28 research outputs found
Steroid biosynthesis and the brain-testis axis
The significanee of testicular tunetion was shown as
l early as 1849 by Berthold when he observed atrophy of the
comb in castrated cockerels, which could be restored by
implantation of a testis in the abdomen. It was only in
the beginning of the twentieth century, however, that
Bouin and Ancel postulated the formation of certain horrnonal
principles in distinct cell types of the testis. The
identification of testasterene as the biologically active
andregen of the bull testis in 1935 rnarked the beginning
of biochemical studies of testis function. The measurement
of testicular horrnonal products has been difficult for a
long time because only small quantities are produced.
However, present techniques such as radioimmunoassay permit
the measurement of picogram quantities of testosterone4.
Parallel with the development of these techniques,
insight has been gained into the endocrine function of the
testis
Implications of progesterone metabolism in MA-10 cells for accurate measurement of the rate of steroidogenesis
In virtually all studies with MA-10 cells, progesterone RIAs have been
used to measure steroid synthesis. To test whether progesterone is a
stable end product, we investigated the metabolism of added tritiated
progesterone and pregnenolone in MA-10 cells over a period of 3 h.
Steroids were then extracted, separated by HPLC, and identified by GC/MS.
We found that more than 70% of radiolabeled steroids were converted to at
least five different metabolites. A major metabolite (40%) was 5
alpha-pregnan-3 alpha or 3 beta-ol-20one. Similar studies, using
radiolabeled T, demonstrated conversion to dihydrotestosterone and two
forms of 5 alpha-androstane-diols. These data indicate the presence of
active 5 alpha-reductase and 3 alpha- and/or 3 beta-hydroxysteroid
dehydrogenase activities in MA-10 cells. Because these results suggest
that progesterone is an unstable end product, to gauge the level of active
metabolism, we incubated cells in the presence of inhibitors of
pregnenolone metabolism and assessed pregnenolone levels by RIA. We
discovered that basal levels of steroidogenesis in MA-10 cells were
considerably higher than previously estimated. Moreover, dibutyryl
cAMP-stimulated steroid production was linear over more than 13 h, in
contrast to previous findings that measured progesterone levels. Other
consequences of inaccurate assessment of steroidogenic activity in MA-10
cells because of the application of the progesterone assay are discussed
Low levels of follicle-stimulating hormone receptor-activation inhibitors in serum and follicular fluid from normal controls and anovulatory patients with or without polycystic ovary syndrome
In patients with normogonadotropic anovulation, either with or without
polycystic ovary syndrome (PCOS), factors interfering with FSH action may
be involved in arrested follicle development. The aim of this study is to
assess whether factors inhibiting FSH receptor activation are elevated in
serum or follicular fluid from anovulatory patients, as compared with
regularly cycling women. For this purpose, a Chinese hamster ovary cell
line, stably transfected with the human FSH receptor, has been applied.
FSH-stimulated cAMP secretion in culture medium was measured in the
presence of serum or follicular fluid. Chinese hamster ovary cells were
stimulated with a fixed concentration of FSH (3 or 6 mIU/mL) to mimic FSH
levels in serum or follicular fluid. Samples were added in concentrations
ranging from 3-90% vol/vol to approach protein concentrations occurring in
serum or follicular fluid. In the presence of 10% vol/vol serum from
regularly cycling women (n = 8), FSH-stimulated cAMP production was
inhibited to 42 +/- 2% (mean +/- SEM of 2 experiments, each performed in
duplicate) of cAMP production in the absence of serum, whereas a similar
cAMP level (up to 38 +/- 4% of the serum-free level) was observed at
higher concentrations of serum (30-90% vol/vol). The inhibition of
FSH-stimulated cAMP production in the presence of serum samples from
normogonadotropic anovulatory patients, without (n = 13) or with (n = 16)
PCOS, was similar to controls. Follicular fluid samples (n = 57) obtained
during the follicular phase in 25 regularly cycling women and follicular
fluid samples (n = 25) from 5 PCOS patients were tested in a slightly
modified assay system. In the presence of 10 or 30% (vol/vol) follicular
fluid, FSH-stimulated cAMP levels were decreased to 68 +/- 2% and 55 +/-
2% (mean +/- SEM of a single experiment in triplicate) of the cAMP levels
in the absence of follicular fluid, respectively. There was no correlation
between the degree of cAMP inhibition and follicle size, steroid content
(androstenedione or estradiol concentrations), or menstrual cycle phase.
