Precise coordination between mammalian precursor egg cell (oocyte) and ovarian follicle development is essential in order to provide an egg that is fully competent to undergo fertilization and embryo development. We have discovered a system of metabolic cooperation in which oocytes promote the expression of genes in cumulus cells for processes that oocytes cannot carry out efficiently themselves, such as amino acid transport and glucose oxidation. In other words, the oocyte out-sources these processes to the cumulus cells. In turn, the cumulus cells pass the essential products of these processes to the oocyte for use in development. The same system allows the oocytes to regulate the metabolic pathways in the follicle cells to control the rate of follicular development. We have also described how oocytes provide the cumulus cells with signals that enable the cumulus cells to respond to preovulatory hormonal stimulus by both undergoing processes essential for ovulation and sending a return signal to the oocyte triggering the resumption of meiosis. These remarkable mechanisms coordinate the maturation of both the oocyte and cumulus cells, culminating in the ovulation of an egg ready for fertilization.
Maturation of the Oocyte-Cumulus Cell Complex
For almost a century, it was assumed that granulosa cells in ovarian follicles functioned as nurse cells to support the development of oocytes. However, research since about 1990 has shown that oocytes are not simple recipients of granulosa cell largess during follicular development, but rather oocytes play crucial roles in follicular development beginning with the formation of primordial follicles, promoting the primary to secondary follicle transition, promoting granulosa cell proliferation and differentiation, and enabling cumulus expansion after the preovulatory surge of luteinizing hormone (LH). Many of these functions can be attributed to oocyte-derived members of the TGFb superfamily, particularly growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). These factors are important regulators of follicular function and ovulation in all mammalian species studied including rodents, domestic species, and humans. We have recently shown that mammalian oocytes, to compensate for their metabolic deficiencies, promote the activity of metabolic pathways in cumulus cells, the granulosa cells associated with oocytes in antral follicles. For example, oocytes poorly utilize glucose as an energy source due to deficiencies in components of glycolytic pathways. Thus oocytes require that cumulus cells provide glycolytic metabolites to support their development and maturation. Fully-grown oocytes promote glycolysis in cumulus cells, which then provide the metabolites to the oocytes.
Since BMP15 and GDF9 appear to be key regulators of granulosa cell function, we assessed the effect of these factors on cumulus cell function before the preovulatory surge of LH by analysis of the transcriptomes of cumulus cells from wildtype (WT), Bmp15-/-, and Bmp15-/- Gdf9+/- double mutant (DM) mice using microarray analysis. There were 5,332, 7,640, and 2,651 transcripts identified to be changed significantly in comparisons of Bmp15-/- vs. WT, DM vs. WT, and DM vs. Bmp15-/- cumulus cells, respectively. Ingenuity Pathway Analyses revealed that metabolism was the major theme underlying the commonly changed transcripts in all the three comparisons: glycolysis and sterol biosynthesis were the two most significantly affected pathways. Most of the transcripts encoding enzymes for sterol biosynthesis were down-regulated in both mutant cumulus cells, and in WT cumulus cells after microsurgical removal of oocytes. Similarly, there was a reduction of de novo-synthesized cholesterol in these cumulus cells. This suggests that oocytes regulate cumulus cell metabolism, particularly sterol biosynthesis, by promoting the expression of required transcripts. Furthermore, in WT-mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol, and Ebp, which encode enzymes in the cholesterol biosynthetic pathway, were expressed robustly in cumulus cells, but expression was barely detectable in oocytes. Levels of de novo-synthesized cholesterol were significantly higher in cumulus-enclosed oocytes than denuded oocytes. These results indicate that mouse oocytes are deficient in their ability to synthesize cholesterol and require cumulus cells to provide them with products of the sterol biosynthetic pathway. Oocyte-derived BMP15 and GDF9 probably promote this metabolic pathway in cumulus cells as compensation for their own deficiencies.
How do preantral granulosa cells become cumulus cells during the preantral to antral follicle transition? To address this question, we defined some of the differences between these two subtypes of granulosa cells. The most dramatic of these is that, while cumulus-oocyte complexes undergo expansion in vitro in response to either EGF or FSH, preantral granulosa cell-oocyte complexes do not. Why not? First, although the fully-grown oocytes isolated from antral follicles (normally enclosed by cumulus cells) secrete factors that enable cumulus expansion, the growing oocytes isolated from large preantral follicles do not. Thus, growing oocytes do not secrete sufficient amounts of active cumulus expansion enabling factors. The first explanation for the incompetence of preantral granulosa cell-oocyte complexes to undergo expansion is that the resident growing oocyte does not secrete sufficient active enabling factors. Next, we asked whether preantral granulosa cells would undergo expansion if provided with the enabling factors from fully-grown oocytes. They do not. Thus, although preantral granulosa cells are competent to respond to EGF and oocyte-derived factors in some ways that are similar to cumulus cells, establishment of the full cumulus cell phenotype requires further developmental changes during the preantral to antral follicle transition.
