Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro

Porcine follicular oocytes, collected from antral follicles (2–5 mm in diameter) of gilt ovaries, were matured in vitro with or without porcine follicular fluid (pFF), gonadotrophins (GTH) or fetal calf serum (FCS) for 48 hours at 37°C under 5% CO₂ in air, and their ability of male pronucleus (mPN) formation was examined after in vitro fertilization. Formation of mPN was observed in 38.6% of penetrated oocytes matured in modified Krebs-Ringer bicarbonate solution (TYH) 18 hours after insemination. The addition of GTH into the maturation medium did not improve the proportion of mPN-formed oocytes (20–30%). In contrast, the mPN formation rate elevated significantly (59.5%) when the oocytes were cultured with pFF, and the addition of follicle-stimulating hormone (FSH) enhanced this pFF action (the rate became 81.0%). In the presence of FSH, significant pFF effect was observable at the concentration of 5%, and its efficiency was elevated with the increase of pFF concentration. When the oocytes were matured with FCS, the mPN formation rate was unchanged or decreased rather than improved (0–25%). These results suggest that pFF, but not FCS, have substance(s) stimulating the ability of mPN formation in porcine oocytes.     

Immunohistochemical localization and possible physiological roles of tumor necrosis factor-α in mouse cumulus-oocyte complexes

Tumor necrosis factor-α (TNF-α), a cytokine which is produced by activated macrophages, has been shown to participate in the regulation of ovarian functions. In the course of our investigation on the mechanism of maturation, fertilization and degeneration of mouse oocytes, immunoreactivity to TNF-α was found in the cytoplasm of the cells surrounding the maturing oocytes and of granulosa cells facing the antral cavity. Immunoblot analysis with the specific antibody to TNF-α identified the 17 kDa Mr band in the extract of cumulusoocyte complexes. Various concentrations of TNF-α (mouse, recombinant) and anti TNF-α antiserum (polyclonal rabbit anti-mouse recombinant TNF-α) were then used to determine their effect on the germinal vesicle breakdown (GVBD), polar body extrusion, fertilization and fragmentation of mouse oocytes/eggs. TNF-α at concentrations of 10 ng/mL or less and anti-TNF-α antiserum at concentrations of 10% or less, had no effect on the spontaneous GVBD and polar body extrusion of mouse oocytes in culture. Mouse follicular oocytes cultured for more than 72 h in modified Krebs-Ringer solution in vitro undergo spontaneous fragmentation, which is a degenerative change to form 'blastomeres' with or without nuclear fragments or chromatin. Ghost-like blastomeres were also identified in the space among fragmented 'blastomeres'. The spontaneous fragmentation of mouse follicular oocytes was suppressed in the presence of TNF-α at concentrations of 1 ng/mL or greater. Anti-TNF-α antiserum (1%) accelerated the induction of fragmentation of oocytes cultured in vitro . The addition of anti TNF-α antiserum (10%) to the culture medium did not influence the fertilization rates of the eggs surrounded by the expanded cumulus. These results appear to indicate that the process of degeneration of mouse oocytes/eggs is modulated by TNF-α accumulated in the expanded cumulus during oocyte maturation.     

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Juvenile and adult immature and in vitro matured ovine oocytes evaluated in relation to membrane electrical properties, calcium stores, IP3 sensitivity and apoptosis occurrence in cumulus cells

The analysis of differences between juvenile and adult oocytes may provide useful information on the acquisition of meiotic and developmental competence of the female gamete. In oocytes collected from either ewes or 40-day-old lambs, we evaluated membrane electrical properties, such as resting potential, conductance, activation ion currents, L-type Ca(2+) currents as well as calcium stores and IP3 sensitivity; in addition, the incidence of apoptosis in cumulus cells in these two age categories was compared. The analysis was carried out in oocytes both prior to and after in vitro maturation. Significant differences were found in all the examined parameters in relation to maturational stages whereas minor differences were recorded in relation to age of the donor. IP3 sensitivity strongly increased after in vitro maturation following a dose-dependent pattern from 1 to 500 micromol/L with a significant interaction (P < 0.01) between dose and maturational stage. The incidence of apoptosis in cumulus cells strongly increased after in vitro maturation and was greater in adult than in juvenile cumulus cells (39.2 +/- 5.8% vs. 21.9 +/- 3.5%; P < 0.01). In conclusion, all the examined parameters were greatly affected by the maturational stage, whereas minor differences were due to age-related oocyte quality, that is, at plasma membrane levels to conductance, activation current peaks and calcium currents, at cytosol level to calcium stores and IP3 sensitivity, and to incidence of apoptosis in cumulus cells. These parameters were compared with previous data in bovine to analyze oocyte quality in juvenile and adult individuals or between species.     

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.     

Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes

Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.     

Utility of ultrasound stimulation for activation of pig oocytes matured in vitro

The present study was carried out to examine the development of pig oocytes after exposing to ultrasound under various conditions. When oocytes were exposed to ultrasound in the sorbitol medium, the blastocyst formation rate was significantly (P < 0.01) higher than that of oocytes exposed in HEPES-TLP-PVA. Optison, an echo-contrast microbubble, prevented the development into blastocysts of oocytes exposed to ultrasound in the sorbitol medium (P < 0.01). The mean number of cells in the blastocysts developed from oocytes exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that obtained by using ultrasound with 50% duty cycle. The blastocyst formation rate of oocytes exposed to ultrasound for 30 sec was significantly (P < 0.05) higher than that exposed for 10 sec. There were no significant differences in the rates of oocytes developed to the blastocyst stage and the mean numbers of cells in the blastocysts among different intensities of ultrasound. The pronuclear formation and second polar body extrusion rates of oocytes exposed to ultrasound did not differ from eclectically activated oocytes. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from oocytes activated by exposing to ultrasound was significantly (P < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the nuclear activation and parthenogenetic development of pig oocytes matured in vitro.     