Furthermore, no differences in inhibition were found, comparing PCOS
follicles with size- and steroid content-matched follicles obtained during
the normal follicular phase. It is concluded that inhibition of FSH
receptor activation by proteins present in serum or follicular fluid is
constant (60 and 40%, respectively) and independent from the developmental
stage of the follicle, either during the normal follicular phase or in
patients with normogonadotropic anovulation. Inhibition of FSH recepto
Specific dose-dependent effects of ethane 1,2-dimethanesulfonate in rat and mouse Leydig cells and non-steroidogenic cells on programmed cell death
The mechanism by which ethane 1,2-dimethanesulfonate (EDS) selectively
kills Leydig cells is poorly understood. To characterize further the
cell-specific actions of EDS, we studied biochemical and morphological
changes during apoptosis in different Leydig cell and non-steroidogenic
cell models.Rat testicular and H540 tumor Leydig cells were killed by 1-2
mM EDS, whereas 20 mM EDS were required for MA-10 cells. This higher
concentration of EDS was also necessary for activation of apoptosis in
non-steroidogenic Chinese hamster ovary cells, whereas COS-1 monkey kidney
cells were resistant. These variable effects of EDS on apoptosis were
independent of new protein synthesis and, interestingly, could be delayed
by co-incubation with dibutyrl cyclic AMP. Along with cell death, we also
observed chromosomal fragmentation and other hallmarks indicative of
apoptosis as evidenced by DNA laddering and fluorescent microscopy.
Time-lapse photography with a confocal microscope showed that the time of
onset, duration and even the sequence of apoptotic events between
individual H540 cells was heterogeneous. When the dose of EDS was
gradually increased from 2 to 10 mM, the proportion of cells showing
normal apoptotic features gradually decreased. Intriguingly, treatment
with 10 mM EDS did not result in death for most cells and was marked by an
absence of DNA laddering and ultrastructural features of apoptosis and
necrosis. However, incubation with 20 mM EDS resulted in necrosis.These
results demonstrated that the effects of EDS on cell survival are not
specific to Leydig cells, that different cell types have different
sensitivities to EDS and that stimulation of the cAMP pathway may mitigate
EDS action. The data obtained with H540 cells further revealed that EDS
can induce two types of programmed cell death
Short-term stimulatory effect of Sertoli cell conditioned medium on Leydig cell steroidogenesis is not mediated by inhibin
Abstract
Addition of concentrated rat Sertoli cell conditioned medium (rSCCM) to isolated Leydig cells from immature rats stimulated steroid production more than 13-fold within 4h. LH-stimulated steroidogenesis was not enhanced by addition of rSCCM. The biological activity of the concentrated rSCCM was higher after incubation of Sertoli cells with FSH, whereas FSH alone did not stimulate steroid production. This effect of rSCCM was not due to inhibin, since highly purified 32 kDa rat inhibin, in doses equivalent to those present in rSCCM, had no effect on steroidogenesis during the 4 h incubation period. Furthermore, inhibin could be separated from the Leydig cell stimulating factor by anion-exchange chromatography. These results indicate a short-term paracrine control of Leydig cell steroidogenesis by Sertoli cell derived factors, which differ from inhibin
Calcium confusion--is the variability in calcium response by Sertoli cells to specific hormones meaningful or simply redundant?
When results of more than ten different studies on hormone-induced calcium
signals in Sertoli cells are taken together, a wide variety of responses
emerges. The reported changes range from increased concentrations, via no
response at all, to decreased calcium concentrations. Minor variations in
cell isolation techniques, culture conditions, or techniques for measuring
the intracellular calcium could explain some of these differences.
