Secretory cell types and cell proliferation of human bronchial epithelial cells in an organ-culture system

We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3β-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3β-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3β-HSD.     

Testicular steroidogenic enzymes in the lizard Podarcis sicula during the spermatogenic cycle

Steroidogenic Acute Regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-Reductase (5α-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17β-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3β-HSD, 17β-HSD and 5α-Red, during three significant periods of the reproductive cycle: summer stasis (July–August), autumnal resumption (November) and reproductive period (May–June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5α-Red and P450 could carry out a pivot role.     

Specific neural phase relation of serotonin and dopamine modulate the testicular activity in Japanese quail

Specific phase relation of serotonin and dopamine modulate the hypothalamo–hypophyseal–gonadal axis as well as photosexual responses in Japanese quail, but the effect of these specific phase relations on testicular activity and steroidogenesis is not yet been investigated. We hypothesized that temporal phase relation induced alteration in local testicular gonadotropin-releasing hormone (GnRH)–Gonadotropin-inhibitory hormone (GnIH) and their receptor system may modulate the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we have checked the alterations in the expression of gonadotropin-releasing hormone receptor (GnRH-R), gonadotropin-inhibitory hormone receptor (GnIH-R) messenger RNA (mRNA), growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), cell communication and gap junctional proteins (14-3-3 and connexin-43 [Cnx-43]), steroidogenic factor-1 (SF-1), steroidogenic acute regulatory (StAR) protein, steroidogenic enzyme (3β-hydroxysteroid dehydrogenase [3β-HSD]) in testis as well as androgen receptor (AR) in testis and epididymis of control, 8-, and 12-hr quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β-HSD in testis as well as AR in testis and epididymis in 8-hr quail, while 12-hr quail exhibited the opposite results that is significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3β-HSD in testis as well as AR in testis and epididymis. The significantly increased intratesticular testosterone has been observed in the 12-hr quail while, 8-hr quail showed opposite result. Hence, it can be concluded that 12-hr quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in 8-hr quail.     

Protective Effect of Selenium on Aflatoxin B1-Induced Testicular Toxicity in Mice

Aflatoxins have been considered as one of the major risk factors of male infertility, and aflatoxin B1 (AFB1) is the most highly toxic and prevalent member of the aflatoxins family. Selenium (Se), an essential nutritional trace mineral for normal testicular development and male fertility, has received extensive intensive on protective effects of male reproductive system due to its potential antioxidant and activating testosterone synthesis. To investigate the protective effect of Se on AFB1-induced testicular toxicity, the mice were orally administered with AFB1 (0.75 mg/kg) and Se (0.2 mg/kg or 0.4 mg/kg) for 45 days. We found that that Se elevated testes index, sperm functional parameters (concentration, malformation, and motility), and the level of serum testosterone in AFB1-exposed mice. Moreover, our results showed that Se attenuated the AFB1-induced oxidative stress and the reduction of testicular testosterone synthesis enzyme protein expression such as steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), and 17β-hydroxysteroid dehydrogenase (17β-HSD) in AFB1-exposed mice. These results demonstrated that Se conferred protection against AFB1-induced testicular toxicity and can be attributed to its antioxidant and increased testosterone level by stimulating protein expression of StAR and testosterone synthetic enzymes.     

H2S catalysed by CBS regulates testosterone synthesis through affecting the sulfhydrylation of PDE

