Purified Leydig Cells Research Articles

Isolation, culture, characterization and the functional study of testicular Leydig cells from Hezuo pig.

Testosterone, an important sex hormone, regulates sexual maturation, testicular development, spermatogenesis and the maintenance of secondary sexual characteristics in males. Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1-month-old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3β-hydroxysteroid dehydrogenase antibody. Enzyme-linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured invitro (generations 5-9) ranged between 1.29-1.67 ng/mL. Additionally, the content of the cellular autophagy signature protein microtubule-associated protein 1 light chain 3 was measured at 230-280 pg/mL. Through this study, we established an invitro system for the isolation, purification and characterization of testicular Leydig cells from 1-month-old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.

Kisspeptin/GnRH1 system in Leydig cells of horse (Equus caballus): Presence and function

The objectives of this study were to evaluate in horse testes the expression of kisspeptin (KiSS) and GnRH1 neuropeptides and their cognate receptors, KiSS1R and GnRH1R, as well as their action on testosterone, GnRH1, prostaglandin F2α (PGF2α), and PGE2 synthesis and cyclooxygenase 1 (COX1) and COX2 activity by Leydig cells in vitro. Testes were obtained from 9 sexually mature horses by surgical castration. Immunohistochemistry, evidenced the presence of KiSS, KiSS1R, GnRH, and GnRH1R in Leydig cells, whereas germinal and Sertoli cells were positive only for GnRH1. Transcripts for both neuropeptides and their cognate receptors were revealed in isolated Leydig cells by RT-PCR. Isolated and purified Leydig cells were in vitro cultured with agonists and antagonists of KiSS (KiSS-10 and KiSS-234, respectively) and GnRH1 (buserelin and antide, respectively). KiSS-10 and buserelin increased (P < 0.01) COX1 activity and testosterone and PGF2α basal secretion, while decreased (P < 0.01) that of PGE2. KiSS-10 and buserelin did not affect COX2 activity. GnRH1 basal production was increased (P < 0.01) by KiSS-10, but not by buserelin. Antide counteracted the KiSS and GnRH1 effects, whereas KiSS-234 influence only those of KiSS. Summarizing, the KiSS/GnRH1 system is present in horse Leydig cells and modulates their endocrine activity. In particular, the endocrine effects of KiSS are mediated by GnRH1, so suggesting that hypothalamic-like interaction between KiSS and GnRH1 occurs also in Leydig cells.

Effect of kisspeptin antagonist on goat in vitro Leydig cell steroidogenesis

Kisspeptin is the protein product of Kiss1 gene. In contrast to its fundamental role as GnRH Secretagogue and puberty initiation, its functions in extrahypothalmic tissues such as the testes remain unknown. The objective of this study was to investigate the potential local role of kisspeptin in the Leydig cell culture in goats. Five male Shiba goats were castrated to isolate Leydig cells from the testes. The isolated Leydig cells were then purified and cultured with different concentration of kisspeptin antagonist (0, 5, and 10 nmol; 0KPA, 5KPA, and 10KPA; respectively). After 12 h, 20 IU of hCG was added to some wells (0KPA + hCG, 5KPA + hCG, and 10KPA + hCG; respectively); other wells did not receive hCG (0KPA + Con, 5KPA + Con, and 10KPA + Con; respectively). Media of Leydig cell culture was harvested at 2, 6, 12, and 24 h after addition of hCG for testosterone (T) and estradiol (E2) assays. The contents of some non KPA treated wells (0KPA + Con and 0KPA + hCG wells) were collected and stored at −80 OC for further identification the expression of mRNA encoding Kiss1 and its receptor (GPR54) using real time PCR. Results revealed high expressions of Kiss1/GPR54 mRNAs in the hCG treated (0KPA + hCG) wells compared to non-hCG treated ones (0KPA + Con). KPA significantly reduced the basal T production at 6 h incubation as well as hCG-induced T production in 2, 6, and 24 h incubation. Basal E2 concentrations were significantly lower in the KPA treated wells at 6, and 24 h likewise, KPA significantly attenuated induced E2 production at 12 h compared with its values in non KPA treated wells. In conclusion, kisspeptin antagonist significantly attenuated both basal and hCG-activated T and E2 production by purified Leydig cells in goats. Therefore, involvement of kisspeptin in the steroidogenic capability of Leydig cells is possible in goats.

