Regulation of gene expression in Sertoli cells by follicle-stimulating hormone (FSH): Cloning and characterization of LRPR1, a primary response gene encoding a leucine-rich protein

Searching for hormone-regulated genes in testicular Sertoli cells, we cloned and sequenced a cDNA of 3108 base pairs, named LRPR1 (signifying leucine-rich primary response gene 1). This cDNA sequence has an open reading frame of 2238 base pairs encoding a leucine-rich protein of 746 amino acid residues with a relative molecular mass of 85.6 kDa. As much as 16% of the amino acid residues is leucine. Database analysis revealed significant similarity of LRPR1 to the human brain cDNA sequence EST00443, but not to any other sequences present in databases. The expression of LRPR1 mRNA in Sertoli cells is strongly and rapidly up-regulated by follicle-stimulating hormone (FSH). The level of LRPR1 mRNA was very low in Sertoli cells isolated from 21-day-old rats and cultured for 3 days in the absence of FSH, but LRPR1 mRNA expression was markedly increased within 2 h after addition of FSH to these cultures. A maximal response was reached within 4 h. Dibutyryl-cyclic AMP [(Bu)2cAMP] and forskolin had similar effects compared to FSH, indicating that cAMP acts as a second messenger in the regulation of LRPR1 expression. The up-regulation of LRPR1 mRNA expression by FSH was also observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that FSH regulates LRPR1 mRNA expression through a direct mechanism which does not require de novo protein synthesis. Thus, LRPR1 represents a primary response gene in FSH action on Sertoli cells. The presently available data indicate that LRPR1 mRNA expression is regulated specifically by FSH, since several other hormones and growth factors did not affect LRPR1 mRNA expression in the cultured Sertoli cells. LRPR1 mRNA expression is relatively high in testis, ovary and spleen. A much lower mRNA level was found in brain and lung, and no expression was detected in liver, kidney, heart, muscle, pituitary gland, prostate, epididymis and seminal vesicle. The basal level of testicular LRPR1 expression in intact 21-day-old rats was markedly increased within several hours after a single i.p. injection of FSH, indicating that in vivo LRPR1 mRNA expression may appear to be a useful parameter to evaluate testicular FSH action

Three-color dSTORM Imaging and Analysis of Recombination Foci in Mouse Spread Meiotic Nuclei

During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience.</p

Female Meiotic Sex Chromosome Inactivation in Chicken

During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI) leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW), whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs) may contribute to silencing of Z. Surprisingly, γH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of γH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses γH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis

Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein

In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the alpha-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of beta B-subunit mRNA is not increased after FSH treatment of the cells, and the ratio between bioactive and immunoactive inhibin decreases after stimulation with FSH. These data suggest that the beta B-subunit is the limiting factor in the production of bioactive inhibin. The aim of the present experiments was to investigate the effect of changes in the amount of beta B-subunit mRNA on the production of bioactive and immunoreactive inhibin. During early postnatal testicular development, the relative amounts of the 4.2 kb and 3.5 kb mRNAs encoding the beta B-subunit of inhibin changed markedly. The meaning of this changing ratio between beta B-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of beta B-subunit mRNA correlated with the in vitro production of bioactive inhibin as published earlier. Prolonged stimulation of cultured Sertoli cells from 14-day-old rats with 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the beta B-subunit mRNAs, presumably by down-regulation of protein kinase C. A similar effect was obtained after addition of the calcium ionophore A23187. Concomitantly, a decreased production of bioactive inhibin was observed. Furthermore, Western blotting revealed that secr

A novel approach to differentiate rat embryonic stem cells in vitro reveals a role for RNF12 in activation of X chromosome inactivation

textabstractX chromosome inactivation (XCI) is a mammalian specific, developmentally regulated process relying on several mechanisms including antisense transcription, non-coding RNA-mediated silencing, and recruitment of chromatin remodeling complexes. In vitro modeling of XCI, through differentiation of embryonic stem cells (ESCs), provides a powerful tool to study the dynamics of XCI, overcoming the need for embryos, and facilitating genetic modification of key regulatory players. However, to date, robust initiation of XCI in vitro has been mostly limited to mouse pluripotent stem cells. Here, we adapted existing protocols to establish a novel monolayer differentiation protocol for rat ESCs to study XCI. We show that differentiating rat ESCs properly downregulate pluripotency factor genes, and present female specific Xist RNA accumulation and silencing of X-linked genes. We also demonstrate that RNF12 seems to be an important player in regulation of initiation of XCI in rat, acting as an Xist activator. Our work provides the basis to investigate the mechanisms directing the XCI process in a model organism different from the mouse

Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene

Multi-color dSTORM microscopy in Hormad1^-/spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy, and a mouse model with severe defects in meiotic DSB formation and synapsis (Hormad1-/-) to obtain more insight in the recombinase accumulation patterns in relation to repair progression. First, we used the known reduction in meiotic DSB frequency in Hormad1-/- spermatocytes to be able to conclude that the RAD51/DMC1 nanofoci that preferentially localize at distances of ~300 nm form within a single DSB site, whereas a second preferred distance of ~900 nm, observed only in wild type, represents inter-DSB distance. Next, we asked whether the proposed role of HORMAD1 in repair inhibition affects the RAD51/DMC1 accumulation patterns. We observed that the two most frequent recombinase configurations (1 DMC1 and 1 RAD51 nanofocus (D1R1), and D2R1) display coupled frequency dynamics over time in wild type, but were constant in the Hormad1-/- model, indicating that the lifetime of these intermediates was altered. Recombinase nanofoci were also smaller in Hormad1-/- spermatocytes, consistent with changes in ssDNA length or protein accumulation. Furthermore, we established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. Finally, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting coil formation in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. Using this large super-resolution dataset, we propose models with the D1R1 configuration representing one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or both ends loaded by the two recombinases, but in below-resolution proximity. This may then often evolve into D2R1, then D1R2, and finally back to D1R1, when DNA synthesis has commenced.</p

The ubiquitin-conjugating enzyme HR6B is required for maintenance of X chromosome silencing in mouse spermatocytes and spermatids

<p>Abstract</p> <p>Background</p> <p>The ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids.</p> <p>Results</p> <p>HR6B is enriched on the XY body and on centromeric regions in pachytene spermatocytes. Global gene expression analyses revealed that spermatid-specific single- and multicopy X-linked genes are prematurely expressed in <it>Hr6b </it>knockout spermatocytes. Very few other differences in gene expression were observed in these cells, except for upregulation of major satellite repeat transcription. In contrast, in <it>Hr6b </it>knockout spermatids, 7298 genes were differentially expressed; 65% of these genes was downregulated, but we observed a global upregulation of gene transcription from the X chromosome. In wild type spermatids, approximately 20% of the single-copy X-linked genes reach an average expression level that is similar to the average expression from autosomes.</p> <p>Conclusions</p> <p>Spermatids maintain an enrichment of repressive chromatin marks on the X chromosome, originating from meiotic prophase, but this does not interfere with transcription of the single-copy X-linked genes that are reactivated or specifically activated in spermatids. HR6B represses major satellite repeat transcription in spermatocytes, and functions in the maintenance of X chromosome silencing in spermatocytes and spermatids. It is discussed that these functions involve modification of chromatin structure, possibly including H2B ubiquitylation.</p

A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the mullerian duct

The activin and TGF-beta type II receptors are members of a separate subfamily of transmembrane receptors with intrinsic protein kinase activity, wh

Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase.

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatc