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.

Enhanced Neonatal Pulse Oximetry Sounds for the First Minutes of Life: A Laboratory Trial.

OBJECTIVE: Auditory enhancements to the pulse oximetry tone may help clinicians detect deviations from target ranges for oxygen saturation (SpO2) and heart rate (HR). BACKGROUND: Clinical guidelines recommend target ranges for SpO2 and HR during neonatal resuscitation in the first 10 minutes after birth. The pulse oximeter currently maps HR to tone rate, and SpO2 to tone pitch. However, deviations from target ranges for SpO2 and HR are not easy to detect. METHOD: Forty-one participants were presented with 30-second simulated scenarios of an infant's SpO2 and HR levels in the first minutes after birth. Tremolo marked distinct HR ranges and formants marked distinct SpO2 ranges. Participants were randomly allocated to conditions: (a) No Enhancement control, (b) Enhanced HR Only, (c) Enhanced SpO2 Only, and (d) Enhanced Both. RESULTS: Participants in the Enhanced HR Only and Enhanced SpO2 Only conditions identified HR and SpO2 ranges, respectively, more accurately than participants in the No Enhancement condition, ps < 0.001. In the Enhanced Both condition, the tremolo enhancement of HR did not affect participants' ability to identify SpO2 range, but the formants enhancement of SpO2 may have attenuated participants' ability to identify tremolo-enhanced HR range. CONCLUSION: Tremolo and formant enhancements improve range identification for HR and SpO2, respectively, and could improve clinicians' ability to identify SpO2 and HR ranges in the first minutes after birth. APPLICATION: Enhancements to the pulse oximeter tone to indicate clinically important ranges could improve the management of oxygen delivery to the neonate during resuscitation in the first 10 minutes after birth.

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.

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.

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.

Duplicate: New Chapter for Biology of Reproduction.

The article https://doi.org/10.1093/biolre/iox090 has been withdrawn because it is a duplicate of https://doi.org/10.093/biolre/iox091. The publisher regrets the error.