Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes.

Coordinated regulation of oocyte and ovarian follicular development is essential for fertility. In particular, the progression of meiosis, a germ cell-specific cell division that reduces the number of chromosomes from diploid to haploid, must be arrested until just before ovulation. Follicular somatic cells are well-known to impose this arrest, which is essential for oocyte-follicle developmental synchrony. Follicular somatic cells sustain meiotic arrest via the natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system, and possibly also via high levels of the purine hypoxanthine in the follicular fluid. Upon activation by the ligand NPPC, NPR2, the predominant guanylyl cyclase in follicular somatic cells, produces cyclic guanosine monophosphate (cGMP), which maintains meiotic arrest after transfer to the oocyte via gap junctions. Here we report that both the NPPC/NPR2 system and hypoxanthine require the activity of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme required for the production of guanylyl metabolites and cGMP. Furthermore, oocyte-derived paracrine factors, particularly the growth differentiation factor 9-bone morphogenetic protein 15 heterodimer, promote expression of Impdh and Npr2 and elevate cGMP levels in cumulus cells. Thus, although the somatic compartment of ovarian follicles plays an essential role in the maintenance of oocyte meiotic arrest, as has been known for many years, this function of the somatic cells is surprisingly regulated by signals from the oocyte itself.

Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells.

Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15(-/-) and Bmp15(-/-) Gdf9(+/-) double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production.

Mutation in mouse hei10, an e3 ubiquitin ligase, disrupts meiotic crossing over.

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5' splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I.

Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells.

Mammalian oocytes are deficient in their ability to carry out glycolysis. Therefore, the products of glycolysis that are necessary for oocyte development are provided to oocytes by companion cumulus cells. Mouse oocytes secrete paracrine factors that promote glycolysis in cumulus cells. The objective of this study was to identify paracrine factors secreted by oocytes that promote glycolysis and expression of mRNA encoding the glycolytic enzymes PFKP and LDHA. Candidates included growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and fibroblast growth factors (FGFs). Bmp15-/- and Gdf9+/- Bmp15-/- (double mutant, DM) cumulus cells exhibited reduced levels of both glycolysis and Pfkp and Ldha mRNA, and mutant oocytes were deficient in promoting glycolysis and expression of Pfkp and Ldha mRNA in cumulus cells of wild-type (WT) mice. Alone, neither recombinant BMP15, GDF9 nor FGF8 promoted glycolysis and expression of Pfkp and Ldha mRNA in WT cumulus cells. Co-treatment with BMP15 and FGF8 promoted glycolysis and increased expression of Pfkp and Ldha mRNA in WT cumulus cells to the same levels as WT oocytes; however, the combinations of BMP15/GDF9 or GDF9/FGF8 did not. Furthermore, SU5402, an FGF receptor-dependent protein kinase inhibitor, inhibited Pfkp and Ldha expression in cumulus cells promoted by paracrine oocyte factors. Therefore, oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells.

Transcript profiling during mouse oocyte development and the effect of gonadotropin priming and development in vitro.

The molecular basis for acquisition of meiotic and developmental competence, the two main outcomes of oocyte development and essential for producing an egg capable of being fertilized and supporting development to term, is largely unknown. Using microarrays, we characterized global changes in gene expression in oocytes derived from primordial, primary, secondary, small antral, and large antral follicles and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany these transitions and likely underpin acquisition of meiotic and developmental competence. The greatest degree of change in gene expression occurs during the primordial to primary follicle transition. Of particular interest is that specific chromosomes display significant changes in their overall transcriptional activity and that in some cases these changes are largely confined to specific regions on these chromosomes. We also examined the transcript profile of oocytes that developed in vitro, as well as following eCG priming. Remarkably, the expression profiles only differed by 4% and 2% from oocytes that developed in vivo when compared to oocytes that developed in vitro from either primordial or secondary follicles, respectively. About 1% of the genes were commonly mis-expressed, and EASE analysis revealed there is an over-representation of genes involved in transcription. Developmental competence of oocytes obtained from eCG-primed mice was substantially improved when compared to oocytes obtained from unprimed mice, and this correlated with decreased expression of genes implicated in basal transcription.

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.

Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex.