A remarkable Activity of Steroid Biosynthesis in Captopril Preserved Leydig Cells of Mice Implicated Leukotriene B4 and Gonadotropin Releasing Hormones in vitro

In this study, investigated whether captopril inhibited steroidogenesis and components of the Leukotriene B4 pathways are involved in GnRH agonist (GnRH)-induced testis steroidogenesis in mice Leydig cells. Primary cultures of mice Leydig cells were established. Purified Leydig cells from adult albino mice were incubated with gradual various concentrations of GnRH with and without Captopril, Luteinizing hormone (LH); LTB4, steroidogenic (testosterone) activity and LTB4 concentration were measured after various time intervals and Leydig cell viability. The maneuvers of Leydig cells treated media was covered the singular and dual actions of antisteroidogenic of captopril and the reversible activity by GnRH-LTB4 as well as contribution of LTB4 in Leydig cells testosterone production endpoint. The different treatment media are Medium alone; Medium plus captopril 60 μM, 65 μM, 70 μM, 75 μM and 80 μM 100 μM; Medium plus 2.5mU/ml leukotriene B4; Medium plus 0.1 mM LH; Medium plus 0.1 μM GnRH; Medium plus 65 μM captopril plus 2.5 mU/ml leukotriene B4;Medium plus 65 μM captopril plus 0.1 mM LH; Medium plus 65 μM captopril plus 0.1 mM GnRH; Medium plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4 and Medium plus 65 μM captopril plus 0.1 mM GnRH plus 2.5 mU/ml leukotriene B4. Basal testosterone levels were maximal at 0.1 μM GnRH concentration and superior testosterone yield in Leydig cells incubated 0.1 μM GnRH media than without GnRH media, and the activity profile LTB4 flow up. That comparable result led to highly correlated approved the contribution of LTB4 in GnRH stimulated Leydig cell steroidogenic end point. Furthermore captopril had an abolishment effect partially of testosterone yield and recovered and improved by GnRH and LTB4. The Leydig cells viability results suggest that the major effect of GnRH is probably beyond the LTB4. The entire key; GnRH induced testosterone production and upregulated LTB4 Levels at both the captopril inhibitory LTB4-testesteron Leydig cells culture media and captopril abolished LTB4 levels; it also activated endogenous LTB4, but not LH motivated testosterone pathway. Our data show that GnRH positively regulates steroidogenesis via LTB4 signaling in mice Leydig cells. LTB4 activation by GnRH may be responsible for the induction of Ca++ signaling indirect. Possibility improve the captopril steroidogenic disruption in Leydig cells via LTB4 and/or GnRH induction of endogenous LTB4, likewise the positive maintenance of Leydig cells viability matched induce testosterone synthesis. The LTB4 production, which may ultimately modulate steroidogenesis in mice Leydig cells, and promise new antidotal and preventative of captopril adverse effects.

Prepubertal Buffalo (Bubalus bubalis) Leydig Cells: Isolation, Culture and Characterization

Water buffalo (Bubalus bubalis) is an economically important livestock species in India. Male buffaloes display delayed sexual maturity as compared to the bulls (Bos taurus). Serum testosterone level, the key regulator of sexual maturity of males, is reported to be low in male buffaloes in comparison to bulls. Testosterone secretion and progression of spermatogenesis is mediated essentially by Leydig cells in the males. Establishment of primary culture for buffalo Leydig cells can provide an excellent tool to investigate the factors which regulate testicular steroidogenesis. Therefore, the objectives of the present study were to isolate, culture and characterize buffalo Leydig cells. Immunohistological analysis revealed that cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) specifically mark the Leydig cells in prepubertal buffalo testis. Using enzymatic digestion and Percoll density gradient centrifugation, a cell population that consisted of approximately 95% pure Leydig cells was obtained as indicated by CYP11A1 staining. Purified Leydig cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum (FBS) for 72 h. The cultured Leydig cells proliferated, expressed Leydig-cell specific transcripts (STAR, HSD3B1, HSD3B6, and CYP17A1) and proteins (CYP11A1, HSD3B and LHCGR), and secreted testosterone. It was concluded from the present study that buffalo Leydig cells can be maintained in culture for 72 h. The primary culture of buffalo Leydig cells can be used for studying acute responses, biochemical properties and other factors regulating testicular steroidogenesis, independent of other testicular cell types.

