In vivo reduction of RAD51-mediated homologous recombination triggers aging but impairs oncogenesis.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.

Author Correction: Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Maternal vitamin C regulates reprogramming of DNA methylation and germline development.

Development is often assumed to be hardwired in the genome, but several lines of evidence indicate that it is susceptible to environmental modulation with potential long-term consequences, including in mammals1,2. The embryonic germline is of particular interest because of the potential for intergenerational epigenetic effects. The mammalian germline undergoes extensive DNA demethylation3-7 that occurs in large part by passive dilution of methylation over successive cell divisions, accompanied by active DNA demethylation by TET enzymes3,8-10. TET activity has been shown to be modulated by nutrients and metabolites, such as vitamin C11-15. Here we show that maternal vitamin C is required for proper DNA demethylation and the development of female fetal germ cells in a mouse model. Maternal vitamin C deficiency does not affect overall embryonic development but leads to reduced numbers of germ cells, delayed meiosis and reduced fecundity in adult offspring. The transcriptome of germ cells from vitamin-C-deficient embryos is remarkably similar to that of embryos carrying a null mutation in Tet1. Vitamin C deficiency leads to an aberrant DNA methylation profile that includes incomplete demethylation of key regulators of meiosis and transposable elements. These findings reveal that deficiency in vitamin C during gestation partially recapitulates loss of TET1, and provide a potential intergenerational mechanism for adjusting fecundity to environmental conditions.

Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis.

Macrophages are the principal immune cells of the epididymis and testis, but their origins, heterogeneity, development, and maintenance are not well understood. Here, we describe distinct populations of epididymal and testicular macrophages that display an organ-specific cellular identity. Combining in vivo fate-mapping, chimeric and parabiotic mouse models with in-depth cellular analyses, we found that CD64hiMHCIIlo and CD64loMHCIIhi macrophage populations of epididymis and testis arise sequentially from yolk sac erythro-myeloid progenitors, embryonic hematopoiesis, and nascent neonatal monocytes. While monocytes were the major developmental source of both epididymal and testicular macrophages, both populations self-maintain in the steady-state independent of bone marrow hematopoietic precursors. However, after radiation-induced macrophage ablation or during infection, bone marrow-derived circulating monocytes are recruited to the epididymis and testis, giving rise to inflammatory macrophages that promote tissue damage. These results define the layered ontogeny, maintenance and inflammatory response of macrophage populations in the male reproductive organs.

shani mutation in mouse affects splicing of Spata22 and leads to impaired meiotic recombination.

Recombination is crucial for chromosome pairing and segregation during meiosis. SPATA22, along with its direct binding partner and functional collaborator, MEIOB, is essential for the proper repair of double-strand breaks (DSBs) during meiotic recombination. Here, we describe a novel point-mutated allele (shani) of mouse Spata22 that we isolated in a forward genetic screen. shani mutant mice phenocopy Spata22-null and Meiob-null mice: mutant cells appear to form DSBs and initiate meiotic recombination, but are unable to complete DSB repair, leading to meiotic prophase arrest, apoptosis and sterility. shani mutants show precocious loss of DMC1 foci and improper accumulation of BLM-positive recombination foci, reinforcing the requirement of SPATA22-MEIOB for the proper progression of meiotic recombination events. The shani mutation lies within a Spata22 coding exon and molecular characterization shows that it leads to incorrect splicing of the Spata22 mRNA, ultimately resulting in no detectable SPATA22 protein. We propose that the shani mutation alters an exonic splicing enhancer element (ESE) within the Spata22 transcript. The affected DNA nucleotide is conserved in most tetrapods examined, suggesting that the splicing regulation we describe here may be a conserved feature of Spata22 regulation.

Mouse model of radiation-induced premature ovarian insufficiency reveals compromised oocyte quality: implications for fertility preservation.

RESEARCH QUESTION: What is the impact of radiation exposure on oocyte quality and female fertility? DESIGN: Prepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (n = 36) and sham-treated (n = 8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (n = 10), activation of checkpoint kinase (Chk2) (n = 10) and dynamics of follicular growth (n = 18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (n = 28) and the fetal abortion rate (n = 24). Ploidy of mature oocytes (n = 20) was analysed after CREST immunostaining, and uterine sections were examined. RESULTS: Radiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, P = 0.0096), related to altered ploidy in the surviving oocytes (25.5%, P = 0.0035). CONCLUSIONS: The effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.

Homozygous hypomorphic BRCA2 variant in primary ovarian insufficiency without cancer or Fanconi anaemia trait.

