Bis(2-butoxyethyl) Phthalate Delays Puberty Onset by Increasing Oxidative Stress and Apoptosis in Leydig Cells in Rats

Objective This study investigated the effects of bis(2-butoxyethyl) phthalate(BBOP) on the onset of male puberty by affecting Leydig cell development in rats.Methods Thirty 35-day-old male Sprague-Dawley rats were randomly allocated to five groups mg/kg bw per day that were gavaged for 21 days with BBOP at 0, 10, 100, 250, or 500 mg/kg bw per day. The hormone profiles;Leydig cell morphological metrics;mRNA and protein levels;oxidative stress;and AKT,mTOR, ERK1/2, and GSK3β pathways were assessed.Results BBOP at 250 and/or 500 mg/kg bw per day decreased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels mg/kg bw per day(P < 0.05). BBOP at 500 mg/kg bw per day decreased Leydig cell number mg/kg bw per day and downregulated Cyp11a1, Insl3, Hsd11b1,and Dhh in the testes, and Lhb and Fshb mRNAs in the pituitary gland(P < 0.05). The malondialdehyde content in the testis significantly increased, while Sod1 and Sod2 mRNAs were markedly downregulated, by BBOP treatment at 250–500 mg/kg bw per day(P < 0.05). Furthermore, BBOP at 500mg/kg bw per day decreased AKT1/AKT2, mTOR, and ERK1/2 phosphorylation, and GSK3β and SIRT1levels mg/kg bw per day(P < 0.05). Finally, BBOP at 100 or 500 μmol/L induced ROS and apoptosis in Leydig cells after 24 h of treatment in vitro(P < 0.05).Conclusion BBOP delays puberty onset by increasing oxidative stress and apoptosis in Leydig cells in rats.The graphical abstract is available on the website www.besjournal.com.

Gonadotrophin-releasing hormone-Ⅰ and -Ⅱ stimulate steroidogenesis in prepubertal murine Leydig cells in vitro

Aim： To study the effect and mechanism of gonadotrophin-releasing hormone （GnRH） on murine Leydig cell steroidogenesis. Methods： Purified murine Leydig cells were treated with GnRH-Ⅰ and -Ⅱ agonists, and testosterone production and steroidogenic enzyme expressions were determined. Results： GnRH-Ⅰ and -Ⅱ agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner （P 〈 0.05）. The mRNA expressions of steroidogenic acute regulatory （STAR） protein, P450scc, 3β-hydroxysteroid dehydrogenase （HSD）, but not 17β-hydroxylase or 17β-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase （P 〈 0.05）. However, only 3β-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase （P 〈 0.05）. Conclusion： GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3β-HSD enzyme expression.

Green tea polyphenols inhibit testosterone production in rat Leydig cells

This study investigated the acute effects of green tea extract （GTE） and its polyphenol constituents, （-）-epigallocatechin-3-gallate （EGCG） and （-）-epicatechin （EC）, on basal and stimulated testosterone production by rat Leydig cells in vitro. Leydig cells purified in a Percoll gradient were incubated for 3 h with GTE, EGCG or EC and the testosterone precursor androstenedione, in the presence or absence of either protein kinase A （PKA） or protein kinase C （PKC） activators. The reversibility of the effect was studied by pretreating cells for 15 min with GTE or EGCG, allowing them to recover for 1 h and challenging them for 2 h with human chorionic gonadotropin （hCG）, luteinizing hormone releasing hormone （LHRH）, 22（R）-hydroxycholesterol or androstenedione. GTE and EGCG, but not EC, inhibited both basal and kinase-stimulated testosterone production. Under the pretreatment conditions, the inhibitory effect of the higher concentration of GTE/EGCG on hCG/LHRH-stimulated or 22（R）- hydroxycholesterol-induced testosterone production was maintained, whereas androstenedione-supported testosterone production returned to control levels. At the lower concentration of GTE/EGCG, the inhibitory effect of these polyphenols on 22（R）-hydroxycholesterol-supported testosterone production was reversed. The inhibitory effects of GTE may be explained by the action of its principal component, EGCG, and the presence of a gallate group in its structure seems important for its high efficacy in inhibiting testosterone production. The mechanisms underlying the effects of GTE and EGCG involve the inhibition of the PKA/PKC signalling pathways, as well as the inhibition of P450 side-chain cleavage enzyme and 17β-hydroxysteroid dehydrogenase function.