Testosterone deficiency resulted in increased mortality in men. Our previous work found that hydrogen sulphide (H₂S) significantly alleviated the spermatogenesis disorder. To investigate whether H₂S could regulate testosterone synthesis and the relative signalling pathways. Disorder model of testosterone synthesis was constructed in vitro and in vivo. The cell viability was detected using CCK-8 method. The concentration of H₂S and testosterone were examined using ELISA kits. The relative mRNA and protein expression of CBS, PDE4A, PDE8A and proteins related to testosterone synthesis were detected by RT-qPCR and western blotting. PAS staining was used to detect the inflammatory status of testis. The sulfhydryl level of PDE4A and PDE8A was determined by Biotin Switch Technique. CBS overexpression inhibited while knockdown promoted LPS + H₂O₂ induced injury in testosterone synthesis of MLTC-1 cells, though regulating the level of H₂S. The LPS + H₂O₂ induced inhibition on cAMP and p-PKA was recovered by CBS overexpression, while addition of the specific inhibitor of PKA had opposite effects. CBS overexpression alleviated the inflammation status in testis and promoted the expression of StAR, P450scc, P450c17 and 3β-HSD. CBS could also exhibit its protective role through promoting sulfhydrylation of PDE4A and PDE8A. H₂S catalysed by CBS could recover testosterone synthesis in vitro and in vivo through inhibiting PDE expression via sulfhydryl modification and activating cAMP/PKA pathway.     

The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6–8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10^?7 M), 2-Hf (1.7 × 10^?4 M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3β-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3β-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary.     

Local transfer of testosterone and aromatase activity in the spermatic cord in wild boar/pig hybrids in short-daylight and long-daylight periods

The local transfer of testosterone (T) and immunolocalization of cytochrome P450 aromatase (P450arom) in the spermatic cord vessels of ten male wild boar × domestic pig hybrids were examined in December (short-daylight period) and June (long-daylight period). Total T concentration was determined in the jugular vein (JV) and free T concentration was estimated in the common carotid artery (CA), branches of the testicular artery supplying the testis (TA) and epididymis (EA), as well as in testicular veins draining blood from the testis (TV) and spermatic cord (SV). P450arom was immunolocalized in the arterial and venous vessels of the spermatic cord. The concentrations of total T in the JV and free T in the CA did not differ between the examined periods. However, in December, free T concentrations in the TA and EA were higher (p < 0.01–0.001) than in the CA. In June, free T concentration was higher (p < 0.01) in EA than in CA and TA. The concentrations of free T in the TV and SV were higher (p < 0.001) than in the JV regardless of the period. Also, free T concentration in the SV was higher (p < 0.05) in June than in December. P450arom was expressed in all layers of the arterial and venous vessels of the spermatic cord. In June, the intensity of the P450arom staining was higher than in December. The results suggest that the local supply of the male reproductive organs with steroid hormones operate in the hybrids of wild boar × domestic pig. This supply includes the local transfer of testosterone and the P450arom action.     

The determination of the relationship between p97/VCP,small VCP-interacting protein,two ERAD proteins and steroidogenesis in Leydig cell lines

Background

In recent studies, it was shown that Endoplasmic reticulum-associated degradation (ERAD) is regulated by androgens and small VCP-interacting protein (SVIP) is an ERAD inhibitor. There is no data available about the interactions of ERAD proteins with proteins involved in steroidogenesis. The aim of the study was to investigate the expressions of SVIP, p97/VCP, StAR, CYP17A1 and 3β-HSD in human and mouse.

Methods and results

HLC, TM3 and MA-10 Leydig cell lines were used to determine roles of ERAD proteins in steroidogenesis based on immunofluorescence, Western blot, qRT-PCR, ELISA. Findings showed that StAR, CYP17A1 and 3β-HSD were colocalized with SVIP and p97/VCP in Leydig cells. A decrease in CYP17A1, 3β-HSD and StAR expressions was observed as a result of suppression of SVIP siRNAs and p97/VCP siRNAs expressions in MA10, TM3 and HLC. When siSVIP transfected cells were compared with siSVIP transfected with hCG-exposed cells, SVIP protein expression was significantly increased as compared to the SVIP transfected group in human Leydig cells.