BACKGROUND: Primary ovarian insufficiency (POI) affects 1% of women under 40 years and is a public health problem. The genetic causes of POI are highly heterogeneous with isolated or syndromic forms. Recently, variants in genes involved in DNA repair have been shown to cause POI. Notably, syndromic POI with Fanconi anaemia (FA) traits related to biallelic BRCA2 truncated variants has been reported. Here, we report a novel phenotype of isolated POI with a BRCA2 variant in a consanguineous Turkish family. METHODS: Exome sequencing (ES) was performed in the patient. We also performed functional studies, including a homologous recombination (HR) test, cell proliferation, radiation-induced RAD51 foci formation assays and chromosome breakage studies in primary and lymphoblastoid immortalised cells. The expression of BRCA2 in human foetal ovaries was studied. RESULTS: ES identified a homozygous missense c.8524C>T/p.R2842C-BRCA2 variant. BRCA2 defects induce cancer predisposition and FA. Remarkably, neither the patient nor her family exhibited somatic pathologies. The patient's cells showed intermediate levels of chromosomal breaks, cell proliferation and radiation-induced RAD51 foci formation compared with controls and FA cells. R2842C-BRCA2 only partially complemented HR efficiency compared with wild type-BRCA2. BRCA2 is expressed in human foetal ovaries in pachytene stage oocytes, when meiotic HR occurs. CONCLUSION: We describe the functional assessment of a homozygous hypomorphic BRCA2 variant in a patient with POI without cancer or FA trait. Our findings extend the phenotype of BRCA2 biallelic alterations to fully isolated POI. This study has a major impact on the management and genetic counselling of patients with POI.

Unexpected Interacting Effects of Physical (Radiation) and Chemical (Bisphenol A) Treatments on Male Reproductive Functions in Mice.

For decades, numerous chemical pollutants have been described to interfere with endogenous hormone metabolism/signaling altering reproductive functions. Among these endocrine disrupting substances, Bisphenol A (BPA), a widely used compound, is known to negatively impact germ and somatic cells in the testis. Physical agents, such as ionizing radiation, were also described to perturb spermatogenesis. Despite the fact that we are constantly exposed to numerous environmental chemical and physical compounds, very few studies explore the impact of combined exposure to chemical and physical pollutants on reproductive health. The aim of this study was to describe the impact of fetal co-exposure to BPA and IR on testicular function in mice. We exposed pregnant mice to 10 µM BPA (corresponding to 0.5 mg/kg/day) in drinking water from 10.5 dpc until birth, and we irradiated mice with 0.2 Gy (γ-ray, RAD) at 12.5 days post-conception. Co-exposure to BPA and γ-ray induces DNA damage in fetal germ cells in an additive manner, leading to a long-lasting decrease in germ cell abundance. We also observed significant alteration of adult steroidogenesis by RAD exposure independently of the BPA exposure. This is illustrated by the downregulation of steroidogenic genes and the decrease of the number of adult Leydig cells. As a consequence, courtship behavior is modified, and male ultrasonic vocalizations associated with courtship decreased. In conclusion, this study provides evidence for the importance of broadening the concept of endocrine disruptors to include physical agents, leading to a reevaluation of risk management and regulatory decisions.

The Role of ERα36 in Development and Tumor Malignancy.

Estrogen nuclear receptors, represented by the canonical forms ERα66 and ERß1, are the main mediators of the estrogen-dependent pathophysiology in mammals. However, numerous isoforms have been identified, stimulating unconventional estrogen response pathways leading to complex cellular and tissue responses. The estrogen receptor variant, ERα36, was cloned in 2005 and is mainly described in the literature to be involved in the progression of mammary tumors and in the acquired resistance to anti-estrogen drugs, such as tamoxifen. In this review, we will first specify the place that ERα36 currently occupies within the diversity of nuclear and membrane estrogen receptors. We will then report recent data on the impact of ERα36 expression and/or activity in normal breast and testicular cells, but also in different types of tumors including mammary tumors, highlighting why ERα36 can now be considered as a marker of malignancy. Finally, we will explain how studying the regulation of ERα36 expression could provide new clues to counteract resistance to cancer treatments in hormone-sensitive tumors.

Meiotic onset is reliant on spatial distribution but independent of germ cell number in the mouse ovary.

Mouse ovarian germ cells enter meiosis in a wave that propagates from anterior to posterior, but little is known about contribution of germ cells to initiation or propagation of meiosis. In a Ror2 mutant with diminished germ cell number and migration, we find that overall timing of meiotic initiation is delayed at the population level. We use chemotherapeutic depletion to exclude a profoundly reduced number of germ cells as a cause for meiotic delay. We rule out sex reversal or failure to specify somatic support cells as contributors to the meiotic phenotype. Instead, we find that anomalies in the distribution of germ cells as well as gonad shape in mutants contribute to aberrant initiation of meiosis. Our analysis supports a model of meiotic initiation via diffusible signal(s), excludes a role for germ cells in commencing the meiotic wave and furnishes the first phenotypic demonstration of the wave of meiotic entry. Finally, our studies underscore the importance of considering germ cell migration defects while studying meiosis to discern secondary effects resulting from positioning versus primary meiotic entry phenotypes.