Chronic stress induces ageing-associated degeneration in rat Leydig cells

Several studies have suggested that stress and ageing exert inhibitory effects on rat Leydig cells. In a pattern similar to the normal process of Leydig cell ageing, stress-mediated increases in glucocorticoid levels inhibit steroidogenic enzyme expression that then results in decreased testosterone secretion. We hypothesized that chronic stress accelerates the degenerative changes associated with ageing in Leydig cells. To test this hypothesis, we established a model of chronic stress to evaluate stress-induced morphological and functional alterations in Brown Norway rat Leydig cells; additionally, intracellular lipofuscin levels, reactive oxygen species （ROS） levels and DNA damage were assessed. The results showed that chronic stress accelerated ageing-related changes： ultrastructural alterations associated with ageing, cellular lipofuscin accumulation, increased ROS levels and more extensive DNA damage were observed. Additionally, testosterone levels were decreased. This study sheds new light on the idea that chronic stress contributes to the degenerative changes associated with ageing in rat Leydig cells in vivo.

Morphometric study on Leydig cells in capsulotomized testis of rats

AIM: To further clarify the changes occurred in the testicular capsulotomized rats. METHODS: In testicular capsulotomized and sham-operated rats, the cross sectional area, the nucleus diameter and the number of Leydig cells were morphologically analyzed by the Vidas Image Processing System connected to a microscope. RESULTS: In the capsulotomized animals, the cross sectional area of Leydig cells was gradually increased from 30 days onwards. There was no obvious change in the nucleus diameter of Leydig cells. However, The Leydig cell number was significantly increased from day 30 onwards. CONCLUSION: In rats, testicular capsulotomy may induce hyperplasia/hypertrophy of Leydig cells in the testis.

Inhibitory actions of mibefradil on steroidogenesis in mouse Leydig cells： involvement of Ca^2＋ entry via the T-type Ca^2＋ channel

Intracellular cAMP and Ca^2＋ are involved in the regulation of steroidogenic activity in Leydig cells, which coordinate responses to luteinizing hormone （LH） and human ehorionic gonadotropin （hCG）. However, the identification of Ca^2＋ entry implicated in Leydig cell steroidogenesis is not well defined. The objective of this study was to identify the type of Ca^2＋ channel that affects Leydig cell steroidogenesis. In vitro steroidogenesis in the freshly dissociated Leydig cells of mice was induced by hCG incubation. The effects of mibefradil （a putative T-type Ca^2＋ channel blocker） on steroidogenesis were assessed using reverse transcription （RT）-polymerase chain reaction analysis for the steroidogenic acute regulatory protein （STAR） mRNA expression and testosterone production using radioimmunoassay. In the presence of 1.0 mmol L-1 extracellular Ca^2＋, hCG at 1 to 100 IU noticeably elevated both StAR mRNA level and testosterone secretion （P 〈 0.05）, and the stimulatory effects of hCG were markedly diminished by mibefradil in a dose-dependent manner （P 〈 0.05）. Moreover; the hCG-induced increase in testosterone production was completely removed when external Ca^2＋ was omitted, implying that Ca entry is needed for hCG-induced steroidogenesis. Furthermore, a patch-clamp study revealed the presence of mibefradil-sensitive Ca^24- currents seen at a concentration range that nearly paralleled those inhibiting steroidogenesis. Collectively, Our data provide evidence that hCG-stimulated steroidogenesis is mediated at least in part by Ca^2＋ entry carried out by the T-type Ca^2＋ channel in the Leydig cells of mice.