The Ca2+-activated, large conductance K+-channel (BKCa) is a player in the LH/hCG signaling cascade in testicular Leydig cells

In Leydig cells, hormonal stimulation by LH/hCG entails increased intracellular Ca2+ levels and steroid production, as well as hyperpolarization of the cell membrane. The large-conductance Ca2+-activated K+-channel (BKCa) is activated by raised intracellular Ca2+ and voltage and typically hyperpolarizes the cell membrane. Whether BKCa is functionally involved in steroid production of Leydig cells is not known. In order to explore this point we first investigated the localization of BKCa in human and hamster testes and then used a highly specific toxin, the BKCa blocker iberiotoxin (IbTx), to experimentally dissect a role of BKCa. Immunohistochemistry and RT-PCR revealed that adult Leydig cells of both species are endowed with these channels. Ontogeny studies in hamsters indicated that BKCa becomes strongly detectable in Leydig cells only after they acquire the ability to produce androgens. Using purified Leydig cells from adult hamsters, membrane potential changes in response to hCG were monitored. HCG hyperpolarized the cell membrane, which was prevented by the selective BKCa blocker IbTx. Steroidogenic acute regulatory (StAR) mRNA expression and testosterone production were not affected by IbTx under basal conditions but markedly increased when hCG, in submaximal and maximal concentration or when db-cAMP was added to the incubation media. A blocker of KV4-channels, expressed by Leydig cells, namely phrixotoxin-2 (PhTx-2) was not effective. In summary, the data reveal BKCa as a crucial part of the signaling cascade of LH/hCG in Leydig cells. The hyperpolarizing effect of BKCa in the Leydig cell membrane appears to set in motion events limiting the production of testosterone evoked by stimulatory endocrine mechanisms.

Influence of the photoperiod on TGF-β1 and p15 expression in hamster Leydig cells

Adult hamsters exposed to short photoperiods show a marked atrophy of their internal reproductive organs, including a reduction in size, though not number of Leydig cells. Transforming growth factor-β1 (TGF-β1) is involved in the regulation of growth and proliferation of different cell types. The aim of the present study was to examine the influence of photoperiod on the protein and gene expression of TGF-β1 and its receptors as well as gene expression of p15. The effect of TGF-β1 on the expression of p15 in purified Leydig cells from regressed and non-regressed hamster testes was also tested. Protein and gene expression of TGF-β1 was detected in both regressed and non-regressed testes. In contrast to the activin receptor-like kinase 1 (ALK-1), the TGF-β1, the activin receptor-like kinase 5 (ALK-5) and the co-receptor endoglin all showed a greater basal expression in regressed than non-regressed hamster testes. Melatonin induced the TGF-β1 mRNA expression in purified Leydig cells from non-regressed testes. The p15 mRNA level was greater in regressed than non-regressed testes. A high dose of TGF-β1 during a short incubation period increased the p15 mRNA level in Leydig cells from non-regressed testes. ALK-5 and mitogen-activated protein kinase (MAPK) p38 might have played a role in this process. In regressed hamster testes, the p15 mRNA level increased due to a low dose of TGF-β1 after short incubation periods and to a high dose after longer incubation periods; in both instances, ALK-5, ERK 1/2 and p38 were involved. Collectively, these results suggest that the alterations in p15 expression, mediated by MAPK, are involved in the shift between the active and inactive states in hamster Leydig cells.

Steroidogenesis in amlodipine treated purified Leydig cells

Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced ( P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein.

