55 research outputs found
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
Regulation of gonadotropin receptor gene expression
The receptors for the gonadotropins differ from the other G protein-coupled receptors by having a large extracellular hormone-binding domain, encoded by nine or ten exons. Alternative splicing of the large pre-mRNA of approximately 100 kb can result in mRNA species that encode truncated receptor proteins. In this review we discuss the regulation of gonadotropin receptor mRNA expression and the possible roles of alternative splicing in gonadotropin receptor function
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
Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts
Glycoprotein hormone receptors contain a large extracellular domain that
is encoded by multiple exons, facilitating the possibility of expressing
alternatively spliced transcripts. We have cloned two new splice variants
of the rat follicle-stimulating hormone (FSH) receptor gene: FSH-R1 and
FSH-R2. The splice variant FSH-R1 differs from the full-length FSH
receptor mRNA by the inclusion of a small extra exon between exons 9 and
10. FSH-R2 lacks the first three base pairs o
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
Proteomic analysis of androgen-regulated protein expression in a mouse fetal vas deferens cell line
During sex differentiation, androgens are essential for development of the
male genital tract. The Wolffian duct is an androgen-sensitive target
tissue that develops into the epididymis, vas deferens, and seminal
vesicle. The present study aimed to identify androgen-regulated proteins
that are involved in development of Wolffian duct-derived structures. We
have used male mouse embryos transgenic for temperature-sensitive simian
virus 40 large tumor antigen at 18 d of gestation, to generate
immortalized mouse fetal vas deferens (MFVD) parental and clonal cell
lines. The MFVD parental and clonal cell lines express androgen receptor
protein and show features of Wolffian duct mesenchymal cells. Clonal cell
line MFVD A6 was selected for proteomic analysis and cultured in the
absence or presence of androgens. Subsequently, two-dimensional gel
electrophoresis was performed on total cell lysates. Differentially
expressed proteins were analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry and two
androgen-regulated proteins were identified as mElfin and CArG-binding
factor-A (CBF-A). CBF-A and mElfin are known to bind to cytoskeletal
F-actin. Both proteins appeared to be regulated by androgens at the
posttranslational level, possibly involving phosphorylation.
Posttranslational modification of mElfin and CBF-A by androgens may be
associated with a cytoskeletal change that is involved in
androgen-regulated gene expression
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
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
Phosporylation of androgen receptor isoforms
Phosphorylation of the human AR (androgen receptor) is directly correlated with the appearance of at least three AR isoforms on an SDS/polyacrylamide gel. However, it is still not clear to what extent phosphorylation is involved in the occurrence of isoforms, which sites are phosphorylated and what are the functions of these phosphosites. The human AR was expressed in COS-1 cells and AR phosphorylation was studied further by mutational analyses and by using reversed-phase HPLC and MS. The reversed-phase HPLC elution pattern of the three isoforms revealed that Ser-650 was phosphorylated constitutively. After de novo synthesis, only Ser-650 was phosphorylated in the smallest isoform of 110 kDa and both Ser-650 and Ser-94 were phosphorylated in the second isoform of 112 kDa. The hormone-induced 114 kDa isoform shows an overall increase in phosphorylation of all the isolated peptides. The activities of the Ser-Ala substitution mutant S650A (Ser-650-->Ala) was found to be identical with wild-type AR activation in four different cell lines and three different functional analyses, e.g. transactivation, N- and C-terminal-domain interaction and co-activation by transcriptional intermediary factor 2. This was also found for mutants S94A and S515A with respect to transactivation. However, the S515A mutation, which should eliminate phosphorylation of the potential mitogen-activated protein kinase site, Ser-515, resulted in an unphosphorylated form of the peptide containing Ser-650. This suggests that Ser-515 can modulate phosphorylation at another site. The present study shows that the AR isoform pattern from AR de novo synthesis is directly linked to differential phosphorylation of a distinct set of sites. After mutagenesis of these sites, no major change in functional activity of the AR was observed
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