Studies on LH modulated 8 kDa peptide involved regulation of testosterone production in rat Leydig cells

Aim: To demonstrate the role of the 8 kDa peptide in regulation of testosterone production by mt Leydig cells. Meth-ods: A peptide similar to 8 kDa peptide purified from immature rat Leydig cells was isolated and purified from rat lungcytosol. Immunological and structural similarity between the peptides purified from lung and Leydig cells was estab-lished by Western blot and tryptic map comparison respectively. Results: Addition of the 8 kDa peptide 10, 50, 100;and 150 ug decreased the production of testosterone in Leydig cells dose-dependently. But the addition of the peptide150 ug along with hCG had no effect on hCG-stimulated increase in testosterone production. Conclusion: In vitro ad-dition of the peptide purified from lung cytosol to adult rat Leydig cells resulted in a concentration-dependent decrease inbasal testosterone production although it had no effect on hCG-stimulated testosterone production. (Asian J Androl1999 Dec; 1: 191-194)

Effects of Basella alba and Hibiscus macranthus extracts on testosterone production of adult rat and bull Leydig cells

Aim： To determine the androgenic effects of Basella alba and Hibiscus macranthus extracts in the rat and the bull, and to develop a novel in vitro test system using Leydig cells from bull testes. Methods： The effect of methanol extracts from both plants on testosterone production in isolated Leydig cells from the rat and the bull was analyzed using ^125I-radioimmunoassay （^125I-RIA）. Rat Leydig cells were obtained by common methods, whereas a novel technique was used to purify Leydig cells from bull testes. Results： Bull testes from the slaughter house were a cheap source of pure Leydig cells. In culture, these cells produced testosterone for 5-6 days, which can be stimulated by human chorionic gonadotrophin （hCG）. Basella alba extracts significantly enhanced testosterone production in bull and rat Leydig cells in a concentration-dependent manner. Hibiscus macranthus showed no androgenic effect but was shown to inhibit testosterone production at higher concentrations. Conclusion： Leydig cells purified from bull testes can be used as an alternative tool in experimental animal research. Certain fractions of Basella alba extract demonstrated androgenic potential whereas Hibiscus macranthus extracts did not.

The effects of 2-Bromopropane on Viability and TestosteroneProduction Ability of Rat Leydig Cells in Primary Culture

Epidemiological surveys and animal experiments have shown that 2-bromopropane induces oligozoospermia in exposed workers and inhibits spermatogensis in laborratory animals. However, themechanism by which 2-bromopropane exerts its effects is unknown. To this end, we examined the formation of testosterone by the Leydig cells and their survival of these cells in the Presence of differ-ent concentrations of 2-bromopropane in vitro. Leydig cells were isolated following vascular Perfu-sion, enzymatic dissociation and Percoll gradient centrifugation techniques. The cells were cultured in culture dishes. After 8 h, different cultures were exposed to 2-bromopropane at concentrations of 0.01 mmol/L, 0.10 mmol/L and 1.00 mmol/L. In order to stimulate Leydig cells to secrete testos-terone, human chorionic gonadotropin (hCG) was also added. Cell viability was determined using the trypan blue dye exclusion test and cell numbers were counted by hemocytometer. Testosterone secretion was detected by radioimmunoassay. The cell viability decreased after exposure to 2-bromo-propane in a dose-dependent way, but no morphological change was observed. The cell number de-creased in the 2-bromopropane-treated cultures. The secretion of testosterone did not manifest de -tectable changes in the culture treated with 0.10 mmol/L and 0.01 mmol/L of 2-bromopropane;however, it decreased significantly (P < 0. 02) in the Presence of 1.00 mmol/L. Therefore, ourresults strongly suggest that 2-bromopropane may exert its cytotoxic effects on heydig cells in vitro.We speculate that the decrease in the numbers of Leydig cells caused by 2-bromopropane was medi-ated by a feedback mechanism resulting from a lower testosterone concentration.

Ethane dimethylsulphonate selectively destroys Leydig cells in the adult bonnet monkeys (Macaca radiata)

Aim: To study the effect of intratesticular administration of ethane-1,2-dimethylsulphonate (EDS) which has been exten-sively used to selectively destroy Leydig cells in rats and study the role of gonadotropin in regulation of differentiation ofLeydig cells (LC) in the adult male bonnet monkey. Methods and Results: In vitro studies with cultured interstitialcells isolated from monkey testis revealed an inhibitory effect of EDS on LC as assessed by decrease in testosterone pro-duction. Intratesticular administration of EDS (5, 10, 20, 50 mg/testis) resulted in a dose-dependent rapid decrease inserum testosterone levels, with a 65 % decrease with 5 mg of EDS by the 3rd day, which returned to control levels by the45th day. EDS treatment resulted in a significant decrease in testicular testosterone. In addition a significant decrease in[(125)～I]hCG binding and phenylesterase activity in the interstitial cells was noticed. Histological analysis of the testes onthe 5th day after administration of EDS revealed an interstitium devoid of LC indicating the destructive action of EDS.Conclusion: The monkey LC are sensitive to destructive action of EDS. (Asian J Androl 1999 Sep ; 1 : 115 - 120)

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Late-onset hypogonadism （LOH） is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone （LH）-stimulated steroidogenic acute regulatory （STAR） protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species （ROS） levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H2O2, resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.