Conclusion

We suggest that the suppression of protein expressions by p97/VCP and SVIP siRNAs in Leydig cells, the effects of proteins involved in steroidogenesis (StAR, CYP17A1 and 3β-HSD) have proven to be originating from p97/VCP and SVIP which were playing a role in the steroidogenesis process. Additionally, it was demonstrated that testosterone levels decreased after transfection with p97/VCP siRNA and SVIP siRNA, p97/VCP and SVIP created an effect on testosterone synthesis while taking place in the steps of testosterone synthesis. Further, it was determined in the study that the SVIP was affected by hCG stimulations.

     

Inhibition of 3β- and 17β-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid

Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in rat testis microsomes and Leydig cells. The IC₅₀s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3β-HSD with an IC₅₀ of 53.2 ± 25.9 μM and 17β-HSD3 with an IC₅₀ 17.7 ± 6.8 μM. PFOA inhibited intact Leydig cell 3β-HSD with an IC₅₀ of 146.1 ± 0.9 μM and 17β-HSD3 with an IC₅₀ of 194.8 ± 1.0 μM. The inhibitions of 3β-HSD and 17β-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3β-HSD and 17β-HSD3 in rat Leydig cells.     

Expression of Cytochrome P450 Side-Chain Cleavage Enzyme and 3β-Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side-chain cleavage enzyme (P450_SCC) and 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerase (3β-HSD) has been studied in the rat brain. P450_SCC transforms cholesterol into pregnenolone and 3β-HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non-steroidogenic tissue lung have been used as controls. The expression of P450_SCC and 3β-HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450_SCC and 3β-HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β-HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450_SCC and 3β-HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450_SCC and 3β-HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.     

Growth factor modulation of steroidogenic acute regulatory protein and luteinization in the pig ovary.

In vivo and in vitro luteinization were investigated in the porcine ovary, with emphasis on expression of steroidogenic acute regulatory protein (StAR). StAR mRNA and protein as well as cytochrome P450 side-chain cleavage mRNA (P450scc) increased during the luteal phase in the corpus luteum (CL) and were absent in regressed CL. Cytochrome P450 aromatase mRNA (P450arom) was not detectable at any time in CL. In vitro luteinization of granulosa cells occurred over 96 h in culture, during which P450arom mRNA was present at 1 h after cell isolation but not detectable at 6 h; and P450scc and StAR mRNAs were first detectable at 6 h and 48 h, respectively. Incubation of cultures with insulin-like growth factor I (IGF-I, 10 ng/ml), dibutyryl cAMP (cAMP, 300 microM), or their combination, induced measurable StAR mRNA at 24 h (p < 0.05), increased progesterone accumulation at 48 h, and elevated both StAR and P450scc expression through 96 h. Incubation of luteinized granulosa cells with epidermal growth factor (EGF, 10 nM) changed their phenotype from epithelioid to fibroblastic, eliminated steady-state StAR expression, and interfered with cAMP induction of StAR mRNA and progesterone accumulation. EGF had little apparent effect on P450scc mRNA abundance. It is concluded that StAR expression characterizes luteinization, and early luteinization is induced by cAMP and IGF-I in vitro. Further, EGF induces a morphological and functional phenotype that appears similar to an earlier stage of granulosa cell function.     

Castration inhibits biliary proliferation induced by bile duct obstruction: novel role for the autocrine trophic effect of testosterone

