Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.     

TAF4b, a TBP associated factor, is required for oocyte development and function

Development of a fertilizable oocyte is a complex process that relies on the precise temporal and spatial expression of specific genes in germ cells and in surrounding somatic cells. Since female mice null for Taf4b, a TBP associated factor, are sterile, we sought to determine when during follicular development this phenotype was first observed. At postnatal day 3, ovaries of Taf4b null females contained fewer (P < 0.01) oocytes than ovaries of wild type and heterozygous Taf4b mice. However, expression of only one somatic cell marker Foxl2 was reduced in ovaries at day 15. Despite the reduced number of follicles, many proceed to the antral stage, multiple genes associated with granulosa cell differentiation and oocyte maturation were expressed in a normal pattern, and immature Taf4b null females could be hormonally primed to ovulate and mate. However, the ovulated cumulus oocyte complexes from the Taf4b null mice had fewer (P < 0.01) cumulus cells, and the oocytes were functionally abnormal. GVBD and polar body extrusion were reduced significantly (P < 0.01). The few oocytes that were fertilized failed to progress beyond the two-cell stage of development. Thus, infertility in Taf4b null female mice is associated with defects in early follicle formation, oocyte maturation, and zygotic cleavage following ovulation and fertilization.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice

Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab-knockout mice. Although Epab(-/-) males and Epab(+/-) of both sexes were fertile, Epab(-/-) female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab(-/-) oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Ccnb1 (cyclin B1) and Dazl (deleted in azoospermia-like) mRNAs. Microinjection of Epab mRNA into Epab(-/-) germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab(-/-) mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding the EGF (epidermal growth factor)-like growth factors Areg (amphiregulin), Ereg (epiregulin) and Btc (betacellulin), and their downstream regulators, Ptgs2 (prostaglandin synthase 2), Has2 (hyaluronan synthase 2) and Tnfaip6 (tumour necrosis factor α-induced protein 6). The findings from the present study indicate that EPAB is necessary for oogenesis, folliculogenesis and female fertility in mice.     

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.     

Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.     

Interactions between somatic cells and germ cells throughout mammalian oogenesis

Oocytes and their companion somatic cells maintain a close association throughout oogenesis and this association is essential for normal oocyte and follicular development. This review summarizes current concepts of the role of the somatic cells in the regulation of mammalian oocyte growth, the maintenance of meiotic arrest, the induction of oocyte maturation, and the acquisition of full embryonic developmental competence during oocyte maturation in vitro. Gap junctions appear to mediate these regulatory processes. The regulatory interaction of oocytes and somatic cells, however, is not unidirectional; the oocyte participates in the proliferation, development, and function of the follicular somatic cells. The oocyte secretes factors that enable the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in response to hormonal stimulation. In addition, the oocyte produces factors that promote the proliferation of granulosa cells. These interactions in vitro do not appear to require the mediation of gap junctions. The oocyte also promotes the differentiation of granulosa cells into functional cumulus cells, but this function of the oocyte appears to require the continued presence and close association of the oocyte and granulosa cells. Therefore, oocytes and follicular somatic cells are interdependent for development and function.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Term development of caprine embryos derived from immature oocytes in vitro

Ovaries were surgically removed from female goats (Toggenburg, Nubian and Saanen breeds). Oocytes were collected by follicular aspiration or after ovaries were minced, then matured in mTCM-199 with 100 mug LH + 0.5 mug FSH + 1.0 mug estradiol 17-beta/ml for 27 h prior to in vitro fertilization (17). Although more oocytes were made available by mincing than by aspiration, higher proportions of aspirated oocytes were fertilized and developed to morulae. Proportions that fertilized and reached morulae were 82 102 (80.4%) and 50 102 (49.0%) versus 77 126 (61.1%) and 27 126 (21.4%) for oocytes obtained by aspiration and after ovarian mincing, respectively (P<0.05). Proportions of inseminated ova undergoing cleavage and continuing development to the morula stage differed significantly (P<0.05) among 5 co-culture treatment groups, with higher proportions of cleavage (23 27 , 85.2%) and morulae (14 27 , 51.9%) obtained by co-culture on caprine cumulus cells (cCC). Some oocytes reached the blastocyst stage (4 54 , 7.4%) following oocyte collection by aspiration and culture on caprine oviduct epithelial cells (cOEC). After 4- and 8-cell stage embryos obtained by aspiration and culture on cCC were transferred pregnancy resulted. Twin male kids (developed from different embryos) were born on August 6, 1993, and have developed into normal bucks. Conditions reported here provided an adequate environment for support of oocyte maturation, fertilization and early embryonic development in vitro (IVMFC) with normal development after embryo transfer.     

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.     

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.     

Betacellulin overexpression in the mouse ovary leads to MAPK3/MAPK1 hyperactivation and reduces litter size by impairing fertilization

The epidermal growth factor receptor (EGFR) and its ligands are emerging as key molecules in regulating female reproduction. Here, we used a transgenic mouse model to evaluate whether and at which level of the reproduction cascade higher-than-normal levels of the EGFR ligand betacellulin (BTC) in the reproductive organs affect fertility. Western blots and immunohistochemistry revealed increased BTC levels in uterus and ovaries from transgenic females, particularly evident in granulosa cells of antral follicles. Onset of puberty, estrous cyclicity, and the anatomy and histology of reproductive organs at puberty were not altered as compared to control females. Fertility tests revealed a reduction (~50%) in litter size as the major reproductive deficit of transgenic females. Embryo implantation was delayed in transgenic females, but this was not the reason for the reduced litter size. Transgenic females produced a normal number of oocytes after natural ovulation. The in vivo fertilization rate was significantly reduced in untreated transgenic females but returned to normal levels after superovulation. Impaired oocyte fertilization in the absence of superovulation treatment was associated with MAPK3/MAPK1 hyperactivation in BTC transgenic ovaries, whereas similar levels of MAPK3/MAPK1 activation were detected in transgenic and control ovaries after superovulation treatment. Thus, tight regulation of MAPK3/MAPK1 activity appears to be essential for appropriate granulosa cell function during oocyte maturation. Our study identified hitherto unknown effects of BTC overabundance in reproduction and suggests BTC as a novel candidate protein for the modulation of fertility.