Effects of varicocele on testosterone, apoptosis and expression of StAR mRNA in rat Leydig cells

The aim of this study was to explore the effects of varicocele on the morphology and function of Leydig cells in the rat testis. Forty male Sprague-Dawley rats were divided into two groups： the experimental group underwent surgery to create a left varicocele （VC）, and the control group underwent a sham operation. Serum testosterone and intratesticular testosterone levels were measured using a radioimmunoassay after 4 and 8 weeks of operation. Leydig cells were studied for apoptosis and expression of steroidogenetic acute regulatory （STAR） protein mRNA levels. Serum testosterone levels declined after 4 and 8 weeks of operation but were not significant （P〉0.05）. However, the intratesticular testosterone levels after 8 weeks were significantly decreased compared with the control group （P〈0.01）. The mean apoptosis index of Leydig cells in the experimental group was significantly higher than that in the control group after 4 or 8 weeks （P〈0.01）. StAR mRNA levels in the Leydig cells of the experimental group were significantly lower compared to those of the control group （P〈0.01）. Our data show that varicocele did impair Leydig cell function by increasing apoptosis and suppressing the expression of the StAR protein.

Cox7a2 mediates steroidogenesis in TM3 mouse Leydig cells

Aim： To investigate the regulatory function of Cox7a2 on steroidogenesis and the mechanism involved in TM3 mouse Leydig cells. Methods： The cDNA of Cox7a2 was cloned from TM3 mouse Leydig cells. It was subcloned to pDsRed- Express-N 1 and transfected back into TM3 mouse Leydig cells for Cox7a2 overexpression by transient gene transfection. Steroidogenesis affected by overexpressed Cox7a2 was studied by ELISA. To elicit the mechanism of this effect, expression of steroidogenic acute regulatory （STAR） protein and reactive oxygen species （ROS） were examined by Western blot and fluorometer, respectively. Results： The cDNA of Cox7a2 （249 bp） was cloned from Leydig cells and confirmed by DNA sequencing. After constructed pDsRed-Express-N1-Cox7a2 was transfected back into TM3 mouse Leydig cells, Cox7a2 inhibited not only luteinizing hormone （LH）-induced secretion of testosterone but also the expression of StAR protein. At the same time, Cox7a2 increased the activity of ROS in TM3 mouse Leydig cells. Conclusion： Cox7a2 inhibited LH-induced StAR protein expression, and consequent testosterone production, at least in part, by increasing ROS activity in TM3 mouse Leydig cells.

Effects of luteinizing hormone and androgen on the development of rat progenitor Leydig cells in vitro and in vivo

Progenitor Leydig cells are derived from stem cells. The proliferation and differentiation of progenitor Leydig cells significantly contributes to Leydig cell number during puberty. However, the regulation of these processes remains unclear. The objective of the present study was to determine whether luteinizing hormone （LH） or androgen contributes to the proliferation and differentiation of progenitor Leydig cells. Fourteen-day-old male Sprague-Dawley rats were treated for 7 days with NalGlu, which is a gonadotropin- releasing hormone antagonist, to reduce the secretion of LH in the pituitary and thus, androgen in the testis. Rats were co-administered with LH or 7a-methyl-nortestosterone （MENT）, which is an androgen resistant to metabolism by 5a-reductase 1 in progenitor Leydig cells, and the subsequent effects of LH or androgen were measured. 3H-Thymidine was also intravenously injected into rats to study thymidine incorporation in progenitor Leydig cells. Progenitor Leydig cells were examined. NalGlu administration reduced progenitor Leydig cell proliferation by 83%. In addition, LH or MENT treatment restored Leydig cell proliferative capacity to 73% or 50% of control, respectively. The messenger RNA levels of proliferation-related genes were measured using real-time PCR. The expression levels of Igfl, Lifr, Pdgfra, Bcl2, Ccnd3and Pcnawere upregulated by MENT, and those of Pdgfra, Ccnd3and Pcnawere upregulated by LH. Both LH and MENT stimulated the differentiation of progenitor Leydig cells in vitro. We concluded that both LH and MENT were involved in regulating the development of progenitor Leydig cells.