The development of meiotic competence in the rat: Role of hormones and of the stage of follicular development

Hypophysectomy of 15-day-old rats (hypox) markedly reduced the normal development of meiotic competence and abolished the development of antral follicles between days 21 and 31 postpartum (pp). Here the correlation among age of the rats, stage of follicular development, and meiotic competence was examined. Administration of pregnant mare's serum gonadotropin (PMSG-3IU) or insertion of an estradiol-17β (E₂) capsule to hypox rats induced the development of meiotic competence provided the treatment started after day 20 pp. Hormonal treatments at an earlier age were not effective in inducing meiotic competence in hypox rats. The induction of meiotic competence by PMSG or E₂ was associated with an increase in the number of granulosa cells and formation of follicular antrum. The finding that PMSG and E₂ failed to induce meiotic competence when administered prior to day 21 pp suggests that the development of meiotic competence is an age-dependent process. When the hormonal treatments commenced after day 21, both follicular development and meiotic competence were induced.     

Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation

Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were designed to examine effects of intracellular calcium elevating agents such as calcium ionophore A23187 (CaA) and ethanol, or protein synthesis and phosphorylation inhibitors such as cycloheximide (CH) and 6-dimethylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matured bovine oocytes. Oocytes were matured for 24 hr in M-199 supplemented with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39°C in humidified air. They were then activated by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8–15% gradient SDS-PAGE and silver stained. Results demonstrated that none of the chemical agents—CaA, ethanol, 6-DMAP, or cycloheximide—could effectively induce parthenogenetic development of young bovine oocytes. When compared with the single treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased the rates of cleavage (78–82% versus 3–13%) and blastocyst development (31–40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P > 0.05). Supplementation with CD to the combined CaA and CH treatment improved rates of cleavage and blastocyst development versus without CD supplementation (31% versus 7%; P < 0.05). Fluorescent microscopy revealed that 95% (n = 40) of oocytes treated with CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), while 88% (n = 40) in the CaA plus cycloheximide–treated group had one PN and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocytes (n = 40) arrested at a metaphase stage with two PBs (named as metaphase III or MIII). Protein patterns were similar for chemically activated and in vitro–fertilized (IVF) oocytes in that the 138- and 133-kDa proteins, whose functions are not yet known, were present in the metaphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocytes can be obtained by calcium ionophore treatment followed by incubation in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes. Mol. Reprod. Dev. 49:298–307, 1998. © 1998 Wiley-Liss, Inc.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of β-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, β-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing. © 1996 Wiley-Liss, Inc.     

Mechanistic insights into the reduced developmental capacity of in vitro matured oocytes and importance of cumulus cells in oocyte quality determination

In vitro maturation of oocytes is a promising assisted reproductive technology (ART) for infertility treatment, although it is still not a routine technique for human ART due to reduced embryonic development. The aim of the present study was to clarify the possible reasons for reduced capacity of in vitro matured oocytes. Our results showed that the oocytes matured in vitro displayed increased abnormal mitochondrial distribution, reduced mitochondrial membrane potential, and increased reactive oxygen species levels when compared to in vivo matured oocytes. These results were not different in oocytes matured in vitro with or without cumulus cells. Notably, in vitro matured oocytes displayed increased mitochondrial DNA numbers probably due to functional compensation. In vitro matured oocytes showed significantly lower activation and embryonic development rates, and their ability to produce Ca²⁺ oscillations was much lower in response to parthenogenetic activation, especially in oocytes matured in vitro without cumulus cells with nearly half of them failing to produce calcium waves upon strontium chloride stimulation. These data are important for understanding the reasons for reduced developmental potential of in vitro matured oocytes and the importance of cumulus cells for oocyte quality.     

Maturation of pig and rat oocytes transplanted into surrogate pig follicles in vitro

Pig oocytes obtained from slaughterhouse material and rat oocytes obtained from PMSG-treated immature females were incubated as isolated oocytes or injected into explanted pig follicles (5–8 mm). Free oocytes of both species, with or without their cumulus investment or gonadotropins during culture, matured at high rates after 30 hr or 9–10 hr of culture, respectively. Gonadotropic stimulation was necessary for maturation of both the native and injected cumulus-intact pig oocytes in follicle culture. Cumulus-free pig oocytes injected into follicle failed to mature in response to gonadotropic stimulation, suggesting an inability to perceive or respond to stimulation. Injected rat oocytes, however, matured irrespective of cumulus investment or gonadotropic stimulation. Their maturation was delayed and reduced at 9 hr. These results in the rat suggest that the pig follicular environment is incapable of regulating rat oocyte maturation but rather presents a permissive or supportive environment for their maturation. The explanted surrogate follicles from the pig or other species may provide a useful model for the study of oocyte-follicle interactions in oocyte maturation within or between species.