scholarly journals Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 851-858 ◽  
Author(s):  
P J O'Shaughnessy ◽  
I D Morris ◽  
P J Baker
Keyword(s):  
Cytochrome P450 ◽  
Cell Signaling ◽  
Germ Cell ◽  
Cell Population ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Signaling Molecules ◽  
Generation Process ◽  
Peritubular Cells

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3β-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17β-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place.

scholarly journals Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary–gonadal axis and Leydig cell steroidogenesis

2005 ◽  
Vol 187 (1) ◽  
pp. 117-124 ◽  
Author(s):  
K Svechnikov ◽  
V Supornsilchai ◽  
M-L Strand ◽  
A Wahlgren ◽  
D Seidlova-Wuttke ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Serum Level ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Ex Vivo ◽  
Elevated Serum ◽  
Seminiferous Tubules ◽  
Dibutyryl Camp

Procymidone is a fungicide with anti-androgenic properties, widely used to protect fruits from fungal infection. Thereby it contaminates fruit products prepared for human consumption. Genistein-containing soy products are increasingly used as food additives with health-promoting properties. Therefore we examined the effects of long-term dietary administration (3 months) of the anti-androgen procymidone (26.4 mg/animal per day) or the phytoestrogen genistein (21.1 mg/animal per day) to rats on the pituitary-gonadal axis in vivo, as well as on Leydig cell steroidogenesis and on spermatogenesis ex vivo. The procymidone-containing diet elevated serum levels of LH and testosterone and, furthermore, Leydig cells isolated from procymidone-treated animals displayed an enhanced capacity for producing testosterone in response to stimulation by hCG or dibutyryl cAMP, as well as elevated expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450 scc) and cytochrome P450 17α (P450c17). In contrast, the rate of DNA synthesis during stages VIII and IX of spermatogenesis in segments of seminiferous tubules isolated from genistein-treated rats was decreased without accompanying changes in the serum level of either LH or testosterone. Nonetheless, genistein did suppress the ex vivo steroidogenic response of Leydig cells to hCG or dibutyryl cAMP by down-regulating their expression of P450 scc. Considered together, our present findings demonstrate that long-term dietary administration of procymidone or genistein to rats exerts different effects on the pituitary–gonadal axis in vivo and on Leydig cell steroidogenesis ex vivo. Possibly as a result of disruption of hormonal feedback control due to its anti-androgenic action, procymidone activates this endocrine axis, thereby causing hyper-gonadotropic activation of testicular steroidogenesis. In contrast, genistein influences spermatogenesis and significantly inhibits Leydig cell steroidogenesis ex vivo without altering the serum level of either LH or testosterone.

scholarly journals Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads

2001 ◽  
Vol 171 (2) ◽  
pp. 373-383 ◽  
Author(s):  
G Pelletier ◽  
S Li ◽  
V Luu-The ◽  
Y Tremblay ◽  
A Belanger ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Steroid Hormones ◽  
Adrenal Cortex ◽  
Leydig Cells ◽  
Ultrastructural Localization ◽  
Hydroxysteroid Dehydrogenase ◽  
Cytochrome P450 Enzymes ◽  
Steroidogenic Enzymes ◽  
Adrenocortical Cells ◽  
Theca Interna

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

scholarly journals Regulation of cytochrome P450 aromatase gene expression in adult rat Leydig cells: comparison with estradiol production

2001 ◽  
Vol 168 (1) ◽  
pp. 95-105 ◽  
Author(s):  
C Genissel ◽  
J Levallet ◽  
S Carreau
Keyword(s):  
Gene Expression ◽  
Cytochrome P450 ◽  
Cyclic Amp ◽  
Leydig Cell ◽  
Half Life ◽  
Leydig Cells ◽  
Aromatase Activity ◽  
Mrna Levels ◽  
Low Concentrations ◽  
Cytochrome P450 Aromatase

