161 research outputs found
Hormonal control of spermatogenesis: expression of FSJH receptor and androgen receptor genes
FSH and testosterone are the main hormonal regulators of spermatogenesis. The actions of
androgens and FSH are mediated by their respective receptors. Receptor gene expression
(mRNA and protein). is an important determinant of hormone action. Biochemical aspects of
the regulation of androgen and FSH receptor gene expression in the testis were chosen as
the subject of the studies described in this thesis. Regulation of the expression of the receptor
genes was studied at the level of gene transcription, and at the level of mRNA and protein
expression.
In Chapters 2-4, a detailed characterization is given of the effects of FSH on androgen
and FSH receptor mRNA and protein expression in cultured immature Sertoli cells. For the
androgen receptor, these findings were extended by measurements of androgen receptor
gene transcription initiation rate in cultured immature Sertoli cells and LNCaP Oymph node
carcinoma of the prostate) cells (Chapter 5).
Preliminary results concerning a putative paracrine factor, produced by Sertoli cells and
affecting androgen receptor mRNA expression in peritubular myoid cells, are presented in Chapter 6.
The effects of testosterone deprivation iD. vivo on androgen receptor mRNA and protein
expression in the adult rat testis were examined as described in Chapter 7.ffi vitro effects of
testosterone on androgen receptor gene expression in cultured testicular cells and LNCaP
cells are described in the Chapters 2, 3 and 5.
In the General Discussion (Chapter 8) we have considered some aspects of regulation
of spermatogenesis by FSH and testosterone and have discussed them in relation to our
experimental data as well as in a broader perspective. This way, we hope that the results
which we have presented, and discussions which we have tried to initiate, may contribute to
research concerning hormonal control of spermatogenesis, now and in the futur
Wnt/Β-Catenin and Sex Hormone Signaling In Endometrial Homeostasis and Cancer
A delicate balance between estrogen and progestagen signaling underlies proper functioning of the female reproductive tract and, in particular, the monthly re- and degenerative phases characteristic of the menstrual cycle. Here, we propose that the canonical Wnt/β-catenin signaling pathway may underlie this finely tuned hormonal equilibrium in endometrial homeostasis and, upon its constitutive activation, lead to neoplastic transformation of the endometrium. During the menstrual cycle, estradiol will enhance Wnt/β-catenin signaling in the proliferative phase, while progesterone inhibits Wnt/β-catenin signaling, thus restraining estrogens' proliferative actions, during the secretory phase. In case of enhanced or unopposed estrogen signaling, constitutive activation of Wnt/β-catenin signaling will trigger endometrial hyperplasia, which may develop further into endometrial cancer
Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP
Elevation of intracellular calcium levels in the presence of normal
androgen levels has been implicated in apoptotic prostate cell death.
Since the androgen receptor (AR) plays a critical role in the regulation
of growth and differentiation of the prostate, it was of interest to
determine whether Ca2+ would affect the expression of androgen receptor
messenger RNA (mRNA) and protein, thus affecting the ability of androgens
to control prostate function. AR-positive human prostate cancer cells,
LNCaP, were incubated with either the calcium ionophore A23187 or the
intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin.
Subsequently, AR mRNA and protein levels were assessed by Northern and
Western blot analysis. Both A23187 and thapsigargin were found to
down-regulate steady state AR mRNA levels in a time- and dose-dependent
manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M
A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of
incubation, respectively. In contrast, control mRNA (glyceraldehyde
3-phosphate dehydrogenase) did not change significantly during the
treatments with either A23187 or thapsigargin. AR protein levels were
found to be decreased after 12 h of incubation with either 10(-6) M A23187
or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to
precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was
found to alter cell morphology within the treatment time. Cycloheximide
and actinomycin D were unable to change the calcium-mediated decrease in
AR mRNA, ruling out the necessity for de novo protein synthesis or a
change in mRNA stability. Moreover, the decrease in AR mRNA induced by
calcium does not seem to involve protein kinase C- or calmodulin-dependent
pathways, since inhibitors of these cellular components had no effect.
Nuclear run-on assays demonstrated little or no effects of either A23187
or thapsigargin treatment on AR gene transcription (8 h and 10 h). In
conclusion, these studies show that intracellular calcium seems to be a
potent regulator of AR gene expression in LNCaP cells
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
Activin is produced by rat Sertoli cells in vitro and can act as an autocrine regulator of Sertoli cell function
Regulation of androgen receptor (AR) mRNA expression was studied in
Sertoli cells and peritubular myoid cells isolated from immature rat
testis, and in the lymph node carcinoma cell line derived from a human
prostate (LNCaP). Addition of dibutyryl-cyclic AMP (dbcAMP) to Sertoli
cell cultures resulted in a rapid transient decrease in AR mRNA expression
(5 h), which was followed by a gradual increase in AR mRNA expression
(24-72 h). This effect of dbcAMP mimicked follicle-stimulating hormone
(FSH) action. In peritubular myoid cells, there was only a moderate but
prolonged decrease during incubation in the presence of dbcAMP, and in
LNCaP cells no effect of dbcAMP on AR mRNA expression was observed. When
Sertoli cells or peritubular myoid cells were cultured in the presence of
androgens, AR mRNA expression in these cell types did not change. This is
in contrast to LNCaP cells, that showed a marked reduction of AR mRNA
expression during androgen treatment. In the present experiments,
transcriptional regulation of AR gene expression in Sertoli cells and
LNCaP cells was also examined. Freshly isolated Sertoli cell clusters were
transfected with a series of luciferase reporter gene constructs, driven
by the AR promoter. It was found that addition of dbcAMP to the
transfected Sertoli cells resulted in a small but consistent increase in
reporter gene expression (which was interpreted as resulting from AR
promoter activity); a construct that only contained the AR 5' untranslated
region of the cDNA sequence did not show such a regulation. The same
constructs, transfected into LNCaP cells, did not show any transcriptional
down-regulation when the synthetic androgen R1881 was added to the cell
cultures. A nuclear transcription elongation experiment (run-on), however,
demonstrated that androgen-induced AR mRNA down-regulation in LNCaP cells
resulted from an inhibition of AR gene transcription. The present results
indicate that in Sertoli cells and LNCaP cells, hormonal effects on AR
gene transcription play a role in regulation of AR expression. However, AR
gene transcription in these cells is differentially regulated
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
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