Anti-Müllerian 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

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

Transcriptional regulation of androgen receptor gene expression in Sertoli cells and other cell types

Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cell

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

The rat androgen receptor gene promoter

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. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcriptio

Repair of exogenous DNA double-strand breaks promotes chromosome synapsis in SPO11-mutant mouse meiocytes, and is altered in the absence of HORMAD1

Repair of SPO11-dependent DNA double-strand breaks (DSBs) via homologous recombination (HR) is essential for stable homologous chromosome pairing and synapsis during meiotic prophase. Here, we induced radiation-induced DSBs to study meiotic recombination and homologous chromosome pairing in mouse meiocytes in the absence of SPO11 activity (Spo11YF/YF model), and in the absence of both SPO11 and HORMAD1 (Spo11/Hormad1 dko). Within 30 min after 5 Gy irradiation of Spo11YF/YF mice, 140–160 DSB repair foci were detected, which specifically localized to the synaptonemal complex axes. Repair of radiation-induced DSBs was incomplete in Spo11YF/YF compared to Spo11+/YF meiocytes. Still, repair of exogenous DSBs promoted partial recovery of chromosome pairing and synapsis in Spo11YF/YF meiocytes. This indicates that at least part of the exogenous DSBs can be processed in an interhomolog recombination repair pathway. Interestingly, in a seperate experiment, using 3 Gy of irradiation, we observed that Spo11/Hormad1 dko spermatocytes contained fewer remaining DSB repair foci at 48 h after irradiation compared to irradiated Spo11 knockout spermatocytes. Together, these results show that recruitment of exogenous DSBs to the synaptonemal complex, in conjunction with repair of exogenous DSBs via the homologous chromosome, contributes to homology recognition. In addition, the data suggest a role for HORMAD1 in DNA repair pathway choice in mouse meiocytes

Live cell analyses of synaptonemal complex dynamics and chromosome movements in cultured mouse testis tubules and embryonic ovaries

During mammalian meiotic prophase, homologous chromosomes connect through the formation of the synaptonemal complex (SC). SYCP3 is a component of the lateral elements of the SC. We have generated transgenic mice expressing N- or C-terminal fluorescent-tagged SYCP3 (mCherry-SYCP3 (CSYCP) and SYCP3-mCherry (SYCPC)) to study SC dynamics and chromosome movements in vivo. Neither transgene rescued meiotic aberrations in Sycp3 knockouts, but CSYCP could form short axial element-like structures in the absence of endogenous SYCP3. On the wild-type background, both fusion proteins localized to the axes of the SC together with endogenous SYCP3, albeit with delayed initiation (from pachytene) in spermatocytes. Around 40% of CSYCP and SYCPC that accumulated on the SC was rapidly exchanging with other tagged proteins, as analyzed by fluorescent recovery after photobleaching (FRAP) assay. We used the CSYCP transgenic mice for further live cell analyses and observed synchronized bouquet configurations in living cysts of two or three zygotene oocyte nuclei expressing CSYCP, which presented cycles of telomere clustering and dissolution. Rapid chromosome movements were observed in both zygotene oocytes and pachytene spermatocytes, but rotational movements of the nucleus were more clear in oocytes. In diplotene spermatocytes, desynapsis was found to proceed in a discontinuous manner, whereby even brief chromosome re-association events were observed. Thus, this live imaging approach can be used to follow changes in the dynamic behavior of the nucleus and chromatin, in normal mice and different infertile mouse models

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