What factors drive the differentiation of preantral granulosa cells to cumulus cells? The two most obvious suspects are FSH and oocyte-derived factors. Our results show that both participate in the transition. These experiments utilized a culture system that we devised for oocyte-granulosa cell complexes isolated from the preantral follicles of 12-day-old mice. This culture system produces oocytes that are competent to undergo meiotic maturation, fertilization, and development to live offspring. The 10-day culture period coincides with the preantral to antral follicle transition in vivo which takes place during approximately days 14 - 16 after birth, hence days 2 to 4 of culture. In initial experiments, complexes were grown in medium without (control) or with a low dose (0.005 IU/ml) of recombinant FSH. The results showed that acquisition of competence to undergo expansion by the oocyte-granulosa cell complex is probably due to both the developing ability of oocytes in the cultured complexes to secrete the expansion enabling factor and also to the developing ability of the granulosa cells to respond to EGF and the oocyte-derived enabling factor(s).
When the growing oocytes were removed from the preantral complexes before culture, the granulosa cells did not develop competence to undergo expansion even when cultured with FSH. However, co-culture of preantral granulosa cells with fully-grown oocytes promoted full competence to undergo expansion, and express expansion-related transcripts, in response to EGF. The acquisition of competence to respond to EGF occurred whether the granulosa cells were grown in low dose FSH-containing medium or not. Therefore, oocytes are necessary for the acquisition of competence to undergo expansion in response to EGF, but FSH is not.
Associate Research Scientists: You-Qiang Su, Ph.D., Kyungbon Lee, Ph.D.
Research Assistant III: Karen Wigglesworth, M.S.
Research Administrative Assistant: Maxine Friend
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Su YQ, Sugiura, K, Woo Y, Wigglesworth K, Kamdar S, Affourtit J, Eppig JJ. 2006. Selective degradation of transcripts during meiotic maturation of mouse oocytes. Dev Biol. 302:104-117.
Eviskov AV, Graber JH, Brockman MJ, Hampl A, Holbrook AE, Singh P, Eppig JJ, Solter D, Knowles BB. 2006. Cracking the egg: Molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo. Genes Dev. 20:2713-2727.
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Handel MA, Lessard C, Reinholdt L, Schimenti J, Eppig JJ. 2006. Mutagenesis as an unbiased approach to identify novel contraceptive targets. Mol Cell Endocrinol 250:201-205.
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Tanaka M, Kihara M, Hennebold JD, Eppig JJ, Viveiros MM, Emery BR, Carrell DT, Kirkman NJ, Meczekalski B, Zhou J, Bondy CA, Becker M, Schultz RM, Misteli T, De La Fuente R, King GJ, Adashi EY. 2005. H1FOO is coupled to the initiation of oocytic growth. Biol Reprod. 72:135-142.
Mehlmann LM, Saeki Y, Tanaka S, Brennan TJ, Evsikov AV, Pendola FL, Knowles BB, Eppig JJ, Jaffe LA. 2004. The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes. Science 306:1647-1950.
Su YQ, Wu X, O'Brien MJ, Pendola FL, Denagre JA, Matzuk MM, Eppig JJ. 2004. 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. Dev Biol 276:64-73.
De La Fuente R, Viveiros MM, Burns KH, Adashi EY, Matzuk MM, Eppig JJ. 2004. Major chromatin remodeling in the germinal vesicle (GV) of mammalian oocytes is dispensable for global transcriptional silencing but required for centromeric heterochromatin function. Dev. Biol. 275:447-458.
Viveiros MM, O'Brien MJ, Eppig JJ. 2004. Protein kinase C activity regulates the onset of anaphase I in mouse oocytes. Biol Reprod 71:1525-1532.
Lindenthal B, O'Brien MJ, Wigglesworth K, Blume T, Grondahl C, Eppig JJ. 2004. A synthetic analogue of meiosis-activating sterol (FF-MAS) is a potent agonist promoting meiotic maturation and preimplantation development of mouse oocytes maturing in vitro. Hum Reprod 19:2340-2344.
De La Fuente R, Viveiros MM, Wigglesworth K, Eppig JJ. 2004. ATRX, a member of the SNF2 family of helicase/ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes. Dev Biol 272:1-14.
Cho YS, Chennathukuzhi VM, Handel MA, Eppig JJ, Hecht NB. 2004. The relative levels of TRAX and TBRBP determine their nucleo-cytoplasmic distribution in male germ cells. J Biol Chem 279:31514-31523.
Latham KE, Wigglesworth K, McMenamin M, Eppig JJ. 2004. Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: Advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles. Biol Reprod 70:1253-1262.
Grondahl C, Murray A, Blume T, Su YQ, Eppig JJ. 2004. Meiosis-activating sterol promotes the metaphase I to metaphase II transition and preimplantation developmental competence of mouse oocytes maturing in vitro. Biol Reprod 70:1458-1464.
Lessard C, Pendola JK, Hartford SA, Schimenti JC, Handel MA, Eppig JJ. 2004. New mouse genetic models for human contraceptive development. Cytogenet Genome Res 105:222-227.