Naked mole rat ovaries allow investigation of ovarian reserve, in vitro germ cell expansion, and oocyte in vitro maturation within a single sample.

Recently, we described that in the naked mole rat ovary it is possible to study the ovarian reserve and the mitotic expansion of the germ cell postnatally. Herein, we show oocyte in vitro maturation and in vitro germ cell expansion using the same ovary.

Germ cell nests in adult ovaries and an unusually large ovarian reserve in the naked mole-rat.

The naked mole-rat (NMR, Heterocephalus glaber) is renowned for its eusociality and exceptionally long lifespan (> 30 y) relative to its small body size (35-40 g). A NMR phenomenon that has received far less attention is that females show no decline in fertility or fecundity into their third decade of life. The age of onset of reproductive decline in many mammalian species is closely associated with the number of germ cells remaining at the age of sexual maturity. We quantified ovarian reserve size in NMRs at the youngest age (6 months) when subordinate females can begin to ovulate after removal from the queen's suppression. We then compared the NMR ovarian reserve size to values for 19 other mammalian species that were previously reported. The NMR ovarian reserve at 6 months of age is exceptionally large at 108,588 ± 69,890 primordial follicles, which is more than 10-fold larger than in mammals of a comparable size. We also observed germ cell nests in ovaries from 6-month-old NMRs, which is highly unusual since breakdown of germ cell nests and the formation of primordial follicles is generally complete by early postnatal life in other mammals. Additionally, we found germ cell nests in young adult NMRs between 1.25 and 3.75 years of age, in both reproductively activated and suppressed females. The unusually large NMR ovarian reserve provides one mechanism to account for this species' protracted fertility. Whether germ cell nests in adult ovaries contribute to the NMR's long reproductive lifespan remains to be determined.

Telomeric repeat-containing RNA and telomerase in human fetal oocytes.

STUDY QUESTION: What is the distribution of telomeric repeat-containing RNA (TERRA) and of telomerase in human fetal oocytes? SUMMARY ANSWER: TERRA forms discrete foci at telomeres of human fetal oocytes and it co-localizes with both the shelterin component telomeric repeat-binding factor 2 (TRF2) and the catalytic subunit of human telomerase at the telomeres of meiotic chromosomes. WHAT IS KNOWN ALREADY: TERRA is a structural element of the telomeric chromatin that has been described in somatic cells of many different eukaryote species. The telomerase enzyme is inactive in adult somatic cells but is active in germ cells, stem cells and in the majority of tumors; however, its distribution in oocytes is still unknown. STUDY DESIGN, SIZE, DURATION: For this study, ovarian samples from four euploid fetuses of 22 gestational weeks were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Hospital de la Vall d'Hebron. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analyzed the distribution of TERRA and telomerase in cells derived from human fetal ovaries. The co-localization of TERRA, telomerase and telomeres was performed by optimizing a combination of immunofluorescence (IF) and RNA-fluorescent in situ hybridization (RNA-FISH) techniques. The synaptonemal complex protein 3 (SYCP3), TRF2 and protein component of telomerase [telomerase reverse transcriptase (TERT)] were detected by IF, whereas TERRA was revealed by RNA-FISH using a (CCCTAA)(3) oligonucleotide. SYCP3 signals allowed us to identify oocytes that had entered meiosis and classify them into the different stages of prophase I, whereas TRF2 indicated the telomeric regions of chromosomes. MAIN RESULTS AND THE ROLE OF CHANCE: We show for the first time the presence of TERRA and the intracellular distribution of telomerase in human fetal ovarian cells. TERRA is present, forming discrete foci, in 75% of the ovarian tissue cells and most of TERRA molecules (≈ 83%) are at telomeres (TRF2 co-localization). TERRA levels are higher in oocytes than in ovarian tissue cells (P = 0.00), and do not change along the progression of the prophase I stage (P = 0.37). TERRA is present on ≈ 23% of the telomeres in all cell types derived from human fetal ovaries. Moreover, ≈ 22% of TERRA foci co-localize with the protein component of telomerase (TERT). LIMITATIONS, REASONS FOR CAUTION: We present a descriptive/qualitative study of TERRA in human fetal ovarian tissue. Given the difficult access and manipulation of fetal samples, the number of fetal ovaries used in this study was limited. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report on TERRA expression in oocytes from human fetal ovaries. The presence of TERRA at the telomeres of oocytes from the leptotene to pachytene stages and its co-localization with the telomerase protein component suggests that this RNA might participate in the maintenance of the telomere structure, at least through the processes that take place during the female meiotic prophase I. Since telomeres in oocytes have been mainly studied regarding the bouquet structure, our results introduce a new viewpoint of the telomeric structure during meiosis.

Characterization of the Postnatal Naked Mole-Rat Ovary: From Primordial Germ Cells to Meiotic Prophase I Oocytes.

The mammalian reproductive cycle, including those of humans and mice, begins very early in development. In utero, the ovaries become populated with primordial germ cells (PGCs) that will generate the oogonia. First, these cells proliferate mitotically, and then they trigger the meiotic program and initiate meiotic prophase I. Since these processes happen during gestation, their study had been very limited and challenging. Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the PGCs, and the initiation of the meiotic program occurs postnatally. In this chapter, we present a comprehensive collection of protocols that permit the analysis of naked mole-rat germ cells, from PGCs to oocytes, in meiotic prophase I, using in vivo and in vitro approaches.