Luteinizing hormone (LH) induces maturational processes in oocyte-cumulus cell complexes (OCC) of preovulatory follicles that include both resumption of meiosis in the oocyte and expansion (mucification) of the cumulus oophorus. Both processes require activation of mitogen-activated protein kinase (MAPK) in granulosa cells. Here, it is reported that inhibition of MAPK activation prevented gonadotropin-stimulated resumption of meiosis as well as the rise in expression of two genes whose products are necessary for normal cumulus expansion, Has2 and Ptgs2. However, inhibition of MAPK did not block gonadotropin-induced elevation of granulosa cell cAMP, indicating that the activation of MAPK required for inducing GVB and cumulus expansion is downstream of cAMP. Moreover, activation of MAPK in cumulus cells requires one or more paracrine factors from the oocyte to induce GVB and cumulus expansion; MAPK activation alone is not sufficient to initiate these maturational processes. This study demonstrates a remarkable interaction between the oocyte and cumulus cells that is essential for gonadotropin-induced maturational processes in OCC. By enabling gonadotropin-dependent MAPK activation in granulosa cells, oocytes promote the generation of a return signal from these cells that induces the resumption of meiosis. It also appears that an oocyte-dependent pathway downstream from oocyte-enabled activation of MAPK, and distinct from that promoting the resumption of meiosis, governs cumulus expansion.

A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence.

The objective of this study was to improve the conditions for oocyte development in vitro beginning with the primordial follicles of newborn mice. Previous studies showed that oocytes competent of meiotic maturation, fertilization, and preimplantation could develop in vitro from primordial follicles. However, the success rates were low and only one live offspring was produced (0.5% of embryos transferred). A revised protocol was compared with the original protocol using oocyte maturation and preimplantation development as end points. The percentage of oocytes maturing to metaphase II and developing to the blastocyst stage was significantly improved using the revised protocol. In addition, we compared the production of offspring from two-cell stage embryos derived from in vitro-grown and in vivo-grown oocytes. Of 1160 transferred two-cell stage embryos derived from in vitro-grown oocytes, 66 (5.7%) developed to term and 7 pups (10.6%) died at birth. The remaining 59 pups (27 females, 32 males) survived to adulthood. By comparison, of 437 transferred two-cell stage embryos derived from in vivo-grown oocytes, 76 (17.4%) developed to term and 4 (5.3%) died at birth. The remaining 72 pups (35 females, 37 males) survived to adulthood. These studies provide proof of the principle that fully competent mammalian oocytes can develop in vitro from primordial follicles and present a significant advance in oocyte culture technology.

Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse.

This study investigated the participation of MAPK in the resumption of meiosis [germinal vesicle breakdown (GVB)] in oocytes and cumulus expansion using oocyte-cumulus cell complexes (OCC) from Mos-null mice (Mos(tm1Ev)/Mos(tm1Ev), hereafter Mos(-/-)). MAPK activity was not detected in Mos(-/-) oocytes whether they matured in vivo or in vitro, with or without gonadotropin stimulation. Therefore, there are no pathways independent of MOS that activate MAPK during gonadotropin-induced maturation. In contrast, MAPK activity was always detected coincident with GVB in Mos(+/+) oocytes. Moreover, MAPK activity was detected in cumulus cells before gonadotropin-induced GVB in OCC regardless of genotype. A specific inhibitor (U0126) of MEK, a MAPKK required for MAPK activity, inhibited gonadotropin-induced GVB in OCC of both Mos(+/+) and Mos(-/-) mice. Activation of MAPK was downstream of elevation of cAMP. U0126 also inhibited cumulus expansion stimulated by FSH, epidermal growth factor, 8-bromo-cAMP, and recombinant growth differentiation factor-9. It is concluded that under the in vitro conditions used here, gonadotropin-induced GVB requires the participation of MAPK activity in the cumulus cells, but not in the oocyte. Moreover, the induction of cumulus expansion also requires the participation of MAPK, and this action is downstream of both elevation of cAMP and growth differentiation factor-9.

The mouse meiotic mutation mei1 disrupts chromosome synapsis with sexually dimorphic consequences for meiotic progression.

mei1 (meiosis defective 1) is the first meiotic mutation in mice derived by phenotype-driven mutagenesis. It was isolated by using a novel technology in which embryonic stem (ES) cells were chemically mutagenized and used to generate families of mice that were screened for infertility. We report here that mei1/mei1 spermatocytes arrest at the zygotene stage of meiosis I, exhibiting failure of homologous chromosomes to properly synapse. Notably, RAD51 failed to associate with meiotic chromosomes in mutant spermatocytes, despite evidence for the presence of chromosomal breaks. Transcription of genes that are markers for the leptotene and zygotene stages, but not genes that are markers for the pachytene stage, was observed. mei1/mei1 females are sterile, and their oocytes also show severe synapsis defects. Nevertheless, unlike arrested spermatocytes, a small number of mutant oocytes proved capable of progressing to metaphase I and attempting the first meiotic division. However, their chromosomes were unpaired and were not organized properly at the metaphase plate or along the spindle fibers during segregation. mei1 was genetically mapped to chromosome (Chr) 15 in an interval that is syntenic to human Chr 22q13. This region, which has been completely sequenced, contains no known homologs of genes specifically required for meiosis in model organisms. Thus, mei1 may be a novel meiotic gene.