Expression and characterisation of Fmr1 splice variants during folliculogenesis in the rat.

CONTEXT: The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5'UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. AIMS: To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. METHODS: We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. KEY RESULTS: We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. CONCLUSIONS: The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. IMPLICATIONS: Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI.

TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41.

Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms.

Meiotic gatekeeper STRA8 suppresses autophagy by repressing Nr1d1 expression during spermatogenesis in mice.

The transition from mitotic to meiotic cell cycles is essential for haploid gamete formation and fertility. Stimulated by retinoic acid gene 8 (Stra8) is an essential gatekeeper of meiotic initiation in vertebrates; yet, the molecular role of STRA8 remains principally unknown. Here we demonstrate that STRA8 functions as a suppressor of autophagy during spermatogenesis in mice. Stra8-deficient germ cells fail to enter meiosis and present aberrant upregulation of autophagy-lysosome genes, commensurate with autophagy activation. Biochemical assays show that ectopic expression of STRA8 alone is sufficient to inhibit both autophagy induction and maturation. Studies also revealed that, Nr1d1, a nuclear hormone receptor gene, is upregulated in Stra8-deficient testes and that STRA8 binds to the Nr1d1 promoter, indicating that Nr1d1 is a direct target of STRA8 transcriptional repression. In addition, it was found that NR1D1 binds to the promoter of Ulk1, a gene essential for autophagy initiation, and that Nr1d1 is required for the upregulated Ulk1 expression in Stra8-deficient testes. Furthermore, both genetic deletion of Nr1d1 and pharmacologic inhibition of NR1D1 by its synthetic antagonist SR8278 exhibit rescuing effects on the meiotic initiation defects observed in Stra8-deficient male germ cells. Together, the data suggest a novel link between STRA8-mediated autophagy suppression and meiotic initiation.

Distribution of FMR1 and FMR2 Repeats in Argentinean Patients with Primary Ovarian Insufficiency.

The premutation state of FMR1 (Fragile X Mental Retardation 1) has been associated with primary ovarian insufficiency (POI), and is the most common known genetic cause for 46,XX patients. Nevertheless, very few studies have analyzed its frequency in Latin American populations. Additionally, a relationship between alleles carrying a cryptic microdeletion in the 5'UTR of FMR2 and the onset of POI has only been studied in one population. Our aim was to analyze the incidence of FMR1 premutations and putative microdeletions in exon 1 of FMR2 in a cohort of Argentinean women with POI. We studied 133 patients and 84 controls. Fluorescent PCR was performed, and the FMR2 exon 1 was further sequenced in samples presenting less than 11 repeats. We found the frequency of FMR1 premutations to be 6.7% and 2.9% for familial and sporadic patients, respectively. Among controls, 1/84 women presented a premutation. In addition, although we did not find microdeletions in FMR2, we observed a change (T >C) adjacent to the repeats in two sisters with POI. Given the repetitive nature of the sequence involved, we could not ascertain whether this represents a single nucleotide polymorphism (SNP) or a deletion. Therefore, a relationship between FMR2 and POI could not be established for our population.

Quantitative analysis of male germline stem cell differentiation reveals a role for the p53-mTORC1 pathway in spermatogonial maintenance.

p53 protects cells from DNA damage by inducing cell-cycle arrest upon encountering genomic stress. Among other pathways, p53 elicits such an effect by inhibiting mammalian target of rapamycin complex 1 (mTORC1), the master regulator of cell proliferation and growth. Although recent studies have indicated roles for both p53 and mTORC1 in stem cell maintenance, it remains unclear whether the p53-mTORC1 pathway is conserved to mediate this process under normal physiological conditions. Spermatogenesis is a classic stem cell-dependent process in which undifferentiated spermatogonia undergo self-renewal and differentiation to maintain the lifelong production of spermatozoa. To better understand this process, we have developed a novel flow cytometry (FACS)-based approach that isolates spermatogonia at consecutive differentiation stages. By using this as a tool, we show that genetic loss of p53 augments mTORC1 activity during early spermatogonial differentiation. Functionally, loss of p53 drives spermatogonia out of the undifferentiated state and causes a consistent expansion of early differentiating spermatogonia until the stage of preleptotene (premeiotic) spermatocyte. The frequency of early meiotic spermatocytes is, however, dramatically decreased. Thus, these data suggest that p53-mTORC1 pathway plays a critical role in maintaining the homeostasis of early spermatogonial differentiation. Moreover, our FACS approach could be a valuable tool in understanding spermatogonial differentiation.

Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

Like stem cells in other tissues, spermatogonia, including spermatogonial stem cells (SSCs) at the foundation of differentiation hierarchy, undergo age-related decline in function. The promyelocytic leukemia zinc finger (Plzf) protein plays an essential role in spermatogonia maintenance by preventing their differentiation. To evaluate whether there is an age-related change in Plzf expression, we found that aged mouse testes exhibited a robust "Plzf overexpression" phenotype, in that they showed not only a higher frequency of Plzf-expressing cells but also an increased level of Plzf expression in these cells. Moreover, some Plzf-expressing cells in aged testes even aberrantly appeared in the differentiating spermatogonia compartment, which is usually low or negative for Plzf expression. Importantly, ectopic Plzf expression in F9 cells suppressed retinoic acid (RA)-induced Stra8 activation, a gene required for meiosis initiation. These data, together with our observation of a lack of meiosis-initiating spermatocytes associated with high Plzf-expressing spermatogonia in the aged testes, particularly in the degenerative seminiferous tubules, suggest that age-related increase in Plzf expression represents a novel molecular signature of spermatogonia aging by functionally arresting their differentiation.

Expression of fragile X mental retardation protein and Fmr1 mRNA during folliculogenesis in the rat.

Fragile X mental retardation protein (FMRP) belongs to a small family of RNA-binding proteins. Its absence or inactivity is responsible for fragile X syndrome, the most common cause of inherited mental retardation. Despite its ubiquitous expression, FMRP function and expression remain almost understudied in non-neuronal tissues, though previous studies on germline development during oogenesis may suggest a special function of this protein also in ovarian tissue. In addition, the well-documented association of FMR1 premutation state with fragile X-related premature ovarian insufficiency adds interest to the role of FMRP in ovarian physiology. The aim of the present work was to investigate the expression of Fmr1 mRNA and its protein, FMRP, at different stages of rat follicular development. By immunohistochemical studies we demonstrated FMRP expression in granulosa, theca and germ cells in all stages of follicular development. In addition, changes in Fmr1 expression, both at the protein and mRNA levels, were observed. FMRP levels increased upon follicular development while preantral and early antral follicles presented similar levels of Fmr1 transcripts with decreased expression in preovulatory follicles. These observations suggest that Fmr1 expression in the ovary is regulated at different and perhaps independent levels. In addition, our results show expression of at least four different isoforms of FMRP during all stages of follicular growth with expression patterns that differ from those observed in brain and testis. Our study shows a regulated expression of Fmr1, both at mRNA and protein levels, during rat follicular development.