C17,20 Lyase: Inhibition of Rat Testicular Microsomes and Purified Leydig Cells by Aminoglutethimide In-Vitro

C17,20 Lyase: Inhibition of Rat Testicular Microsomes and Purified Leydig Cells by Aminoglutethimide In-Vitro

Structural complexity of the testis and PKG I / StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

Sildenafil treatment in vivo stimulates Leydig cell steroidogenesis via the cAMP/cGMP signaling pathway

Sildenafil citrate (Viagra), a cGMP-selective phosphodiesterase (PDE) inhibitor, is widely used to treat erectile dysfunction and pulmonary arterial hypertension. In contrast to its well established action on erectile dysfunction, little is known on the action of sildenafil on cGMP/cAMP signaling and testicular steroidogenesis. This study was designed to assess the effects of prolonged sildenafil treatment on NO synthase-dependent signaling and steroidogenic function of rat Leydig cells. Male adult rats were treated with Viagra (1.25 mg/kg body wt) daily for 30 days. In our studies, serum testosterone and ex vivo testosterone production significantly increased in sildenafil-treated animals. Human chorionic gonadotropin-stimulated testosterone production and cAMP accumulation were also significantly higher in Leydig cells obtained from sildenafil-treated rats. The expression of soluble guanylyl cyclase (GUCY1) subunits (Gucy1a1, Gucy1b1) significantly increased; cAMP-specific Pde4a, cGMP-specific Pde6c, and dual Pde1c and Nos2 were inhibited and expression of Nos3, protein kinase G1 (Pkg1), and Pde5 remained unchanged. Treatment of purified Leydig cells with NO donor caused a dose-dependent increase in both testosterone and cGMP production. Testosterone and cGMP production was significantly higher in Leydig cells obtained from sildenafil-treated animals. The stimulatory effect of NO donor was significantly enhanced by saturating concentrations of hCG in both Leydig cells obtained from control and sildenafil-treated animals. Occurrence of mature steroidogenic acute regulatory protein also increased in sildenafil treated animals in accord with increased cAMP and cGMP production. In summary, inhibition of PDE activity during prolonged sildenafil treatment increased serum testosterone level and Leydig cells' steroidogenic capacity by coordinated stimulatory action on cAMP and cGMP signaling pathway.

Testosterone-Induced Modulation of Nitric Oxide-cGMP Signaling Pathway and Androgenesis in the Rat Leydig Cells1

Testosterone, acting as a systemic and local factor, is one of the major regulatory molecules that initiate and maintain testicular function. In the present study, different experimental approaches were used to evaluate the role of testosterone in regulation of the nitric oxide (NO)-cGMP pathway in Leydig cells derived from normal and hypogonadotropic male rats treated with testosterone for 24 h and 2 wk. Real-time quantitative PCR and Western blot analysis revealed increased inducible NO synthase (NOS2) expression followed by increased NO secretion from Leydig cells ex vivo after continuous treatment with testosterone for 2 wk in vivo. The cGMP-specific phosphodiesterases Pde5, Pde6, and Pde9 were up-regulated, whereas PRKG1 protein was decreased after a 2-wk testosterone treatment. Induction of Nos2 and Pde5 in Leydig cells was blocked by androgen receptor antagonist. In experimental hypogonadotropic hypogonadism, expression of NOS2 was significantly reduced, and treatment with testosterone increased NOS2 expression above control levels. PDE5 protein level was unchanged in hypogonadal rats, whereas treatment of hypogonadal rats with testosterone significantly increased it. In contrast, hypogonadism and testosterone replacement reduced PRKG1 protein in Leydig cells. In vitro treatment with testosterone caused gradually increased Nos2 gene expression followed by increased nitrite and cGMP production by purified Leydig cells. In summary, testosterone up-regulated NO signaling via increased NOS2 expression and contributed to down-regulation of cGMP signaling in Leydig cells. Thus, testosterone-induced modulation of NO-cGMP signaling may serve as a potent autocrine regulator of testicular steroidogenesis.

