similar to the control (70.29% and 75.49%, respectively). In conclusion, it is recommended the use of TCM199 medium supplemented with pFF and hormonal treatment with 10 ng EGF/ml, 10 UI hCG/mland 10 UI eCG/ml during the first 22 h and more 22 h with 10 ng EGF/ml for the pig oocytes maturation. Butyrolactone I and cycloheximide effectively arrested/resumpted maturation; however, the oocytes percentages in metaphase II was the same for both cycloheximide and the control groups.     

The effect of growth hormone (GH) and insulin-like growth factor-I (IGF-I) on in vitro maturation of equine oocytes

Summary The objective of this study was to test the hypothesis that equine growth hormone (eGH), in combination with insulin growth factor-I (IGF-I), influences positively in vitro nuclear and cytoplasmic maturation of equine oocytes. Cumulus-oocyte complexes were recovered from follicles that were < 25 mm in diameter, characterized by morphology and were allocated randomly as follow: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 400 ng/ml anti-IGF-I antibody. Oocytes were matured for 30 h at 38.5°C in air with 5% CO2 and then stained with 10 μg/ml propidium iodide (PI) to evaluate nuclear status and 10 μg/ml Lens culinaris agglutinin-fluorescein complex (FITC-LCA) to assess cortical granule migration by confocal microscopy. The proportion of immature oocytes that developed to the metaphase II (MII) stage in the eGH + IGF-I group (15 of 45) was greater than in the groups that were treated only with IGF-I (7 of 36, p = 0.03). Oocytes that reached MII in the control group (20 of 56; 35.7%) showed a tendency to be different when compared with eGH + IGF-I group (15 of 45; 33.3%, p = 0.08). The treated group that contained anti-IGF-I (15 of 33; 45.4%) decreased the number of oocytes reaching any stage of development when compared with eGH (47 of 72; 65.3%) and eGH + IGF-I (33 of 45; 73.3%) groups (p = 0.05) when data from MI and MII were combined. We concluded that the addition of eGH to in vitro maturation (IVM) medium influenced the in vitro nuclear and cytoplasmic maturation of equine oocytes. The use of GH and IGF-I in vitro may represent a potential alternative for IVM of equine oocytes.     

In vitro maturation and ultrastructural observation of cryopreserved minke whale (Balaenoptera acutorostrata) follicular oocytes

Minke whale (Balaenoptera acutorostrata) follicular oocytes were cryopreserved by a slow-step freezing procedure using ethylene glycol. The morphologically viable proportion of postthawed minke whale follicular oocytes was 39.7%. The maturity of the animals (immature and mature whales) or the presence or absence of cumulus cells (CC) did not affect the proportion of morphologically viable oocytes. Postthawed oocytes were examined for nuclear status after in vitro maturation. The presence of CC (29.1%) significantly enhanced (P < 0.05) the proportion of oocytes at metaphase I/anaphase I/telophase I stages compared to results with the absence of CC (13.5%). A total of 4 of 194 postthawed oocytes matured to the second metaphase stage after culture for 5.5 days with or without CC. The cryopreserved immature oocytes obtained from immature and mature whales were processed to examine the ultrastructure by transmission electron microscopy. Varying ultrastructural damage to the cytoplasm was observed as a result of the cryopreservation procedures. These results show that 20-30% of cryopreserved minke whale follicular oocytes can resume meiosis in vitro, but damage induced by the freezing and thawing procedures was observed.