Increased cholangiocyte growth is critical for the maintenance of biliary mass during liver injury by bile duct ligation (BDL). Circulating levels of testosterone decline following castration and during cholestasis. Cholangiocytes secrete sex hormones sustaining cholangiocyte growth by autocrine mechanisms. We tested the hypothesis that testosterone is an autocrine trophic factor stimulating biliary growth. The expression of androgen receptor (AR) was determined in liver sections, male cholangiocytes, and cholangiocyte cultures [normal rat intrahepatic cholangiocyte cultures (NRICC)]. Normal or BDL (immediately after surgery) rats were treated with testosterone or antitestosterone antibody or underwent surgical castration (followed by administration of testosterone) for 1 wk. We evaluated testosterone serum levels; intrahepatic bile duct mass (IBDM) in liver sections of female and male rats following the administration of testosterone; and secretin-stimulated cAMP levels and bile secretion. We evaluated the expression of 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3, the enzyme regulating testosterone synthesis) in cholangiocytes. We evaluated the effect of testosterone on the proliferation of NRICC in the absence/presence of flutamide (AR antagonist) and antitestosterone antibody and the expression of 17β-HSD3. Proliferation of NRICC was evaluated following stable knock down of 17β-HSD3. We found that cholangiocytes and NRICC expressed AR. Testosterone serum levels decreased in castrated rats (prevented by the administration of testosterone) and rats receiving antitestosterone antibody. Castration decreased IBDM and secretin-stimulated cAMP levels and ductal secretion of BDL rats. Testosterone increased 17β-HSD3 expression and proliferation in NRICC that was blocked by flutamide and antitestosterone antibody. Knock down of 17β-HSD3 blocks the proliferation of NRICC. Drug targeting of 17β-HSD3 may be important for managing cholangiopathies.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

Alterations in luteal production of androstenedione,testosterone, and estrone,but not estradiol,during mid- and late pregnancy in pigs: Effects of androgen deficiency

Recently, we have found that flutamide-induced androgen deficiency altered progesterone production in the porcine corpus luteum (CL) during mid- and late pregnancy. Herein, we tested whether flutamide administration subsequently influences androgen and estrogen metabolism in the CL of pregnancy. Pregnant gilts were treated with flutamide between Days 43 and 49 (GD50F), 83 and 89 (GD90F), or 101 and 107 (GD108F) of gestation. Corpora lutea (CLs) were collected from treated and nontreated (control) pigs. The concentrations of androstenedione (A4), testosterone (T), estrone (E1), and estradiol (E2) together with the levels of expression of mRNAs and proteins for cytochrome P450 17α-hydroxylase/c17-20 lyase (CYP17A1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), cytochrome P450 aromatase (CYP19A1), and 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7) were measured in the CL of control and flutamide-treated animals. Steroidogenic enzymes were also immunolocalized in luteal tissues. The luteal concentrations of A4 and T were higher in the GD50F (P = 0.006, P = 0.03) and GD108F (P = 0.005, P = 0.035) groups, but lower in the GD90F (P = 0.004, P = 0.014) group. The E1 level was greater only in the GD90F (P = 0.03) and GD108F (P = 0.035) groups, whereas E2 concentration was not affected by flutamide treatment. Increased luteal CYP17A1 mRNA and protein expression was found in the GD50F (P = 0.002, P = 0.03) and GD108F (P = 0.0026, P = 0.03) groups, but reduced in the GD90F (P = 0.002, P = 0.03) group. mRNA of 17β-HSD1 was upregulated in the GD50F (P = 0.0005) group, but downregulated in the GD90F (P = 0.002) and GD108F (P = 0.0005) groups. In contrast, 17β-HSD1 protein expression was higher in the GD50F and GD108F (P = 0.03) groups, but lower in the GD90F (P = 0.03) group. Both CYP19A1 mRNA and protein levels were greater in the GD90F (P = 0.001, P = 0.028) and GD108F (P = 0.005, P = 0.03) groups. Neither 17β-HSD7 mRNA nor protein level were affected by flutamide exposure. Both CYP17A1 and 17β-HSD1 were immunolocalized exclusively in small luteal cells, whereas CYP19A1 and 17β-HSD7 were found in large luteal cells of control and flutamide-treated CLs. Overall, flutamide administration led to the alterations in A4, T, and E1, but not in E2, production in the CL of pregnancy in pigs, probably because of disrupted steroidogenic enzymes expression. These changes suggest that androgens are important modulators of luteal function during pregnancy in pigs.     

