MYO10 promotes transzonal projection-dependent germ line-somatic contact during mammalian folliculogenesis†.

Granulosa cells of growing ovarian follicles elaborate filopodia-like structures termed transzonal projections (TZPs) that supply the enclosed oocyte with factors essential for its development. Little is known, however, of the mechanisms underlying the generation of TZPs. We show in mouse and human that filopodia, defined by an actin backbone, emerge from granulosa cells in early stage primary follicles and that actin-rich TZPs become detectable as soon as a space corresponding to the zona pellucida appears. mRNA encoding Myosin10 (MYO10), a motor protein that accumulates at the base and tips of filopodia and has been implicated in their initiation and elongation, is present in granulosa cells and oocytes of growing follicles. MYO10 protein accumulates in foci located mainly between the oocyte and innermost layer of granulosa cells, where it colocalizes with actin. In both mouse and human, the number of MYO10 foci increases as oocytes grow, corresponding to the increase in the number of actin-TZPs. RNAi-mediated depletion of MYO10 in cultured mouse granulosa cell-oocyte complexes is associated with a 52% reduction in the number of MYO10 foci and a 28% reduction in the number of actin-TZPs. Moreover, incubation of cumulus-oocyte complexes in the presence of epidermal growth factor, which triggers a 93% reduction in the number of actin-TZPs, is associated with a 55% reduction in the number of MYO10 foci. These results suggest that granulosa cells possess an ability to elaborate filopodia, which when directed toward the oocyte become actin-TZPs, and that MYO10 increases the efficiency of formation or maintenance of actin-TZPs.

Transzonal projections: Essential structures mediating intercellular communication in the mammalian ovarian follicle.

The development of germ cells relies on contact and communication with neighboring somatic cells that provide metabolic support and regulatory signals. In females, contact is achieved through thin cytoplasmic processes that project from follicle cells surrounding the oocyte, extend through an extracellular matrix (ECM) that lies between them, and reach its surface. In mammals, the ECM is termed the zona pellucida and the follicular cell processes are termed transzonal projections (TZPs). TZPs become detectable when the zona pellucida is laid down during early folliculogenesis and subsequently increase in number as oocyte growth progresses. They then rapidly disappear at the time of ovulation, permanently breaking germ-soma contact. Here we review the life cycle and functions of the TZPs. We begin with an overview of the morphology and cytoskeletal structure of TZPs, in the context of actin- and tubulin-based cytoplasmic processes in other cell types. Next, we review the roles played by TZPs in mediating progression through successive stages of oocyte development. We then discuss two mechanisms that may generate TZPs-stretching at pre-existing points of granulosa cell-oocyte contact and elaboration of new processes that push through the zona pellucida-as well as gene products implicated in their formation or function. Finally, we describe the signaling pathways that cause TZPs to be retracted in response to signals that also trigger meiotic maturation and ovulation of the oocyte. The principles and mechanisms that govern TZP behavior may be relevant to understanding communication between physically separated cells in other physiological contexts.

Germ cells of the mammalian female: A limited or renewable resource?†.

In many non-mammalian organisms, a population of germ-line stem cells supports continuing production of gametes during post-natal life, and germ-line stem cells are also present and functional in male mammals. Traditionally, however, they have been thought not to exist in female mammals, who instead generate all their germ cells during fetal life. Over the last several years, this dogma has been challenged by several reports, while being supported by others. We describe and compare these conflicting studies with the aim of understanding how they came to opposing conclusions. We first consider studies that, by examining marker-gene expression, the fate of genetically marked cells, and consequences of depleting the oocyte population, addressed whether ovaries of post-natal females contain oogonial stem cells that give rise to new oocytes. We next discuss whether ovaries contain cells that, even if inactive under physiological conditions, nonetheless possess oogonial stem cell properties that can be revealed through cell culture. We then examine studies of whether cells harvested after long-term culture of cells obtained from ovaries can, following transplantation into ovaries of recipient females, give rise to oocytes and offspring. Finally, we note studies where somatic cells have been re-programmed to acquire a female germ-cell fate. We conclude that the weight of evidence strongly supports the traditional interpretation that germ-line stem cells do not exist post-natally in female mammals. However, the ability to generate germ cells from somatic cells in vitro establishes a method to generate new gametes from cells of post-natal mammalian females.