Postnatal oogenesis leads to an exceptionally large ovarian reserve in naked mole-rats.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

Unraveling mitochondrial piRNAs in mouse embryonic gonadal cells.

Although mitochondria are widely studied organelles, the recent interest in the role of mitochondrial small noncoding RNAs (sncRNAs), miRNAs, and more recently, piRNAs, is providing new functional perspectives in germ cell development and differentiation. piRNAs (PIWI-interacting RNAs) are single-stranded sncRNAs of mostly about 20-35 nucleotides, generated from the processing of pre-piRNAs. We leverage next-generation sequencing data obtained from mouse primordial germ cells and somatic cells purified from early-differentiating embryonic ovaries and testis from 11.5 to 13.5 days postcoitum. Using bioinformatic tools, we elucidate (i) the origins of piRNAs as transcribed from mitochondrial DNA fragments inserted in the nucleus or from the mitochondrial genome; (ii) their levels of expression; and (iii) their potential roles, as well as their association with genomic regions encoding other sncRNAs (such as tRNAs and rRNAs) and the mitochondrial regulatory region (D-loop). Finally, our results suggest how nucleo-mitochondrial communication, both anterograde and retrograde signaling, may be mediated by mitochondria-associated piRNAs.

Meiotic Cohesin and Variants Associated With Human Reproductive Aging and Disease.

Successful human reproduction relies on the well-orchestrated development of competent gametes through the process of meiosis. The loading of cohesin, a multi-protein complex, is a key event in the initiation of mammalian meiosis. Establishment of sister chromatid cohesion via cohesin rings is essential for ensuring homologous recombination-mediated DNA repair and future proper chromosome segregation. Cohesin proteins loaded during female fetal life are not replenished over time, and therefore are a potential etiology of age-related aneuploidy in oocytes resulting in decreased fecundity and increased infertility and miscarriage rates with advancing maternal age. Herein, we provide a brief overview of meiotic cohesin and summarize the human genetic studies which have identified genetic variants of cohesin proteins and the associated reproductive phenotypes including primary ovarian insufficiency, trisomy in offspring, and non-obstructive azoospermia. The association of cohesion defects with cancer predisposition and potential impact on aging are also described. Expansion of genetic testing within clinical medicine, with a focus on cohesin protein-related genes, may provide additional insight to previously unknown etiologies of disorders contributing to gamete exhaustion in females, and infertility and reproductive aging in both men and women.

Sex- and Age-Specific Impact of ERK Loss Within the Pituitary Gonadotrope in Mice.

Extracellular signal-regulated kinase (ERK) signaling regulates hormone action in the reproductive axis, but specific mechanisms have yet to be completely elucidated. In the current study, ERK1 null and ERK2 floxed mice were combined with a gonadotropin-releasing hormone receptor (GnRHR)-internal ribosomal entry site-Cre (GRIC) driver. Female ERK double-knockout (ERKdko) animals were hypogonadotropic, resulting in anovulation and complete infertility. Transcript levels of four gonadotrope-specific genes (GnRHR and the three gonadotropin subunits) were reduced in pituitaries at estrus in ERKdko females, and the postcastration response to endogenous GnRH hyperstimulation was blunted. As females aged, they exhibited abnormal ovarian histology, as well as increased body weight. ERKdko males were initially less affected, showing moderate subfertility, up to 6 months of age. Male ERKdko mice also displayed a blunted response to endogenous GnRH following castration. By 12 months of age, ERKdko males had reduced testicular weights and sperm production. By 18 months of age, the ERKdko males displayed reduced testis and seminal vesicle weights, marked seminiferous tubule degeneration, and a 77% reduction in sperm production relative to controls. As the GRIC is also active in the male germ line, we examined the specific role of ERK loss in the testes using the stimulated by retinoic acid 8 (Stra8)-Cre driver. Whereas ERK loss in GRIC and Stra8 males resulted in comparable losses in sperm production, seminiferous tubule histological degeneration was only observed in the GRIC-ERKdko animals. Our data suggest that loss of ERK signaling and hypogonadotropism within the reproductive axis impacts fertility and gonadal aging.

Endocrine disrupters, microRNAs, and primordial germ cells: a dangerous cocktail.

Endocrine-disrupting chemicals (EDCs) are environmental pollutants that may change the homeostasis of the endocrine system, altering the differentiation of germ cells with consequences for reproduction. In mammals, germ cell differentiation begins with primordial germ cells (PGCs) during embryogenesis. Primordial germ cell development and gametogenesis are genetically regulated processes, in which the posttranscriptional gene regulation could be mediated by small noncoding RNAs (sncRNAs) such as microRNAs (miRNAs). Here, we review the deleterious effects of exposure during fetal life to EDCs mediated by deregulation of ncRNAs, and specifically miRNAs on PGC differentiation. Moreover, the environmental stress induced by exposure to some EDCs during the embryonic window of development could trigger reproductive dysfunctions transgenerationally transmitted by epigenetic mechanisms with the involvement of miRNAs expressed in germ line cells.

Meiosis in a bottle: new approaches to overcome Mammalian meiocyte study limitations.

The study of meiosis is limited because of the intrinsic nature of gametogenesis in mammals. One way to overcome these limitations would be the use of culture systems that would allow meiotic progression in vitro. There have been some attempts to culture mammalian meiocytes in recent years. In this review we will summarize all the efforts to-date in order to culture mammalian sperm and oocyte precursor cells.