The role of phosphoenolpyruvate carboxykinase in neuronal steroidogenesis under acute inflammation

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme found in many tissues throughout the body including brain. In the present study, we have investigated the effect of bacterial lipopolysaccharide (LPS) on PEPCK and its role in neuronal steroidogenesis. Adult female albino rats were administered LPS (5 mg/kg body weight) to induce acute inflammation. LPS administration resulted in a significant increase of PEPCK mRNA expression with concomitant increase in mRNA levels of steroidogenic acute regulatory (StAR) protein and other steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and aromatase in brain tissue. Further, the inhibition of PEPCK expression by glipizide significantly decreased the mRNA expression of steroidogenic proteins and concurrently increased the mRNA levels of proinflammatory cytokines under LPS administration. The results of this study suggest a novel finding that PEPCK may have an important role in neuronal steroidogenesis; which serves as an adaptive response under inflammation.     

The expression of CKLFSF2B is regulated by GATA1 and CREB in the Leydig cells,which modulates testicular steroidogenesis

CKLFSF is a protein family that serves as a functional bridge between chemokines and members of the transmembrane 4 superfamily (TM4SF). In the course of evolution, CKLFSF2 has evolved as two isoforms, namely CKLFSF2A and CKLFSF2B, in mice. CKLFSF2A, also known as CMTM2A and ARR19, is expressed in the testis and is important for testicular steroidogenesis. CKLFSF2B is also known to be highly expressed in the testis. In the prepubertal stage, CKLFSF2B is expressed only in Leydig cells, but it is highly expressed in haploid germ cells and Leydig cells in adult testis. CKLFSF2B is naturally processed inside the cell at its C-terminus to yield smaller proteins compared to its theoretical size of ≈25?kDa. The Cklfsf2b gene is regulated by GATA-1 and CREB protein, binding to their respective binding elements present in the 2-kb upstream promoter sequence. In addition, the overexpression of CKLFSF2B inhibited the activity of the Nur77 promoter, which consequently represses the promoter activity of Nur77-target steroidogenic genes such as P450c17, 3β-HSD, and StAR in MA-10 Leydig cells. Adenovirus-mediated overexpression of CKLFSF2B in primary Leydig cells isolated from adult mice shows a repression of steroidogenic gene expression and consequently testosterone production. Moreover, intratesticular injection of CKLFSF2B-expressing adenovirus in adult mice clearly had a repressive effect compared to the control injected with only GFP-expressing adenovirus. Altogether, these findings suggest that CKLFSF2B might be involved in the development and function of Leydig cells and regulate testicular testosterone production by fine-tuning the expression of steroidogenic genes.     

Rôle du peptide vasoactif intestinal (VIP) dans la stéroïdogenèse et le vieillissement testiculaire

VIP (vasoactive intestinal peptide) neuropeptide has long been considered to be putative regulator of testicular functions.In vitro evidence suggests that VIP could play an important role in testosterone biosynthesis. However, the endogenous role of VIP on testicular functions remained to be demonstrated. In C57BL/6 mice exhibiting complete disruption of the VIP gene, the authors observed that male fertility remained intact but serum testosterone levels were lower than those of WT littermates. At the age of 4 months, this phenotype was accompanied by reduced steroidogenesis due to inhibition of the expression of StAR (steroidogenic acute regulatory protein) and 3ßHSD (3ß-hydroxysteroid dehydrogenase) in the testis. In addition, serum levels of FSH (Follicle-stimulating hormone) but not LH (Luteinizing hormone) were reduced in young KO males. Testicular anatomy also revealed a subtle but significantly higher percentage of degenerated seminiferous tubules in 4-month-old VIP-/-animals compared to WT. In aging animals (15 months old), control males showed typical testicular aging including severe degeneration of seminiferous tubules, a dramatic decrease in serum testosterone levels and a reduction in StAR and 3ß-HSD gene expression. In age-matched VIP-/-males, serum levels of testosterone and steroidogenic enzymes were still very low. Interestingly, in contrast with young mice, testicular degeneration at 15 months was significantly less severe marked in VIP-/-mice than in WT mice. Altogether, these results suggest that: 1) VIP is an important factor for regulating testosterone biosynthesis and FSH secretion and 2) VIP regulates testicular aging.