Ablation of TGFBR3 (betaglycan) in oocytes does not affect fertility in female mice.

Ovarian follicle development is regulated by locally produced TGFß superfamily members. The TGFß type III receptor (TGFBR3, or betaglycan), which regulates the actions of diverse TGFß ligands, including inhibins, is expressed in different ovarian cell types. However, its functional roles in the ovary have not been investigated in vivo. Here, we ablated Tgfbr3 in murine oocytes using the Cre-loxP system. Oocyte-specific Tgfbr3 knockout (cKO) females were fertile, producing litters of similar size and frequency as controls. Their ovarian weights and histology were also normal. Though we confirmed efficient recombination of the floxed alleles, we did not detect Tgfbr3 mRNA in purified oocytes from superovulated cKO or control mice. These results challenge earlier observations of betaglycan protein expression in this cell type. Regardless, Tgfbr3 in the murine oocyte is clearly dispensable for female fertility.

Epigenetic inheritance through the female germ-line: The known, the unknown, and the possible.

Although genetic mutations have long been known to influence gene expression and individual phenotype, studies emerging over the past decade indicate that such changes can also be induced in the absence of alterations in base-sequence. Epigenetically driven changes in gene expression or phenotype, when they are transmitted to succeeding generations, represent an entirely new mechanism that could generate heritable variation in a population. To understand the mechanistic basis of epigenetic inheritance, it is essential to learn how these changes may be transmitted through the germ-line to the next generation. Here, we review the process of female germ cell specification, oocyte growth, and meiotic maturation. We discuss what is known of the activity and role of three principal candidates to transmit epigenetic information--DNA methylation, histone post-translational modifications, and short non-coding RNAs--in the developing oocyte. We then consider intergenerational inheritance and true transgenerational inheritance and, in the case of the latter, compare examples in which insertional mutations have driven the heritable epigenetic phenotype with examples of environmentally induced epigenetic inheritance for which the mechanism remains to be identified.

Follicle-stimulating hormone regulates expression and activity of epidermal growth factor receptor in the murine ovarian follicle.

Fertility depends on the precise coordination of multiple events within the ovarian follicle to ensure ovulation of a fertilizable egg. FSH promotes late follicular development, including expression of luteinizing hormone (LH) receptor by the granulosa cells. Expression of its receptor permits the subsequent LH surge to trigger the release of ligands that activate EGF receptors (EGFR) on the granulosa, thereby initiating the ovulatory events. Here we identify a previously unknown role for FSH in this signaling cascade. We show that follicles of Fshb(-/-) mice, which cannot produce FSH, have a severely impaired ability to support two essential EGFR-regulated events: expansion of the cumulus granulosa cell layer that encloses the oocyte and meiotic maturation of the oocyte. These defects are not caused by an inability of Fshb(-/-) oocytes to produce essential oocyte-secreted factors or of Fshb(-/-) cumulus cells to respond. In contrast, although expression of both Egfr and EGFR increases during late folliculogenesis in Fshb(+/-) females, these increases fail to occur in Fshb(-/-) females. Remarkably, supplying a single dose of exogenous FSH activity to Fshb(-/-) females is sufficient to increase Egfr and EGFR expression and to restore EGFR-dependent cumulus expansion and oocyte maturation. These studies show that FSH induces an increase in EGFR expression during late folliculogenesis and provide evidence that the FSH-dependent increase is necessary for EGFR physiological function. Our results demonstrate an unanticipated role for FSH in establishing the signaling axis that coordinates ovulatory events and may contribute to the diagnosis and treatment of some types of human infertility.

Multiple Mechanisms Cooperate to Constitutively Exclude the Transcriptional Co-Activator YAP from the Nucleus During Murine Oogenesis.

