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.

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.

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.

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.

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.

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.

Growth In Vitro of Granulosa Cell-Oocyte Complexes of the Mouse.

Analysis of the mechanisms that drive the growth and meiotic maturation of the female germ cell, the oocyte, has been greatly facilitated by the development of conditions that support these processes in vitro. Easily identified signposts of oocyte differentiation enable the ability of specific culture conditions to recapitulate normal oocyte development to be robustly assayed. Here we describe a technique for deriving complexes consisting of an oocyte surrounded by somatic granulosa cells from follicles and growing these granulosa cell-oocyte complexes in vitro. Such culture systems are useful for uncovering the principles of germ cell development and for improving our ability to preserve human and animal fertility through assisted reproduction.

CNOT6 regulates a novel pattern of mRNA deadenylation during oocyte meiotic maturation.

In many cell types, the length of the poly(A) tail of an mRNA is closely linked to its fate - a long tail is associated with active translation, a short tail with silencing and degradation. During mammalian oocyte development, two contrasting patterns of polyadenylation have been identified. Some mRNAs carry a long poly(A) tail during the growth stage and are actively translated, then become deadenylated and down-regulated during the subsequent stage, termed meiotic maturation. Other mRNAs carry a short tail poly(A) tail and are translationally repressed during growth, and their poly(A) tail lengthens and they become translationally activated during maturation. As well, a program of elimination of this 'maternal' mRNA is initiated during oocyte maturation. Here we describe a third pattern of polyadenylation: mRNAs are deadenylated in growing oocytes, become polyadenylated during early maturation and then deadenylated during late maturation. We show that the deadenylase, CNOT6, is present in cortical foci of oocytes and regulates deadenylation of these mRNAs, and that PUF-binding elements (PBEs) regulate deadenylation in mature oocytes. Unexpectedly, maintaining a long poly(A) tail neither enhances translation nor inhibits degradation of these mRNAs. Our findings implicate multiple machineries, more complex than previously thought, in regulating mRNA activity in oocytes.

Mammalian Oocytes Locally Remodel Follicular Architecture to Provide the Foundation for Germline-Soma Communication.

Germ cells develop in a microenvironment created by the somatic cells of the gonad [1-3]. Although in males, the germ and somatic support cells lie in direct contact, in females, a thick extracellular coat surrounds the oocyte, physically separating it from the somatic follicle cells [4]. To bypass this barrier to communication, narrow cytoplasmic extensions of the follicle cells traverse the extracellular coat to reach the oocyte plasma membrane [5-9]. These delicate structures provide the sole platform for the contact-mediated communication between the oocyte and its follicular environment that is indispensable for production of a fertilizable egg [8, 10-15]. Identifying the mechanisms underlying their formation should uncover conserved regulators of fertility. We show here in mice that these structures, termed transzonal projections (TZPs), are specialized filopodia whose number amplifies enormously as oocytes grow, enabling increased germ-soma communication. By creating chimeric complexes of genetically tagged oocytes and follicle cells, we demonstrate that follicle cells elaborate new TZPs that push through the extracellular coat to reach the oocyte surface. We further show that growth-differentiation factor 9, produced by the oocyte, drives the formation of new TZPs, uncovering a key yet unanticipated role for the germ cell in building these essential bridges of communication. Moreover, TZP number and germline-soma communication are strikingly reduced in reproductively aged females. Thus, the growing oocyte locally remodels follicular architecture to ensure that its developmental needs are met, and an inability of somatic follicle cells to respond appropriately to oocyte-derived cues may contribute to human infertility.

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic Development > Gametogenesis.

History, origin, and function of transzonal projections: the bridges of communication between the oocyte and its environment.

Development and differentiation of a functional oocyte that following fertilization is able to give rise to a new individual requires continuous physical contact with the supporting somatic cells of the ovarian follicle. As the oocyte is surrounded by a thick extracellular coat, termed the zona pellucida, this essential contact is mediated through thin cytoplasmic filaments known as transzonal projections (TZPs) that project from the somatic granulosa cells adjacent to the oocyte and penetrate through the zona pellucida to reach the oocyte. Gap junctions assembled where the tips of the TZPs contact the oocyte plasma membrane, and other contact-dependent signaling may also occur at these sites. Here, I describe early studies of TZPs, which were first identified in the late 19th century, discuss their similarities with classical filopodia, review their structure and function, and compare two models that could account for their origin. Possible priorities and directions for future studies close this contribution.

Control of Oocyte Growth and Development by Intercellular Communication Within the Follicular Niche.

In the mammalian ovary, each oocyte grows and develops within its own structural and developmental niche-the follicle. Together with the female germ cell in the follicle are somatic granulosa cells, specialized companion cells that surround the oocyte and provide support to it, and an outer layer of thecal cells that serve crucial roles including steroid synthesis. These follicular compartments function as a single physiological unit whose purpose is to produce a healthy egg, which upon ovulation can be fertilized and give rise to a healthy embryo, thus enabling the female germ cell to fulfill its reproductive potential. Beginning from the initial stage of follicle formation and until terminal differentiation at ovulation, oocyte and follicle growth depend absolutely on cooperation between the different cellular compartments. This cooperation synchronizes the initiation of oocyte growth with follicle activation. During growth, it enables metabolic support for the follicle-enclosed oocyte and allows the follicle to fulfill its steroidogenic potential. Near the end of the growth period, intra-follicular interactions prevent the precocious meiotic resumption of the oocyte and ensure its nuclear differentiation. Finally, cooperation enables the events of ovulation, including meiotic maturation of the oocyte and expansion of the cumulus granulosa cells. In this chapter, we discuss the cellular interactions that enable the growing follicle to produce a healthy oocyte, focusing on the communication between the germ cell and the surrounding granulosa cells.

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.

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 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.