Regulation of aromatase gene expression in purified rat Leydig cells has not yet been investigated. Therefore, using a highly specific quantitative RT-PCR method, we have measured the amount of cytochrome P450 aromatase (P450arom) mRNA and aromatase activity in mature rat Leydig cells submitted to various treatments during 24 h. Estradiol production was enhanced in a dose-related manner in the presence of testosterone, the maximum (28% increase) being obtained with 200 ng/ml. Related to the P450arom mRNA levels, a decrease was observed in the presence of low concentrations (50 and 100 ng/ml) of testosterone, then a 20% increase of the amount of transcripts was recorded for the higher concentrations (200-500 ng/ml). The same result was obtained in the presence of 5alpha-dihydrotestosterone (an androgen resistant to aromatase activity). The addition of ovine LH (oLH; 0.1-50 ng/ml) to the Leydig cell culture medium induced a dose-related augmentation of estradiol output up to 10 ng/ml oLH, although a decrease was observed with 50 ng/ml when compared with maximal values. mRNA levels slightly decreased in the presence of low concentrations (0.1-1 ng/ml) of oLH, an effect that was abolished by the addition of testosterone; mRNA levels were increased by oLH (5-10 ng/ml) 35 and 75% respectively in the absence and presence of testosterone (when compared with Leydig cells incubated without treatment). With 50 ng/ml oLH, a large augmentation (twofold) of the P450arom mRNA level either without or with testosterone was observed. Dibutyryl cyclic AMP (1 mM) mimicked the effect of oLH. The half-life of the P450arom mRNAs was twofold increased in the presence of testosterone and oLH when compared with the half-life in the absence of treatment (5.8+/-0.6 h). Taken together, our data have demonstrated that, in freshly isolated Leydig cells from mature rat testes, the regulation of aromatase expression and enzymatic activity is under LH (through cyclic AMP) and steroid control; moreover seminiferous tubule-secreted factor(s) are also involved. Therefore, rat Leydig cell aromatase is controlled at both transcriptional and post-transcriptional steps by endocrine and/or locally produced modulators.

scholarly journals 11-Ketotestosterone Is a Major Androgen Produced in Human Gonads

2016 ◽  
Vol 101 (10) ◽  
pp. 3582-3591 ◽  
Author(s):  
Yoshitaka Imamichi ◽  
Koh-ichi Yuhki ◽  
Makoto Orisaka ◽  
Takeshi Kitano ◽  
Kuniaki Mukai ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Cytochrome P450 ◽  
Androgen Receptor ◽  
Leydig Cells ◽  
Plasma Concentrations ◽  
Hydroxysteroid Dehydrogenase ◽  
Reproductive Age ◽  
Luciferase Reporter ◽  
Mcf 7

Context: 11-ketotestosterone (11-KT) is a novel class of active androgen. However, the detail of its synthesis remains unknown for humans. Objective: The objective of this study was to clarify the production and properties of 11-KT in human. Design, Participants, and Methods: Expression of cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 (key enzymes involved in the synthesis of 11-KT) were investigated in human gonads. The production of 11-KT was investigated in Leydig cells. Plasma concentrations of testosterone and 11-KT were measured in 10 women and 10 men of reproductive age. Investigation of its properties was performed using breast cancer-derived MCF-7 cells. Results: Cytochrome P450 and 11β-hydroxysteroid dehydrogenase types 1 and 2 were detected in Leydig cells and theca cells. Leydig cells produced 11-KT, and relatively high levels of plasma 11-KT were measured in both men and women. There was no sexual dimorphism in the plasma levels of 11-KT, even though testosterone levels were more than 20 times higher in men than in women. It is noteworthy that the levels of testosterone and 11-KT were similar in women. In a luciferase reporter system, 11-KT activated human androgen receptor-mediated transactivation. Conversely, 11-KT did not activate estrogen receptor-mediated transactivation in aromatase-expressed MCF-7 cells, whereas testosterone did following conversion to estrogen. 11-KT did not affect the estrogen/estrogen receptor -mediated cell proliferation of MCF-7 cells. Furthermore, it significantly inhibited cell proliferation when androgen receptor was transfected into MCF-7 cells. Conclusions: The current study indicates that 11-KT is produced in the gonads and represents a major androgen in human. It can potentially serve as a nonaromatizable androgen.