Altered Expression of NDRG2 in the Testes of Experimental Rat Model of Cryptorchidism

To examine the expression of N-Myc downstream regulated gene 2 (NDRG2) in the cryptorchid testes and its potential association with germ cell apoptosis in a rat model of cryptorchidism. Groups of pubertal Sprague-Dawley rats were subjected to surgical procedure for the induction of experimental unilateral cryptorchidism. The weight of the abdominal and scrotal testes was measured and their apoptotic cells and NDRG2 expression were examined longitudinally by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, immunohistochemistry, and immunofluorescence analyses. The expression of NDRG2 and p53 in the purified germ cells and Leydig cells was determined by Western blot assay. Compared with the control scrotal testes, the mean weights of the abdominal testes were significantly reduced at 7 and 14 days after surgery, accompanied by significantly increased numbers of apoptotic germ cells. Although the NDRG2 expression was detected in Leydig cells and spontaneous apoptotic germ cells in the scrotal testes, it was significantly upregulated predominately in apoptotic spermatocytes and round spermatids in the abdominal testes. However, the NDRG2 expression was downregulated in Leydig cells in the abdominal testes. Western blot analysis revealed that the expression of NDRG2 and p53 was upregulated significantly in purified germ cells 7 days after surgery, whereas NDRG2 was downregulated in the purified Leydig cells from the abdominal testes 7 and 14 days after surgery. The upregulated expression of NDRG2 was associated with germ cell apoptosis in the cryptorchid testes, suggesting that the NDRG2 may be a positive regulator for germ cell apoptosis in rat cryptorchidism.

TGF-.BETA.1 System in Leydig Cells. Part I: Effect of hCG and Progesterone

Transforming growth factor beta 1 (TGF-beta1) modulates male reproductive function. Genetically modified mice overexpressing alpha/beta subunits of hCG (hCG+) show Leydig cell hyperplasia/hypertrophy at prepuberty that disappears as the mice approach adulthood. In this study we analyzed the gene expression of TGF-beta1, its specific receptors, type II (TGF-betaRII) and type I (activin receptor-like kinase 1 and 5: ALK1 and ALK5), and co-receptor endoglin (CD105) in purified Leydig cells from hCG+ and wild-type mice at 3 and 8 weeks of age and the occurrence of TGF-beta1, ALK1 and ALK5 by immunohistochemistry. The expression of TGF-beta1 was higher in hCG+ mice at both ages studied, and no changes were observed in TGF-betaRII. ALK5 diminished with age in wild-type mice, whereas ALK1 decreased in hCG+ mice at 8 weeks of age. Endoglin expression showed a marked increase in 3-week-old hCG+ animals. In vitro incubation of Leydig cells from wild-type animals with hCG (10 IU/ml) increased TGF-beta1 and ALK5 expression. Progesterone (10(-6) M) induced endoglin expression. These studies provide novel evidence for differential gene and protein expression of ALK1 and ALK5 at different ages and endoglin expression and hormonal, in purified Leydig cells.

TGF-.BETA.1 System in Leydig Cells. Part II: TGF-.BETA.1 and Progesterone, Through Smad1/5, are Involved in the Hyperplasia/hypertrophy of Leydig Cells

Several reports indicate that transforming growth factor beta1 (TGF-beta1) participates in the regulation of cell cycle progression. In this work, we analyzed the in vitro effect of TGF-beta1 on Leydig cell proliferation markers and the in vivo effect of this cytokine in Leydig cell hyperplasia/hypertrophy. The in vitro effect of TGF-beta1 (1 ng/ml) plus progesterone (10(-6) M) on purified Leydig cells from 3 week-old mice increased the immunocytochemically detected PCNA and stimulated the phosphorylation of Smad 1/5. Progesterone (10(-6) M) in the presence or absence of TGF-beta1 diminished the ratio Bax/Bcl-2. Morphometric testicular studies of mice treated with progesterone (s.c.) plus TGF-beta1 (intratesticular), showed an increase in interstitial volume and a decrease in tubular volume. Furthermore, the cytoplasmic volume of Leydig cells showed an increment in this experimental group with a diminution in nuclear volume. Thus, it turned out that the administration of progesterone and TGF-beta1 augmented the volume of Leydig cells. These results indicate a clear effect of TGF-beta1 in the hypertrophy/hyperplasia of Leydig cells.