Ultrastructural and G-6-Pase Cytochemical Studies of Injurious Action of Cadmium and Protection of Zinc on Rat Leydig Cells

Objective To investigate ultrastructural effects of cadmium(Cd) and zinc(Zn) on rat Leydig cells (LCs) and the possible mechanisms of Cd induced injury. Methods The Wistar rats were injected with low dose cadmium chloride (CdCl 2, 2 mg/kg body weight).The specimens obtained from 1 h to 60 d after dosing were studied by using transmission electron microscope (TEM) combined with a quantitative analysis of glucose 6 phosphatase(G 6 Pase) cytochemistry. Meanwhile, the protective effects of Zn on Cd induced injury were observed. Results The ultrastructural changes of LCs were detected at 4 h after Cd treatment and became more serious after 24 h. The main alterations were dilatation of smooth endoplasmic reticulum (SER), increasing of lipid droplets and myelin figures as well as appearing of vacuoles in the endothelial cell of lymphatic and blood capillaries. At 3,7 and 15 d, the degeneration above mentioned was most prominent, numerous necrotic LCs and flocculent densities in mitochondria were observed. After 30 d, the injuries of LCs appeared to be alleviated. But most of LCs still not recovered to normal after 60 d. However, the G 6 Pase reaction products was found to be reduced at 1 h after Cd treatment, and such decrease was most pronounced within 3～15 d. After 30 d, there was an obviously recovery of the G 6 Pase reaction product. The injuries of LCs of Zn protected groups were gentle and the G 6 Pase reaction products were more than that of Cd treated groups at the same time. Conclusions The early injuries of LCs were related to the direct action of Cd; the effects of Cd on the G 6 Pase activities occured earlier than the morphological alterations; the damage of lymphatic and blood capillaries as well as interstitial fibrosis might accelerate the degeneration and Zn could protect obviously LCs from damage by Cd.

Expression of 11β-Hydroxysteroid Dehydrogenase mRNA in Rat Leydig Cells

11β-hydroxysteroid dehydrogenase (11β-HSD) in Leydig cells regulates sterodogenesis by controlling intra cellular glucocorticoid (corticosterone, B, in rat) concentration.Prior to the 26th postnatal day, 11β-HSD is absent from rat immature Leydig cells. Asthe Leydig cells are matured, the enzyme is gradually produced. The highest levels of11β-HSD activity are present in adult rat Leydig cells. 11β-HSD controls the intracellular glucocorticoid concentration in Leydig cells and the glucocorticoids at the physiologicallevels also regulate levels of 11β-HSD activity in Leydig cells. The expressions of 11βHSD mRNA in Leydig cells from three different age groups of rats and adrenalectomizedrats (ADX), with and without B replacement were Observed in this study. The steady statelevels of 11β-HSD mRNA could not be detected in Leydig cells from immature rats aged21 days, but this could be detected in those aged 45 days. The highest levels of expressionOf 11β-HSD mRNA were found in adult Leydig cells. The mRNA expression of 11β-HSD was declined in Leydig cells after adrenalectomy, and this decline was prevented byB replacement (the levels were restored to control). The results indicated that 11β-HSDmRNA leVels expressed in three different age groups of rats are parallel with those ofantigen by immunohistochemical analysis and with those of enzyme activity by biochemicalmeasurement in previous studies. Similarly, the effect of B at physiological and endogenous levels on the expressions Of 11β-HSD mRNA corresponded to that on enzyme activity.