Some histochemical and ultrastructural observations on the early foetal pig testis

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 261-277
Author(s):  
C. J. A. H. V. van Vorstenbosch ◽  
C. M. J. E. van Rossum-Kok ◽  
B. Colenbrander ◽  
C. J. G. Wensing
Keyword(s):  
Endoplasmic Reticulum ◽  
Leydig Cell ◽  
Sertoli Cells ◽  
Leydig Cells ◽  
Tunica Albuginea ◽  
Hydroxysteroid Dehydrogenase ◽  
Mesenchymal Cells ◽  
Mitochondrial Complex ◽  
Sustentacular Cells

Testes of foetal pigs between 26 to 35 days post coitum (p.c.) were investigated histochemically and ultrastructurally. Diaphorase and Δ5-3β-hydroxysteroid dehydrogenase activities were studied using, respectively, NADH and pregnenolone and dihydroxy androsterone as substrates. Ultrastructurally, attention was focused on the development of mesenchymal cells and on the sustentacular cells in the primitive sex cords in an attempt to detect the origin of Ley dig cells. Histochemically there is a concentration of activity toward the interstitium with increasing age. Also the reactions increase in intensity. Ultrastructurally no evidence for Leydig cell development from Sertoli cells could be observed. Mesenchymal cells between the sex cords show a development toward Leydig cells. This is absent in mesenchymal cells in the future tunica albuginea. Before 30 days p.c. no ‘true’ Leydig cells can be observed morphologically. The role of the rough endoplasmic reticulum/mitochondrial complex, which is present in many mesenchymal and sustentacular cells, is discussed.

scholarly journals Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment

Reproduction ◽  
2013 ◽  
Vol 145 (4) ◽  
pp. 371-380 ◽  
Author(s):  
Jingjing Guo ◽  
Hongyu Zhou ◽  
Zhijian Su ◽  
Bingbing Chen ◽  
Guimin Wang ◽  
...  
Keyword(s):  
Leydig Cell ◽  
Leydig Cells ◽  
Pubertal Development ◽  
Cell Lineage ◽  
Hydroxysteroid Dehydrogenase ◽  
Mrna Levels ◽  
Steroidogenic Enzymes ◽  
Isolated Cells ◽  
Adult Leydig Cells

The objective of this study was to purify cells in the Leydig cell lineage following regeneration after ethane dimethanesulfonate (EDS) treatment and compare their steroidogenic capacity. Regenerated progenitor (RPLCs), immature (RILCs), and adult Leydig cells (RALCs) were isolated from testes 21, 28 and 56 days after EDS treatment respectively. Production rates for androgens including androsterone and 5α-androstane-17β, 3α-diol (DIOL), testosterone and androstenedione were measured in RPLCs, RILCs and RALCs in media after 3-h in vitro culture with 100 ng/ml LH. Steady-state mRNA levels of steroidogenic enzymes and their activities were measured in freshly isolated cells. Compared to adult Leydig cells (ALCs) isolated from normal 90-day-old rat testes, which primarily produce testosterone (69.73%), RPLCs and RILCs primarily produced androsterone (70.21%) and DIOL (69.79%) respectively. Leydig cells isolated from testes 56 days post-EDS showed equivalent capacity of steroidogenesis to ALCs and primarily produced testosterone (72.90%). RPLCs had cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1 and 17α-hydroxylase but had almost no detectable 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities, while RILCs had increased 17β-hydroxysteroid dehydrogenase 3 and 11β-hydroxysteroid dehydrogenase 1 activities. Because RPLCs and RILCs had higher 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase activities they produced mainly 5α-reduced androgens. Real-time PCR confirmed the similar trends for the expressions of these steroidogenic enzymes. In conclusion, the purified RPLCs, RILCs and RALCs are similar to those of their counterparts during rat pubertal development.

scholarly journals Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors

Reproduction ◽  
2010 ◽  
Vol 139 (1) ◽  
pp. 177-184 ◽  
Author(s):  
P J O'Shaughnessy ◽  
A Monteiro ◽  
G Verhoeven ◽  
K De Gendt ◽  
M H Abel
Keyword(s):  
Germ Cell ◽  
Leydig Cell ◽  
Sertoli Cells ◽  
Leydig Cells ◽  
Androgen Receptors ◽  
Round Spermatid ◽  
Cell Number ◽  
Cell Numbers ◽  
No Formation ◽  
Testicular Morphology