RPA homologs and ssDNA processing during meiotic recombination.

Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

The zinc-finger protein basonuclin 2 is required for proper mitotic arrest, prevention of premature meiotic initiation and meiotic progression in mouse male germ cells.

Absence of mitosis and meiosis are distinguishing properties of male germ cells during late fetal and early neonatal periods. Repressors of male germ cell meiosis have been identified, but mitotic repressors are largely unknown, and no protein repressing both meiosis and mitosis is known. We demonstrate here that the zinc-finger protein BNC2 is present in male but not in female germ cells. In testis, BNC2 exists as several spliced isoforms and presumably binds to DNA. Within the male germ cell lineage, BNC2 is restricted to prospermatogonia and undifferentiated spermatogonia. Fetal prospermatogonia that lack BNC2 multiply excessively on embryonic day (E)14.5 and reenter the cell cycle prematurely. Mutant prospermatogonia also engage in abnormal meiosis; on E17.5, Bnc2(-/-) prospermatogonia start synthesizing the synaptonemal protein SYCP3, and by the time of birth, many Bnc2(-/-) prospermatogonia have accumulated large amounts of nonfilamentous SYCP3, thus appearing to be blocked at leptonema. Bnc2(-/-) prospermatogonia do not undergo proper male differentiation, as they lack almost all the mRNA for the male-specific methylation protein DNMT3L and have increased levels of mRNAs that encode meiotic proteins, including STRA8. Bnc2(-/-) prospermatogonia can produce spermatogonia, but these enter meiosis prematurely and undergo massive apoptotic death during meiotic prophase. This study identifies BNC2 as a major regulator of male germ stem cells, which is required for repression of meiosis and mitosis in prospermatogonia, and for meiosis progression during spermatogenesis. In view of the extreme evolutionary conservation of BNC2, the findings described here are likely to apply to many species.

Human foetal ovary shares meiotic preventing factors with the developing testis.

STUDY QUESTION: How can pre-meiotic germ cells persist in the human foetal ovary? SUMMARY ANSWER: Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. WHAT IS KNOWN ALREADY: The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. STUDY DESIGN SIZE, DURATION: In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. PARTICIPANTS/MATERIALS SETTING, METHODS: Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. MAIN RESULTS AND THE ROLE OF CHANCE: Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. WIDER IMPLICATIONS OF THE FINDINGS: We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Université Paris Diderot-Paris 7 and Université Paris-Sud, CEA, INSERM, and Agence de la Biomédecine. The authors declare no conflict of interest.

MEIOB targets single-strand DNA and is necessary for meiotic recombination.

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/-) spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/-) meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

Msx1 and Msx2 promote meiosis initiation.

The mechanisms regulating germ line sex determination and meiosis initiation are poorly understood. Here, we provide evidence for the involvement of homeobox Msx transcription factors in foetal meiosis initiation in mammalian germ cells. Upon meiosis initiation, Msx1 and Msx2 genes are strongly expressed in the foetal ovary, possibly stimulated by soluble factors found there: bone morphogenetic proteins Bmp2 and Bmp4, and retinoic acid. Analysis of Msx1/Msx2 double mutant embryos revealed a majority of undifferentiated germ cells remaining in the ovary and, importantly, a decrease in the number of meiotic cells. In vivo, the Msx1/Msx2 double-null mutation prevented full activation of Stra8, a gene required for meiosis. In F9 cells, Msx1 can bind to Stra8 regulatory sequences and Msx1 overexpression stimulates Stra8 transcription. Collectively, our data demonstrate for the first time that some homeobox genes are required for meiosis initiation in the female germ line.

Involvement of doublesex and mab-3-related transcription factors in human female germ cell development demonstrated by xenograft and interference RNA strategies.

We identified three doublesex and mab-3-related transcription factors (DMRT) that were sexually differentially expressed in human fetal gonads and present in the ovaries at the time of meiotic initiation. These were also identified in murine embryonic female germ cells. Among these, we focused on DMRTA2 (DMRT5), whose function is unknown in the developing gonads, and clarified its role in human female fetal germ cells, using an original xenograft model. Early human fetal ovaries (8-11 weeks post-fertilization) were grafted into nude mice. Grafted ovaries developed normally, with no apparent overt changes, when compared with ungrafted ovaries at equivalent developmental stages. Appropriate germ cell density, mitotic/meiotic transition, markers of meiotic progression and follicle formation were evident. Four weeks after grafting, mice were treated with siRNA, specifically targeting human DMRTA2 mRNA. DMRTA2 inhibition triggered an increase in undifferentiated FUT4-positive germ cells and a decrease in the percentage of meiotic γH2AX-positive germ cells, when compared with mice that were injected with control siRNA. Interestingly, the expression of markers associated with pre-meiotic germ cell differentiation was also impaired, as was the expression of DMRTB1 (DMRT6) and DMRTC2 (DMRT7). This study reveals, for the first time, the requirement of DMRTA2 for normal human female embryonic germ cell development. DMRTA2 appears to be necessary for proper differentiation of oogonia, prior to entry into meiosis, in the human species. Additionally, we developed a new model of organ xenografting, coupled with RNA interference, which provides a useful tool for genetic investigations of human germline development.

Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors.

Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.

Direct and indirect consequences on gene expression of a thyroid hormone receptor alpha 1 mutation restricted to Sertoli cells.

Thyroid hormone is required for the timely transition of Sertoli cells from proliferative to differentiating and maturing. This transition takes place during a critical developmental period in mammals, which in mice is the first post-natal week. In order to identify the underlying molecular mechanisms of this differentiation process, we used Cre/loxP technology to selectively block the function of the thyroid hormone receptor TRα1 in Sertoli cells. We then used RNA-seq to analyze the changes in gene expression induced in the post-natal testis. This differential analysis provides genetic clues to the initial testicular defects resulting from disrupted thyroid hormone signaling, and suggests that Sertoli cells influence germ cells soon after their birth.

Foetal exposure to the bisphenols BADGE and BPAF impairs meiosis through DNA oxidation in mouse ovaries.

Many endocrine disruptors have been proven to impair the meiotic process which is required for the production of healthy gametes. Bisphenol A is emblematic of such disruptors, as it impairs meiotic prophase I and causes oocyte aneuploidy following in utero exposure. However, the mechanisms underlying these deleterious effects remain poorly understood. Furthermore, the increasing use of BPA alternatives raises concerns for public health. Here, we investigated the effects of foetal exposure to two BPA alternatives, bisphenol A Diglycidyl Ether (BADGE) and bisphenol AF (BPAF), on oogenesis in mice. These compounds delay meiosis initiation, increase the number of MLH1 foci per cell and induce oocyte aneuploidy. We further demonstrate that these defects are accompanied by changes in gene expression in foetal premeiotic germ cells and aberrant mRNA splicing of meiotic genes. We observed an increase in DNA oxidation after exposure to BPA alternatives. Specific induction of oxidative DNA damage during foetal germ cell differentiation causes similar defects during oogenesis, as observed in 8-oxoguanine DNA Glycosylase (OGG1)-deficient mice or after in utero exposure to potassium bromate (KBrO3), an inducer of oxidative DNA damage. The supplementation of BPA alternatives with N-acetylcysteine (NAC) counteracts the effects of bisphenols on meiosis. Together, our results propose oxidative DNA lesion as an event that negatively impacts female meiosis with major consequences on oocyte quality. This could be a common mechanism of action for numerous environmental pro-oxidant pollutants, and its discovery, could lead to reconsider the adverse effect of bisphenol mixtures that are simultaneously present in our environment.

Thyroid hormone limits postnatal Sertoli cell proliferation in vivo by activation of its alpha1 isoform receptor (TRalpha1) present in these cells and by regulation of Cdk4/JunD/c-myc mRNA levels in mice.

Hypo- and hyperthyroidism alter testicular functions in the young. Among T3 receptors, TRalpha1 is ubiquitous, and its previously described knockout leads to an increase in testis weight and sperm production. We tested, for the first time, the hypothesis that TRalpha1-dependent regulation of Sertoli cell (SC) proliferation was directly regulated by TRalpha1 present in these cells. Thus, after crossing with the AMH-Cre line, we generated and analyzed a new line that expressed a dominant-negative TRalpha1 isoform (TRalpha(AMI)) in SCs only. So-called TRalpha(AMI)-SC (TRalpha(AMI/+) Cre(+)) mice exhibited similar phenotypic features to the knockout line: heavier testicular weight and higher sperm reserve, in comparison with their adequate controls (TRalpha(AMI/+) Cre(-)). SC density increased significantly as a result of a higher proliferative index at ages Postnatal Day (P) 0 and P3. When explants of control testes were cultured (at age P3), a significant decrease in the proliferation of SCs was observed in response to an excess of T3. This response was not observed in the TRalpha(AMI)-SC and knockout lines. Finally, when TRalpha(AMI) is present in SCs, the phenotype observed is similar to that of the knockout line. This study demonstrates that T3 limits postnatal SC proliferation by activation of TRalpha1 present in these cells. Moreover, quantitative RT-PCR provided evidence that regulation of the Cdk4/JunD/c-myc pathway was involved in this negative control.