Reproduction depends on the generation of healthy oocytes. Improving therapeutic strategies to prolong or rescue fertility depends on identifying the inter- and intracellular mechanisms that direct oocyte development under physiological conditions. Growth and proliferation of multiple cell types is regulated by the Hippo signaling pathway, whose chief effectors are the transcriptional co-activator YAP and its paralogue WWTR1. To resolve conflicting results concerning the potential role of Hippo in mammalian oocyte development, we systematically investigated the expression and localization of YAP in mouse oocytes. We report that that YAP is expressed in the germ cells beginning as early as Embryonic Day 15.5 and subsequently throughout pre- and postnatal oocyte development. However, YAP is restricted to the cytoplasm at all stages. YAP is phosphorylated at serine-112 in growing and fully grown oocytes, identifying a likely mechanistic basis for its nuclear exclusion, and becomes dephosphorylated at this site during meiotic maturation. Phosphorylation at serine-112 is regulated by a mechanism dependent on cyclic AMP and protein kinase A, which is known to be active in oocytes prior to maturation. Growing oocytes also contain a subpopulation of YAP, likely dephosphorylated, that is able enter the oocyte nucleus, but it is not retained there, implying that oocytes lack the cofactors required to retain YAP in the nucleus. Thus, although YAP is expressed throughout oocyte development, phosphorylation-dependent and -independent mechanisms cooperate to ensure that it does not accumulate in the nucleus. We conclude that nuclear YAP does not play a significant physiological role during oocyte development in mammals.

Follicle-Stimulating Hormone Increases Gap Junctional Communication Between Somatic and Germ-Line Follicular Compartments During Murine Oogenesis.

Germ cells develop in intimate contact and communication with somatic cells of the gonad. In female mammals, oocyte development depends crucially on gap junctions that couple it to the surrounding somatic granulosa cells of the follicle, yet the mechanisms that regulate this essential intercellular communication remain incompletely understood. Follicle-stimulating hormone (FSH) drives the terminal stage of follicular development. We found that FSH increases the steady-state levels of mRNAs encoding the principal connexins that constitute gap junctions and cadherins that mediate cell attachment. This increase occurs both in granulosa cells, which express the FSH-receptor, and in oocytes, which do not. FSH also increased the number of transzonal projections that provide the sites of granulosa cell-oocyte contact. Consistent with increased connexin expression, FSH increased gap junctional communication between granulosa cells and between the oocyte and granulosa cells, and it accelerated oocyte development. These results demonstrate that FSH regulates communication between the female germ cell and its somatic microenvironment. We propose that FSH-regulated gap junctional communication ensures that differentiation processes occurring in distinct cellular compartments within the follicle are precisely coordinated to ensure production of a fertilizable egg.

Identification of a ß-galactosidase transgene that provides a live-cell marker of transcriptional activity in growing oocytes and embryos.

Identifying the events and molecular mechanisms that regulate oocyte growth has emerged as a key objective of research in human fertility, fuelled by evidence from human and animal studies indicating that disease and environmental factors can act on oocytes to affect the health of the resulting individual and by efforts to grow oocytes in vitro to enable fertility preservation of cancer survivors. Techniques that monitor the development of growing oocytes would be valuable tools to assess the progression of growth under different conditions. Most methods used to assess oocytes grown in vitro are indirect, however, relying on characteristics of the somatic compartment of the follicle, or compromise the oocyte, preventing its subsequent culture or fertilization. We investigated the utility of T-cell factor/lymphoid enhancer-binding factor (TCF/Lef)-LacZ transgene expression as a predictor of global transcriptional activity in oocytes and early embryos. Using a fluorescent ß-galactosidase substrate combined with live-cell imaging, we show that TCF/Lef-LacZ transgene expression is detectable in growing oocytes, lost in fully grown oocytes and resumes in late two-cell embryos. Transgene expression is likely regulated by a Wnt-independent mechanism. Using chromatin analysis, LacZ expression and methods to monitor and inhibit transcription, we show that TCF/Lef-LacZ expression mirrors transcriptional activity in oocytes and preimplantation embryos. Oocytes and preimplantation embryos that undergo live-cell imaging for TCF/Lef-LacZ expression are able to continue development in vitro. TCF/Lef-LacZ reporter expression in living oocytes and early embryos is thus a sensitive and faithful marker of transcriptional activity that can be used to monitor and optimize conditions for oocyte growth.

The gametic synapse: RNA transfer to the bovine oocyte.