177. ANTIBODIES AGAINST BMPR-IB UNCOVERS A PARACRINE FUNCTION FOR THE RECEPTOR IN MALE MOUSE LEYDIG CELL TESTOSTERONE PRODUCTION IN VITRO

The male reproductive system is regulated by pituitary gonadotrophins and local testicular factors including the transforming growth factor-β superfamily members which up-regulate and modulate testosterone production respectively. Type I and type II bone morphogenetic protein (BMP) receptors have been identified in both steroidogenic and non-steroidogenic cells in the testis and have been reported to impact steroid production via their effect on steroidogenic enzymes. In this study we investigated if BMPR-IB had an autocrine or paracine role in testosterone production using BMPR-IB antibodies in testis tissue and Leydig cell culture in vitro. Immature (21d) and mature (60d) mice were sacrificed by CO2 asphyxiation and the testis removed, decapsulated and cultured in basal and equine chorionic gonadotrophin (eCG)-conditioned media for three hours at 32 °C in 5 % CO2, in the presence and absence of anti-BMPR-IB. Additionally, Leydig cells were Percoll purified from adult mouse testicular interstitial cells and cultured for three hours with and without anti-BMPR-IB under human chorionic gonadotrophin (hCG) stimulated or unstimulated conditions. After three hours of incubation the culture media was aspirated into labeled vials and assayed for testosterone using a radioimmunoassay. In adult testicular slice culture, treatment with anti-BMPR-IB resulted in a significant decrease in basal and eCG-stimulated testosterone production by 37 % and 41 % respectively, while having no significant effect on basal or eCG-stimulated testosterone production by the immature testis. In purified Leydig cell culture from adult male mice BMPR-IB immunization had no effect on testosterone production in basal or hCG-stimulated conditions. In conclusion, anti-BMPR-IB significantly reduced testosterone production in adult testicular slice culture but not in cell culture, demonstrating that BMP paracrine signalling from the seminiferous tubules is likely to be important in modulating testosterone production by Leydig cells. Additionally, the paracrine signalling appears to be developmentally regulated only occurring in the adult testis.

Rapid down-regulation of testicular androgen biosynthesis at increased environmental temperature is due to cytochrome P450c17 (CYP17) thermolability in Leydig cells, but not in endoplasmic reticulum membranes.

To identify possible molecular targets in moderate heat-induced, short-term derangements of rat testicular endocrine function, rates of androgen and precursor biosynthesis and key enzyme concentrations were compared at 38 degrees C (normal body core temperature) and 31 degrees C (normal scrotal temperature) in three in-vitro models of decreasing complexity and increasing specificity. In purified Leydig cells and similarly in decapsulated testes, gross testosterone secretion was by 20% higher at 38 degrees C under basal conditions and during the initial phase of stimulation with hCG or cAMP; longer (> 1 hour) exposure to the elevated temperature resulted in a marked decrease (52% after 3 hours) of testosterone response to hCG or cAMP as compared to the corresponding rates at 31 degrees C. This phenomenon was neither due to the development of hormone resistance at the receptor level nor to restricted cholesterol supply and turnover nor to increased testosterone accumulation. Whereas mitochondrial CYP11A (cytochrome P450cscc: cholesterol monooxygenase) was absolutely temperature-insensitive in all systems tested, CYP17 (cytochrome P450c17: steroid-17 alpha-monooxygenase/C17, 20-aldolase) in the smooth endoplasmic reticulum responded with a 57% loss in whole testes and 39% loss in purified Leydig cells upon a 3-hour temperature elevation from 31 degrees C to 38 degrees C. In contrast, CYP17 was stable (4% loss) when tested directly in microsomal membranes. It is concluded that CYP17, but not CYP11A, is very sensitive towards even moderate elevation of environmental temperature, and that this apparent lability is not an intrinsic property of the enzyme protein but rather mediated by heat-activated intracellular factors.