Exosomes derived from BMSCs ameliorate cyclophosphamide-induced testosterone deficiency by enhancing the autophagy of Leydig cells via the AMPK-mTOR signaling pathway

Cyclophosphamide-induced testosterone deficiency (CPTD) during the treatment of cancers and autoimmune disorders severelyinfluences the quality of life of patients. Currently, several guidelines recommend patients suffering from CPTD receive testosteronereplacement therapy (TRT). However, TRT has many disadvantages underscoring the requirement for alternative, nontoxictreatment strategies. We previously reported bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exos) could alleviatecyclophosphamide (CP)-induced spermatogenesis dysfunction, highlighting their role in the treatment of male reproductive disorders.Therefore, we further investigated whether BMSCs-exos affect autophagy and testosterone synthesis in Leydig cells (LCs). Here,we examined the effects and probed the molecular mechanisms of BMSCs-exos on CPTD in vivo and in vitro by detecting theexpression levels of genes and proteins related to autophagy and testosterone synthesis. Furthermore, the testosterone concentrationin serum and cell-conditioned medium, and the photophosphorylation protein levels of adenosine monophosphate-activatedprotein kinase (AMPK) and mammalian target of rapamycin (mTOR) were measured. Our results suggest that BMSCs-exos couldbe absorbed by LCs through the blood–testis barrier in mice, promoting autophagy in LCs and improving the CP-induced low serumtestosterone levels. BMSCs-exos inhibited cell death in CP-exposed LCs, regulated the AMPK-mTOR signaling pathway to promoteautophagy in LCs, and then improved the low testosterone synthesis ability of CP-induced LCs. Moreover, the autophagy inhibitor,3-methyladenine (3-MA), significantly reversed the therapeutic effects of BMSCs-exos. These findings suggest that BMSCs-exospromote LC autophagy by regulating the AMPK-mTOR signaling pathway, thereby ameliorating CPTD. This study provides novelevidence for the clinical improvement of CPTD using BMSCs-exos.

Fate determination of fetal Leydig cells

Leydig cell(LC)is one of the most important somatic cell types in testis,which localized in the interstitium between seminiferous tubules.The major function of Leydig cells is to produce steroid hormone,androgens.LC differentiation exhibits a biphasic pattern in rodent testes,which are divided into two different temporal mature populations,fetal Leydig cells(FLCs)and adult Leydig cells(ALCs).FLCs are transiently present in fetal testes and undergo involution or degeneration after birth.FLCs are completely devoid and replaced by ALCs in adult testes.Comparing to ALCs,FLCs display unique morphology,ultrastructure and functions.The origin of FLCs has been debated for many years,but it is still a mystery.Many factors have been reported regulating the specification,proliferation and differentiation of FLCs.FLCs degenerate in a few weeks postnatally,however,the underlying mechanism is still unknown.In this review,we will focus on the fate determination of FLCs,and summarize the resent progress on the morphology,ultrastructure,function,origin and involution of FLCs.

An in vitro prototype of a porcine biomimetic testis-like cell culture system： a novel tool for the study of reassembled Sertoli and Leydig cells

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors （testicular cross-talk）, mainly orchestrated by gonadotropins such as luteinizing hormone （LH） and follicle-stimulating hormone （FSH） that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay （ELISA） for anti-Mullerian hormone （AMH）, inhibin B, and testosterone secretion in the medium, and Real-lime PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3βHSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Comparative Potency of Seven Compounds Isolated from Tripterygium Wilfordii on Cultred Rat Leydig Cells and Sertoli Cells

The damaging effect of seven compounds isolated from Triplelygium Wilfordii Hook.F were tested on the rat Leydig cells and Sertoli cells in culture. The reslllts showed thai 10 μg/ ml orthosphenic acid (TW2), 10μg/ ml 39,22α -dihydroxy-△12 ursen- 30-oic acid(TW5), 5.0 μg/ ml 39,22a--dihydroxy-△12-oleallen--29-oic acid(TW6), 5.0 μg/ ml salaspermic acid(TW7), 0.5μg/ ml celastrol(TW27) alld 1.25μg/ ml polpunonic acid (TW28) Ivere all detrimental to Leydig cells. and Sertoli cells. Holvever, 30 μg/ ml wilforlide A (TW1) did not affect the viability of Leydig cells and Sertoli cells significantly.The most toxic compounds was TW27, and the leasl,TW1.These results suggested that the-COOH at the C-20 site of these triterpenoids may be the functional chemical group responsible for the damaging effect on the Leydig cells and Sertoli. cells.