Fetal Ovarian Reserve: the Dynamic Changes in Ubiquitin C-Terminal Hydrolase L1.

The regulation of protein turnover by the unique deubiquitinating enzyme ubiquitin C-terminal hydrolase L1 (UCHL1) is only seen in oocytes, spermatogonia, and neurons. Our objective was to investigate variation in expression of UCHL1 across fetal maturation of oocytes that result in lifelong ovarian reserve. We performed a retrospective cohort study of 25 fetal autopsy specimens from 21 to 36 weeks. This was an IRB-approved protocol with parental permission for use of tissues for research purposes. Tissues were stained for expression of the oocyte-specific protein UCHL1, and expression levels were evaluated using quantitative immunofluorescence across gestational ages after correction for the area and background absorbance. Corrected total cell fluorescence (CTCF) for expression of UCHL1 within human oocytes was compared across fetal gestational ages and oocyte size. Trends were analyzed using a locally weighted scatterplot smoothing algorithm. Local expression of UCHL1 increases in oocytes across ovarian development reaching a plateau at 27 weeks with the maintenance of elevated levels through 36 weeks gestational age. This maturation trend is also evidenced by the increase in protein expression as oocyte area increases (r = 0.5530, p ≤ 0.001) with the largest rise occurring as oocytes are enveloped into primordial follicles. The increase in expression as oocytes transition from oogonia into oocytes in primordial follicles and beyond may be part of the preparation of both oocytes and the surrounding somatic cells for the long-term maintenance of the ovarian reserve.

The requirement of ubiquitin C-terminal hydrolase L1 in mouse ovarian development and fertility†.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Pathways coordinating oocyte attrition and abundance during mammalian ovarian reserve establishment.

The mammalian ovarian reserve is comprised of a finite pool of primordial follicles, representing the lifetime reproductive capacity of females. In most mammals, the reserve is produced during embryonic and early postnatal development with oocyte numbers peaking during mid-to-late gestation, and then experiencing a dramatic decline continuing until shortly after birth. Oocytes remaining after the bulk of this attrition are subsequently surrounded by a layer of somatic pre-granulosa cells with these units then referred to as "primordial follicles." The complex and varied cell death mechanisms intrinsic to this process are not only characteristic of, but also essential for, the proper formation of this pool of follicles, and as a result must be immaculately balanced to ensure long-term fertility and reproductive health. Too few follicles can lead to Primary Ovarian Insufficiency, resulting in fertility loss and other features of aging, such as an overall shorter lifespan. On the other hand, whereas an excess of follicles might extend reproductive lifespan, this might also be the underlying etiology of other ovarian pathologies. The last decade, in particular, has vastly expanded our understanding of oocyte attrition and determinants of ovarian reserve abundance. By continuing to decipher the intricacies underlying the cell death processes and development of the initial primordial follicle pool, we may be in a much better position to understand idiopathic cases of premature follicle depletion and improve ovarian health in reproductive-age women.

Dynamic transcriptome profiles within spermatogonial and spermatocyte populations during postnatal testis maturation revealed by single-cell sequencing.

Spermatogenesis is the process by which male gametes are formed from a self-renewing population of spermatogonial stem cells (SSCs) residing in the testis. SSCs represent less than 1% of the total testicular cell population in adults, but must achieve a stable balance between self-renewal and differentiation. Once differentiation has occurred, the newly formed and highly proliferative spermatogonia must then enter the meiotic program in which DNA content is doubled, then halved twice to create haploid gametes. While much is known about the critical cellular processes that take place during the specialized cell division that is meiosis, much less is known about how the spermatocytes in the "first-wave" in juveniles compare to those that contribute to long-term, "steady-state" spermatogenesis in adults. Given the strictly-defined developmental process of spermatogenesis, this study explored the transcriptional profiles of developmental cell stages during testis maturation. Using a combination of comprehensive germ cell sampling with high-resolution, single-cell-mRNA-sequencing, we have generated a reference dataset of germ cell gene expression. We show that discrete developmental stages of spermatogenesis possess significant differences in the transcriptional profiles from neonates compared to juveniles and adults. Importantly, these gene expression dynamics are also reflected at the protein level in their respective cell types. We also show differential utilization of many biological pathways with age in both spermatogonia and spermatocytes, demonstrating significantly different underlying gene regulatory programs in these cell types over the course of testis development and spermatogenic waves. This dataset represents the first unbiased sampling of spermatogonia and spermatocytes during testis maturation, at high-resolution, single-cell depth. Not only does this analysis reveal previously unknown transcriptional dynamics of a highly transitional cell population, it has also begun to reveal critical differences in biological pathway utilization in developing spermatogonia and spermatocytes, including response to DNA damage and double-strand breaks.

TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis.

TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women.

The developmental origins of the mammalian ovarian reserve.

The adult mammalian ovary is devoid of definitive germline stem cells. As such, female reproductive senescence largely results from the depletion of a finite ovarian follicle pool that is produced during embryonic development. Remarkably, the crucial nature and regulation of follicle assembly and survival during embryogenesis is just coming into focus. This developmental pathway involves the coordination of meiotic progression and the breakdown of germ cell cysts into individual oocytes housed within primordial follicles. Recent evidence also indicates that genetic and environmental factors can specifically perturb primordial follicle assembly. Here, we review the cellular and molecular mechanisms by which the mammalian ovarian reserve is established, highlighting the presence of a crucial checkpoint that allows survival of only the highest-quality oocytes.

TAF4b promotes mouse primordial follicle assembly and oocyte survival.

Primary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development.

Assembly of Drosophila centromeric chromatin proteins during mitosis.

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.