However, erratic variations in response are also observed within research
groups under very similar experimental conditions. Such 'negative'
findings are mainly reported orally and do not further penetrate the
scientific community. As hormone-dependent calcium responses evidently may
depend very much on the context of the cells, calcium transients would
appear to be unreliable bioassay principles with which to detect the
primary actions of FSH and effectors such as androgens on Sertoli cells. A
more important biological question is whether these sometimes opposed
calcium transients are connected with a particular cellular response. To
date there is no evidence for such a tight coupling in Sertoli cells,
implying that, at least under in vitro conditions, calcium signals might
even be redundant altogether. Such calcium variability is probably not
unique to Sertoli cells, and the aim of this commentary is to promote an
open debate that may help to transform the current state of 'calcium
confusion' into a better understanding of the intracellular calcium
language
The stimulatory effect of albumin on luteinizing hormone-stimulated Leydig cell steroid production depends on its fatty acid content and correlates with conformational changes
__Abstract__
The effects of purified albumin species and albumin fragments (0.2–1% w/v) on short-term (4 h) steroid secretion by immature rat Leydig cells, in the presence of a maximally stimulating dose of luteinizing hormone (LH), were investigated. Human albumin and the peptic fragment (comprising residues 1–387) enhanced pregnenolone production in isolated rat Leydig cells, whereas chicken albumin and the tryptic fragment (comprising residues 198–585) were not active.
This stimulatory effect of human albumin and the peptic fragment correlated with the potential of these proteins to undergo a pH-dependent neutral-to-base transition as measured by circular dichroism. The tryptic fragment and chicken albumin did not have the potential to undergo such a transition. The pH-dependent conformational changes of albumin and fragments thereof occurred in parallel with a change in the binding affinity for testosterone and pregnenolone.
The fatty acid oleic acid and the drug suramin, only when present in a molar ligand-to-albumin ratio equal to or higher than 2, inhibited the albumin-mediated stimulation of steroid production.
These data show that the stimulatory effects of albumin species on LH-induced Leydig cell pregnenolone production depend on their fatty acid content and correlate with the potential of these molecules to undergo conformational changes. It is unknown via which mechanisms albumin exerts its stimulatory effect, but the LH action through the cyclic AMP pathway seems not to be affected
Rapid Androgen Actions on Calcium Signaling in Rat Sertoli Cells and Two Human Prostatic Cell Lines: Similar Biphasic Responses Between 1 Picomolar and 100 Nanomolar Concentrations1
Are Polyphosphorylated Phospholipids Involved in the Hormonal Control of Cholesterol Side-Chain Cleavage Activity in Tumour Leydig Cells?
The possible role of LH or dcAMP induced changes in polyphosphorylated phospholipid metabolism in the regulation of cholesterol side-chain cleavage activity has been studied in tumour Leydig cells. Mitochondria isolated from LH-stimulated Leydig cells were 400% more active in pregnenolone production than mitochondria from control cells. Steroid production in isolated mitochondria from control cells could be stimulated only 25% by cytosol fractions from stimulated cells and 100 µM phosphatidyl inositol-4’-phosphate (PtdIns4P). Other polyphosphorylated phospholipids were either inactive or showed aspecific effects. During a preincubation period tumour cells were labelled with [32P]phosphate and steady-state labelling was obtained for the pholyphosphorylated phospholipids after 40-60 min. [32P]Phosphate incorporation in Ptd Ins4P, phosphatidyl inositol (PtdIns), phosphatidyl choline (PtChl), phosphatidyl ethanolamine (PtdEtn) and cardiolipin (CL) was not affected by treatment of the Leydig cells with LH which stimulated (6-fold), or with cycloheximide which suppressed (4-fold) steroid production. A 25% increase of phosphate incorporation by LH was observed only in phosphatidyl inositol-4',5'-biphosphate (PtdIns4,5P2). 32P Incorporation in PtdIns4,5P2, PtdIns, PtdEtn and CL was stimulated by quinacrine 50 µM. Under these conditions the LH-stimulated pregnenolone production but not the 25-hydroxycholesterol dependent pregnenolone production, was completely inhibited. The results obtained with isolated mitochondria and intact cells indicate that increased levels of polyphosphorylated phospholipids are not consistently correlated with increased mitochondrial pregnenolone production. This argues against an important role of polyphosphorylated pohospholipids in the hormonal regulation of cholesterol side-chain cleavage activity in tumour Leydig cells.