Atrazine Oral Exposure of Peripubertal Male Rats Downregulates Steroidogenesis Gene Expression in Leydig Cells

In the present study, we investigated the effects of oral dosing of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) to peripubertal male rats (50 and 200 mg/kg body weight daily from postnatal days 23-50) on ex vivo Leydig cell steroidogenesis. Leydig cells from treated rats were characterised by significant decline in mRNA transcripts of several genes responsible for steroidogenesis: luteinizing hormone receptor (LHR), scavenger receptor-B1, steroidogenic acute regulatory protein, translocator protein, steroidogenic factor-1, phosphodiesterase 4B, 3beta-hydroxysteroid dehydrogenase (HSD), CYP17A1, and 17betaHSD. In the presence of human chorion gonadotropin, the dose-dependent decrease in extracellular cAMP level and accordingly strong inhibition of androgenesis were obtained. The transcription of LHR gene in Leydig cells of atrazine-treated rats was downregulated in a dose-dependent manner, which could be the reason for reduction in cAMP level and expression of cAMP-dependent genes. To clarify the activity of the steroidogenic enzymes responsible for androgenesis, purified Leydig cells were challenged with different steroid substrates (22OH-cholesterol, pregnenolone, progesterone, and Delta(4)-androstenedione), and the obtained results indicated inhibition of androgen production in Leydig cells isolated from atrazine-treated animals in the presence of all those substrates. However, when Leydig cells were challenged with 22OH-cholesterol, the progesterone level in the incubation medium was unchanged, indicating that decrease in cholesterol transport and/or CYP17A1 and 17betaHSD activity are most probably responsible for inhibition of androgen production after the addition of different substrates. Our results demonstrated that in vivo exposure to atrazine affects Leydig cell steroidogenesis via the inhibition of steroidogenesis gene expression, which is accompanied by decreased androgenesis.

Effects of Daidzein on Testosterone Synthesis and Secretion in Cultured Mouse Leydig Cells

The objective of this work was to study the direct effects of daidzein on steroidogenesis in cultured mouse Leydig cells. Adult mouse Leydig cells were purified by Percoll gradient centrifugation, and the cell purity was determined using a 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. The purified Leydig cells were exposed to different concentrations (10 -7 M to 10 -4 M) of daidzein for 24 h under basal and human chorionic gonadotropin (hCG)-stimulated conditions. The cell viability and testosterone production were determined, and the related mechanisms of daidzein action were also evaluated using the estrogen receptor antagonist ICI 182,780 and measuring the mRNA levels of steroidogenic acute regulatory protein (StAR). cholesterol side-chain cleavage enzyme (P450scc), and 3β-HSD-1 involved in testosterone biosynthesis. The results revealed that daidzein did not influence cell viability. Daidzein increased both basal and hCG-stimulated testosterone production in a dose-dependent manner, and this effect was statistically significant at concentrations of 10 -5 M and 10 -4 M daidzein (p<0.05). ICI 182.780 had no influence on daidzein action. RT-PCR results revealed that 10 -5 M and 10 -4 M daidzein did not exert any obvious influence on the mRNA level of P450scc in Leydig cells. However, in the presence of hCG, these concentrations of daidzein significantly increased the StAR and 3β-HSD-1 mRNA levels (p<0.05), but in the absence of hCG, only 10 -5 M and 10 -4 M daidzein up-regulated the StAR and 3β-HSD-1 mRNA expression (p<0.05), respectively. These results suggest that daidzein has direct effect on Leydig cells. Daidzein-induced increase of testosterone production is probably not mediated by the estrogen receptor but correlates with the increased mRNA levels of StAR and 3β-HSD-1.

Substance P-induced inhibition of Leydig cell steroidogenesis in primary culture

The effect of substance P (SP) on hamster Leydig cell steroidogenesis in primary culture was investigated. Purified Leydig cells were cultured with or without SP for 24 h. The levels of testosterone and progesterone in the culture media were 174 +/- 20 pg ml-1 and 105 +/- 12 pg ml-1, respectively. In the presence of SP (10(-7) mol l-1), testosterone concentration significantly decreased to 123 +/- 19 pg ml-1, e.g. with 29.3%. By contrast, at the concentration used, SP had no effect on progesterone secretion. The possible molecular mechanism of the SP action on Leydig cell function was discussed. The reported results indicate that SP can modulate Leydig cell steroidogenesis in culture.