Hormones and the control of biochemical processes in spermatogenesis

The aim of the experiments described in this thesis was to contribute to a better understanding of the relationship, in molecular terms, between hormones and the development of male germinal cells in rats. In this introduction we will discuss some important aspects of spermatogenesis, the hormonal control of spermatogenesis, and the aim and scope of the present thesis

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

Hormonal control of gubernaculum development during testis descent: gubernaculum outgrowth in vitro requires both insulin-like factor and androgen

The gubernaculum connects the gonad to the inguinoscrotal region and is involved in testis descent. It rapidly develops in the male fetus, whereas development in the female fetus is lacking. Possible factors involved in gubernaculum development are androgens, anti-Mullerian hormone (AMH), and insulin-like factor (Insl3). Sexual dimorphism in gubernaculum development correlated with the mitotic activity of cells in the gubernacular bulbs from male and female fetuses. Androgen receptor expression was restricted to the mesenchymal core of the gubernacular bulb, whereas skeletal muscle was detected in its outer layer. In an organ culture system devised to further study gubernaculum development in vitro, morphology of gubernacular explants grown in the presence of testes was comparable with that of gubernacula developed in vivo. Testicular tissue or medium containing R1881, a synthetic androgen, had a growth stimulatory effect on gubernacular explants compared with ovarian tissue or basal medium only. Moreover, Amh-/-, Amh+/-, and Insl3+/- testes stimulated the growth of gubernacular explants to the same extent as control testes. Insl3-/- testes, however, did not produce such an activity. This study reveals an essential role for both androgen and Insl3 in the gubernaculum outgrowth during transabdominal testis descent

Regulation of inhibin βB-subunit mRNA expression in rat Sertoli cells: Consequences for the production of bioactive and immunoreactive inhibin

Abstract In Sertoli cells from 21-day-old rats, the expression of the mRNA encoding the α-subunit of inhibin, and the production of immunoreactive inhibin are stimulated by follicle-stimulating hormone (FSH). In contrast, the amount of βB-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 βB-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 βB-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 βB-subunit of inhibin changed markedly. The meaning of this changing ratio between βB-subunit mRNAs is not clear, since both mRNAs are actively translated, as demonstrated by polysomal analysis. The total amount of βB-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 4β-phorbol 12-myristate 13-acetate (PMA) caused a decreased expression of the βB-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 secretion of the 32 kDa inhibin αβ-dimer was decreased, whereas secretion of the combination of the C-terminal part with the pro-region of the α-subunit was increased. It is concluded that the level of the βB-subunit of inhibin is rate-limiting for the production of bioactive inhibin in cultured Sertoli cells, and that its expression can be influenced by modulation of protein kinase C, and/or intracellular calcium levels

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