165 research outputs found
Anti-MΓΌllerian hormone and androgens: regulation of receptors during sex differentiation and gonadal development
This chapter gives an outline of sex determination, sex differentiation, and gonadal
development in mammalian species. In most studies described herein, rats and mice
were used.
During embryonal development in mammals, sex differentiation is preceded by a
bipotential stage. Indifferent gonads are formed that can develop into eilher testes or
ovaries. The anlagen of the male and female intemal genitalia, which are both present
in embryos of either chromosomal sex, are called the wolffian and the mullerian ducts,
respectively
DNA repair mechanisms and gametogenesis
In mammals, there is a complex and intriguing relationship between DNA
repair and gametogenesis. DNA repair mechanisms are involved not only in
the repair of different types of DNA damage in developing germline cells,
but also take part in the meiotic recombination process. Furthermore, the
DNA repair mechanisms should tolerate mutations occurring during
gametogenesis, to a limited extent. In the present review, several
gametogenic aspects of DNA mismatch repair, homologous recombination
repair and postreplication repair are discussed. In addition, the role of
DNA damage-induced cell cycle checkpoint control is considered briefly. It
appears that many genes encoding proteins that take part in DNA repair
mechanisms show enhanced or specialized expression during mammalian
gametogenesis, and several gene knockout mouse models show male or female
infertility. On the basis of such knowledge and models, future experiments
may provide more information about the precise relationship between DNA
repair, chromatin dynamics, and genomic stability versus instability
during gametogenesis
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, Ξ³H2AX, 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 Ξ³H2AX 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 Ξ³H2AX 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
- β¦