Miguel Angel Brieño-Enríquez, M.D., Ph.D.

Protection of ovarian reserve and reproductive aging in mammals (Naked Mole-Rat Research)

In mammals, it is generally thought that the total number of oocytes and follicles in adult ovaries is established during pre- and perinatal life. However, the number of oocytes at birth can be modulated by any number of endogenous and exogenous factors. During the last two decades, several groups have been investigating and reporting on the presence of postnatal oogenesis in mammals; however, the results of these studies have been heavily debated in the field. Because naked mole-rats (NMRs, Heterocephalus glaber) reportedly demonstrate no decline in fertility and fecundity into their third decade of life, we have recently begun to study NMR ovarian development to elucidate the means by which they accomplish this remarkable feat. These include the possibilities that NMRs generate new oocytes during the postnatal life and/or that they establish a very large ovarian reserve during pre- and perinatal life. To investigate these possibilities, we used an array of approaches, which included Immunohistochemistry for stem cell (SOX2, OCT4), germ cell (VASA), meiotic initiation (STRA8) and meiotic (REC8) markers on ovaries at different times of development (embryonic day 56 (E56), postnatal day (PD) PD5, PD8, PD15, PD28, and 3 months) as well as RNAseq. Collectively, our results show, for first time, that ovarian germ cell development is highly asynchronous in NMRs, which is a developmental characteristic that NMRs share with humans. Because NMRs and humans also both have long reproductive lifespans that comprise decades, the NMR is an excellent laboratory model system for studying the processes that lead to variations in the size of the ovarian reserve and that modulate the age of onset of reproductive senescence.

Impact of SARS-CoV2 in reproduction

The novel coronavirus SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA beta-coronavirus of the family Coronaviridae. The main target of these virus is the lungs, but new reports about COVID-19 indicate its effects on other organs, including kidney, brain and testis. SARS-CoV-2, which uses the angiotensin-converting enzyme 2 (ACE2) receptor and the transmembrane protease serine 2 (TMPRSS2) to invade human cells. Based on ACE2 and TMPRSS2 expression, the testis has been classified as being at high risk of being infected by SARS-CoV-2. Indeed, patients with COVID-19 suffer from testicular discomfort, which can be associated with viral orchitis. Similarly, some studies have reported reduced testosterone levels in the serum from COVID-19 patients. These data suggest that COVID-19 could affect male reproductive function, in fact the presence of SARS-CoV-2 has been detected in the testis of some deceased patients of COVID-19 as well as in some semen samples of patients in the acute and recovery phase of COVID-19. SSCs reside within a specific microenvironment in the testes called “niche” which regulates their properties, such as, self-renewal, pluripotency, quiescence, and their ability to differentiate. However, how SARS-CoV-2 disrupts the SSC niche and its relationship with long term infertility is unknow.

Transgenerational epigenetic effects, PGCs, and non-coding RNAs

In mammals, Primordial Germ Cells (PGCs) give rise to the germ cell pool during fetal development. I investigated the question of whether prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation and development of the male germline and may induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a strong example of a molecule that causes transgenerational effects on germ cells through a mechanism mediated by changes in methylation. My studies in this area showed for very first time that prenatal exposure to vinclozolin in mice induced changes in microRNAs (miRNAs) and their targets across three successive generations. This transgenerational effect was characterized by a reduction in the number of PGCs, increased apoptosis, and decreased fertility rate in adult males across generations. The number and quality of PGCs during embryogenesis depends on a series of factors, such as Blimp1, Lin28, and the miRNA Let7, and all of them are crucial regulators of PGC differentiation. My results showed that exposure to vinclozolin deregulated specific miRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. Taken together, my data showed that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of miRNAs affecting key regulatory pathways of germ cell differentiation without changes in methylation.

Regulation of cohesin removal mediated by NEK1 kinase during meiotic prophase I

The cohesin complex is essential for maintaining sister chromatid cohesion and correcting chromosome segregation in both mitosis and meiosis. In mitosis, cohesin removal is orchestrated in two steps: (1) the prophase pathway and (2) cleavage by separase. The prophase pathway depends on the proper activity of wings apart-like (WAPL). WAPL action is dependent on its interaction with regulator of cohesion maintenance, homolog B (PDS5B) and phosphorylation of sororin. While the regulation of the prophase pathway and mechanisms that control WAPL activity have been extensively studied in mitosis, these processes have not been characterized extensively in meiosis. The serine/threonine and tyrosine dual-specificity NIMA-like kinase-1, NEK1, is highly expressed in testis and is required for accurate cohesin removal at the first meiotic division via mechanisms that were hitherto unknown. My studies demonstrate the regulative role of NEK1 during the meiotic prophase pathway. NEK1 phosphorylates the cohesin SMC3 and REC8 and also regulates the stability and/or action of WAPL. Analysis using mass spectrometry reveals that Nek1 mutants show abnormal phosphorylation of serine 549 of RAD21, serine 1067 on SMC3, and serine 226 on WAPL. Using immunoprecipitation followed by mass spectrometry, I revealed that the protein phosphatase PP1γ interacts with both NEK1 and WAPL. Using PP1γ inhibitors and PP1γ mutant mice, I confirmed that the lack of PP1γ phosphorylation or protein induces a phenotype that mimics the Nek1 mutant. These results also show that NEK1 interacts indirectly with WAPL binding partner PDS5B. Taken together, my data demonstrate that NEK1 is essential for the prophase pathway regulating cohesin phosphorylation and maintaining unphosphorylated WAPL through a mechanism mediated by PP1γ.