Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca2+ induced by inositol 1,4,5-trisphosphate

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca2+]i (4.0-6.0) and reduced rates of activation (31.4-36.8%) and development (10.0-24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9-6.3, P < 0.05). Also, mature oocytes from cumulus-oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca2+]i than that in mature oocytes from COCs cultured with glucose (7.4-6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.     

The secretions of the cumulus-oocyte complex in relation to fertilization and early mouse embryonic development: a histochemical study

Summary A study has been made of the histochemical composition of the murine cumulus—oocyte complex and zona pellucida following treatment of immature females with exogenous gonadotrophins. Selected developmental stages were studied in detail, namely (i) the ovulated and unfertilized egg, (ii) the fertilized oocyte and (iii) the preimplantation embryo. In addition, the histochemical features observed in normal fertilized embryos have been compared with those of haploid and diploid parthenogenetic embryos at comparable stages following activation. Shortly after fertilization, glycosaminoglycans, which form a major component of the extracellular matrix surrounding the cumulus cells, become incorporated into the zona pellucida of the fertilized egg. In oocytes with few or no attendant cumulus cells, there appeared to be a diminished uptake of glycosaminoglycans and a reduced intensity of the zona staining reaction to Alcian Blue. In these oocytes, uptake of glycosaminoglycans appeared to be from the secretions lining the oviduct. There was little incorporation of the glycosaminoglycans from the extra-cellular matrix of the surrounding cumulus cells into the zona pellucida in unfertilized or parthenogenetic eggs despite the activation stimulus. After fertilization or activation, the zona pellucida became increasingly PAS-positive. Enzymic studies clearly indicate that the composition of the zona pellucida of the early embryo is histochemically different from the zona that surrounds the oocyte in the preovulatory follicle. These findings are discussed in relation to the decreased viability of embryos from oocytes which have been ovulated.The death of Mrs Carol Grainge is sadly recorded.     

TGFalpha and EGFR in ovine preimplantation embryos and effects on development

The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFalpha) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0-3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFalpha/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFalpha to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFalpha was a similar to EGFR. However, the staining for TGFalpha slightly increased in the fertilized oocytes (1-2+) as compared to immature and mature oocytes (1+). TGFalpha was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5 ng/ml TGFalpha was increased as compared to other treatments (P<0.05). There was no significant difference in the rate of development of blastocysts cultured with 5 ng/ml TGFalpha, 20 ng/ml EGF, 20 ng/ml EGF+5 ng/ml TGFalpha or the control treatment (P>0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P>0.05). However, TGFalpha alone enhanced cell survival rated (P<0.01) and reduced apoptosis. We concluded that TGFalpha can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro.     

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.     

Cloning the laboratory mouse.

A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two–cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1*b/ of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?     

Activation of human embryonic gene expression in cytoplasmic hybrid embryos constructed between bovine oocytes and human fibroblasts

Cross-species somatic all number transfer (SCNT) provides a potential solution to overcome the problem of oocyte shortage for therapeutic cloning. To further characterize the system, we constructed cytoplasm hybrid embryos between bovine oocytes and human fibroblasts and examined dynamics of human gene activation during preimplantation stages. Data from this study showed that human embryonic genes, OCT4, SOX2, NANOG, E-CADHERIN, as well as beta-ACTIN, were activated by enucleated bovine oocytes. Activation of human genes was correlated with developmental potential of the embryos. The extent of human gene activation varied drastically and was incomplete in a large proportion of the embryos. Activation of human genes in the human-bovine cytoplasm hybrid embryos occurs in a temporal pattern resembling that of the bovine species. Results from this study suggest that human gene products are required for hybrid embryos to develop to later preimplantation stages. Facilitating human genome activation may improve successful rates in cross-species SCNT.     

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.     

The relationship between parthenogenetic embryonic development and cumulus cell-oocyte intercellular coupling during oocyte meiotic maturation

Intercellular coupling between cumulus cells and oocytes persists after oocyte meiotic maturation has been initiated. The experiments described here focus on the relationship between oocyte-cumulus cell intercellular coupling during maturation and the subsequent embryonic development of spontaneous mouse parthenotes. Several lines of evidence suggest that this coupling during oocyte maturation is required for the acquisition of the capacity for spontaneous mouse parthenotes to develop embryologically. First, the period of time that LT/Sv oocytes remained coupled to cumulus cells during oocyte maturation in vivo corresponded to that required for the acquisition of the capacity for parthenogenetic embryonic development. Second, the longer that cumulus cells were present during Fpontaneous oocyte maturation in vitro, the higher was the percentageofova undergoing subsequent parthenogenetic development. Third, cumulus cell-free oocytes cocultured with cumulus cell-enclosed oocytes during the maturation period in vitro did not develop embryologically. Fourth, intercellular coupling between cumulus cells and oocytes persisted throughout the oocyte maturation period in vitro. Fifth, incubation of oocyte-cumulus cell complexes in medium containing follicle-stimulating hormone (FSH) promoted uncoupling and decreased the percentage of ova undergoing parthenogenetic development. Thus, cell-to-cell communication, mediated via the intercellular coupling pathway between cumulus cells and oocytes, plays an important role during oocyte maturation and relates to subsequent preimplantation development.