FSH and androgen act to stimulate and maintain spermatogenesis. FSH acts directly on the Sertoli cells to stimulate germ cell number and acts indirectly to increase androgen production by the Leydig cells. In order to differentiate between the direct effects of FSH on spermatogenesis and those mediated indirectly through androgen action, we have crossed hypogonadal (hpg) mice, which lack gonadotrophins, with mice lacking androgen receptors (AR) either ubiquitously (ARKO) or specifically on the Sertoli cells (SCARKO). These hpg.ARKO and hpg.SCARKO mice were treated with recombinant FSH for 7 days and testicular morphology and cell numbers were assessed. In untreated hpg and hpg.SCARKO mice, germ cell development was limited and did not progress beyond the pachytene stage. In hpg.ARKO mice, testes were smaller with fewer Sertoli cells and germ cells compared to hpg mice. Treatment with FSH had no effect on Sertoli cell number but significantly increased germ cell numbers in all groups. In hpg mice, FSH increased the numbers of spermatogonia and spermatocytes, and induced round spermatid formation. In hpg.SCARKO and hpg.ARKO mice, in contrast, only spermatogonial and spermatocyte numbers were increased with no formation of spermatids. Leydig cell numbers were increased by FSH in hpg and hpg.SCARKO mice but not in hpg.ARKO mice. Results show that in rodents 1) FSH acts to stimulate spermatogenesis through an increase in spermatogonial number and subsequent entry of these cells into meiosis, 2) FSH has no direct effect on the completion of meiosis and 3) FSH effects on Leydig cell number are mediated through interstitial ARs.

scholarly journals Use of antibodies against LH receptor, 3beta-hydroxysteroid dehydrogenase and vimentin to characterize different types of testicular tumour in dogs

Reproduction ◽  
2001 ◽  
pp. 287-296 ◽  
Author(s):  
MA Peters ◽  
KJ Teerds ◽  
I van der Gaag ◽  
DG de Rooij ◽  
FJ van Sluijs
Keyword(s):  
Sertoli Cell ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Histological Diagnosis ◽  
Lh Receptor ◽  
Testicular Tumours ◽  
Sertoli Cell Tumours ◽  
Mixed Tumours ◽  
Leydig Cell Tumours

Testicular tumours in dogs are of Sertoli cell, Leydig cell or germinal origin and mixed tumours are also frequently observed. The cellular components of mixed tumours are usually identified by histological examination but sometimes this is difficult. In this study, a panel of specific antibodies was used to identify the different cell types in testicular tumours by immunohistochemistry. Leydig cells were identified using an antibody against the LH receptor and an antibody against the steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD), both of which are characteristic of Leydig cells in testes. Sertoli cells were identified using an antibody against the intermediate filament vimentin. Seminoma cells did not stain with any of these antibodies. Vimentin was used only in histologically complex cases. Eighty-six tumours, diagnosed histologically as 29 Sertoli cell tumours, 25 Leydig cell tumours, 19 seminomas and 13 mixed tumours, were studied. Feminization was observed in 17 dogs. Leydig cell tumours stained positively with the antibodies against the LH receptor and 3beta-HSD, whereas seminomas and Sertoli cell tumours were negative (unstained). The antibody against vimentin stained both Sertoli and Leydig cells, and tumours arising from these cells, but not seminomas. Immunohistochemistry revealed that three tumours identified histologically as Sertoli cell tumours were actually Leydig cell tumours. In 14 dogs the histological diagnosis appeared to be incomplete, as mixed tumours instead of pure types of tumours were identified in 11 dogs, and in three dogs mixed tumours appeared to be pure types. Hence, the histological diagnosis was insufficient in approximately 20% of dogs. Furthermore, immunohistochemical analysis of testis tumours revealed that feminization occurred in dogs with Sertoli cell tumours or Leydig cell tumours and their combinations, but not in dogs with a seminoma. In conclusion, incubation with antibodies against LH receptor and 3beta-HSD proved to be a consistently reliable method for identification of Leydig cell tumours in dogs. Vimentin can be used to discriminate between Sertoli cell tumours and seminomas. Overall, this panel of antibodies can be very useful for determination of the identity of testicular tumours in which histological characterization is complicated and the pathogenesis of feminization is not clear.

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