Even after several decades of quiescent storage in the ovary, the female germ cell is capable of reinitiating transcription to build the reserves that are essential to support early embryonic development. In the current model of mammalian oogenesis, there exists bilateral communication between the gamete and the surrounding cells that is limited to paracrine signaling and direct transfer of small molecules via gap junctions existing at the end of the somatic cells' projections that are in contact with the oolemma. The purpose of this work was to explore the role of cumulus cell projections as a means of conductance of large molecules, including RNA, to the mammalian oocyte. By studying nascent RNA with confocal and transmission electron microscopy in combination with transcript detection, we show that the somatic cells surrounding the fully grown bovine oocyte contribute to the maternal reserves by actively transferring large cargo, including mRNA and long noncoding RNA. This occurrence was further demonstrated by the reconstruction of cumulus-oocyte complexes with transfected cumulus cells transferring a synthetic transcript. We propose selective transfer of transcripts occurs, the delivery of which is supported by a remarkable synapselike vesicular trafficking connection between the cumulus cells and the gamete. This unexpected exogenous contribution to the maternal stores offers a new perspective on the determinants of female fertility.

SMAD4 promotes somatic-germline contact during murine oocyte growth.

Development of the mammalian oocyte requires physical contact with the surrounding granulosa cells of the follicle, which provide it with essential nutrients and regulatory signals. This contact is achieved through specialized filopodia, termed transzonal projections (TZPs), that extend from the granulosa cells to the oocyte surface. Transforming growth factor (TGFß) family ligands produced by the oocyte increase the number of TZPs, but how they do so is unknown. Using an inducible Cre recombinase strategy together with expression of green fluorescent protein to verify Cre activity in individual cells, we examined the effect of depleting the canonical TGFß mediator, SMAD4, in mouse granulosa cells. We observed a 20-50% decrease in the total number of TZPs in SMAD4-depleted granulosa cell-oocyte complexes, and a 50% decrease in the number of newly generated TZPs when the granulosa cells were reaggregated with wild-type oocytes. Three-dimensional image analysis revealed that TZPs of SMAD4-depleted cells were longer than controls and more frequently oriented towards the oocyte. Strikingly, the transmembrane proteins, N-cadherin and Notch2, were reduced by 50% in SMAD4-depleted cells. SMAD4 may thus modulate a network of cell adhesion proteins that stabilize the attachment of TZPs to the oocyte, thereby amplifying signalling between the two cell types.

Filopodia-like protrusions of adjacent somatic cells shape the developmental potential of oocytes.

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

Follicle-stimulating hormone accelerates mouse oocyte development in vivo.

During folliculogenesis, oocytes grow and acquire developmental competence in a mutually dependent relationship with their adjacent somatic cells. Follicle-stimulating hormone (FSH) plays an essential and well-established role in the differentiation of somatic follicular cells, but its function in the development of the oocyte has still not been elucidated. We report here that oocytes of Fshb(-/-) mice, which cannot produce FSH, grow at the same rate and reach the same size as those of wild-type mice. Consistent with this observation, the granulosa cells of Fshb(-/-) mice express the normal quantity of mRNA encoding Kit ligand, which has been implicated in oocyte growth. Oocytes of Fshb(-/-) mice also accumulate normal quantities of cyclin B1 and CDK1 proteins and mitochondrial DNA. Moreover, they acquire the ability to complete meiotic maturation in vitro and undergo transition from non-surrounded nucleolus to surrounded nucleolus. However, these events of late oocyte development are significantly delayed. Following in vitro maturation and fertilization, only a small number of embryos derived from oocytes of Fshb(-/-) mice reach the blastocyst stage. Administration of equine chorionic gonadotropin, which provides FSH activity, 48 h before in vitro maturation increases the number of blastocysts obtained subsequently. These results indicate that FSH is not absolutely required for oocyte development in vivo but that this process occurs more rapidly in its presence. We suggest that FSH may coordinate the development of the germline and somatic compartments of the follicle, ensuring that ovulation releases a developmentally competent egg.

Regulation of mitochondrial DNA accumulation during oocyte growth and meiotic maturation in the mouse.

Oocytes accumulate an enormous quantity of mitochondrial (mt) DNA, and an insufficient amount of mtDNA may underlie some cases of poor oocyte quality leading to infertility. Little is known, however, about the mechanisms that govern the timing and regulation of mtDNA accumulation during oogenesis. We report, through analysis of the mtDNA content of individual oocytes of the mouse, that mtDNA accumulates steadily during oocyte growth to reach a value of ~175 000 copies per cell. MtDNA content ceases to increase once oocytes reach full size and remains unchanged during meiotic maturation. To test whether mtDNA accumulation depends on oocyte growth, we inhibited growth in vitro in two ways - by exposing complexes comprising partially grown oocytes enclosed by granulosa cells to a chemical inhibitor of the phosphatidylinositol-3-kinase signaling pathway and by removing the surrounding granulosa cells from partially grown oocytes. Under both conditions, the oocytes fail to grow, but mtDNA accumulation is unaffected, indicating that the two processes can be mechanistically uncoupled. Quantitative analysis of the mRNAs encoding proteins required for mtDNA replication revealed that Polg (Polga) (polymerase-γ, α-subunit), Polg2 (Polgb), and Tfam (transcription factor A, mitochondrial) increase during oocyte growth but then decrease after fully grown oocytes become transcriptionally silent as indicated by the non-surrounded nucleolus-to-surrounded nucleolus transition. Thus, there is a correlation between the decline in the quantity of mRNAs encoding mtDNA replication factors in fully grown oocytes and the arrest of mtDNA accumulation in these cells, suggesting that the two events may be causally linked.

Mice lacking the USP2 deubiquitinating enzyme have severe male subfertility associated with defects in fertilization and sperm motility.

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Epidermal growth factor receptor signaling uncouples germ cells from the somatic follicular compartment at ovulation.

Germ cells are physically coupled to somatic support cells of the gonad during differentiation, but this coupling must be disrupted when they are mature, freeing them to participate in fertilization. In mammalian females, coupling occurs via specialized filopodia that project from the ovarian follicular granulosa cells to the oocyte. Here, we show that signaling through the epidermal growth factor receptor (EGFR) in the granulosa, which becomes activated at ovulation, uncouples the germ and somatic cells by triggering a massive and temporally synchronized retraction of the filopodia. Although EGFR signaling triggers meiotic maturation of the oocyte, filopodial retraction is independent of the germ cell state, being regulated solely within the somatic compartment, where it requires ERK-dependent calpain-mediated loss of filopodia-oocyte adhesion followed by Arp2/3-mediated filopodial shortening. By uncovering the mechanism regulating germ-soma uncoupling at ovulation, our results open a path to improving oocyte quality in human and animal reproduction.

Maternally encoded stem-loop-binding protein is degraded in 2-cell mouse embryos by the co-ordinated activity of two separately regulated pathways.

Oocytes accumulate mRNAs and proteins that direct early embryonic development. Although subsequent development requires the timely degradation of these maternal products, little is known of the underlying mechanisms. The stem-loop-binding protein (SLBP), which regulates the stability and translation of mRNAs encoding histones and is synthesized during S-phase and degraded during G2 in somatic cells, accumulates during oogenesis. Maternal SLBP is required for mouse embryos to develop beyond the 2-cell stage, but must be degraded to allow the cell-cycle-regulated expression of somatic cells to be established. We report that the quantity of maternal SLBP changes little following fertilization until 44-52 h post-hCG, corresponding to mid-/late G2 of the 2-cell stage, when it decreases by 75%. Efficient degradation requires two pathways. The first requires activity of cyclin-dependent kinases (cdk) and embryonic transcription, preferentially targets nuclear SLBP, and likely corresponds to the pathway that degrades SLBP at G2 in somatic cells. The second does not require cdk activity or transcription and becomes active at 44-52 h post-hCG independently of cell-cycle progression to mid-/late G2, but is not solely regulated by the time elapsed since hCG injection. Thus, the co-ordinated activity of two separately regulated pathways eliminates maternally encoded SLBP from early mouse embryos.

Proteasomal activity is required to initiate and to sustain translational activation of messenger RNA encoding the stem-loop-binding protein during meiotic maturation in mice.

Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3'-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3'-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation.