Effect of cadmium on the regulatory mechanism of steroidogenic pathway of Leydig cells during spermatogenesis.

Cadmium is a male reproductive toxicant that interacts with a variety of pathogenetic mechanisms. However, the effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis is still ambiguous. Light microscopy, Western blot, immunohistochemistry, immunofluorescence, and quantitative polymerase chain reaction were performed to study the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis. The results indicated that in the control group, Leydig cells showed dynamic immunoreactivity and immunosignaling action with a strong positive significant secretion of 3ß-hydroxysteroid hydrogenase (3ß-HSD) in the interstitial compartment of the testis. Leydig cells showed a high active regulator mechanism of the steroidogenic pathway with increased the proteins and genes expression level of steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol (CYP11A1), cytochrome P450 cholesterol (CYP17A1), 3ß-hydroxysteroid hydrogenase (3ß-HSD) 17ß-hydroxysteroid hydrogenase (17ß-HSD), and androgen receptor (AR) that maintained the healthy and vigorous progressive motile spermatozoa. However, on treatment with cadmium, Leydig cells were irregularly dispersed in the interstitial compartment of the testis. Leydig cells showed reduced immunoreactivity and immunosignaling of 3ß-HSD protein. Meanwhile, cadmium impaired the regulatory mechanism of the steroidogenic process of the Leydig cells with reduced protein and gene expression levels of STAR, CYP11A1, CYP17A1, 3ß-HSD, 17ß-HSD, and AR in the testis. Additionally, treatment with cadmium impaired the serum LH, FSH, and testosterone levels in blood as compared to control. This study explores the hazardous effect of cadmium on the regulatory mechanism of the steroidogenic pathway of Leydig cells during spermatogenesis.

Mouse-human species differences in early testicular development and its implications.

The mouse has been used as a model of human organogenesis with the tacit assumption that morphogenetic and molecular mechanisms in mice are translatable to human organogenesis. While many morphogenetic and molecular mechanisms are shared in mice and humans, many anatomic, morphogenetic, and molecular differences have been noted. Two critical gaps in our knowledge prevent meaningful comparisons of mouse versus human testicular development: (a) human testicular development is profoundly under-represented in the literature, and (b) an absence of a detailed day-by-day ontogeny of mouse testicular development from E11.5 to E16.5 encompassing the ambisexual stage to seminiferous cord formation. To address these deficiencies, histologic and immunohistochemical studies were pursued in comparable stages of mouse and human testicular development with a particular emphasis on Leydig, Sertoli and myoid cells through review of the literature and new observations. For example, an androgen-receptor-positive testicular medulla is present in the developing human testis but not in the developing mouse testis. The human testicular medulla and associated mesonephros were historically described as the source of Sertoli cells in seminiferous cords. Consistent with this idea, the profoundly androgen receptor (AR)-positive human testicular medulla was shown to be a zone of mesenchymal to epithelial transition and a zone from which AR-positive cells appear to migrate into the human testicular cortex. While mouse Sertoli and Leydig cells have been proposed to arise from coelomic epithelium, Sertoli (SOX9) or Leydig (HSD3B1) cell markers are absent from the immediate coelomic zone of the developing human testis, perhaps because Leydig and Sertoli cell precursors are undifferentiated when they egress from the coelomic epithelium. The origin of mouse and human myoid cells remains unclear. This study provides a detailed comparison of the early stages of testicular development in human and mouse emphasizing differences in developmental processes.

Testicular microlithiasis in paediatric patients with Klinefelter syndrome from infancy till adolescence: early start of degenerative process in the testes-preliminary results.

To present the results of testicular ultrasonography supported by clinical and hormonal aspects in paediatric patients with Klinefelter syndrome (KS). Prospective analysis of medical files of 20 patients diagnosed with KS between 2016 and 2022. Assessed data included analysis of causes of referral, ultrasound, and clinical characterisation with hormonal evaluation of serum FSH, LH, testosterone, inhibin B, and anti-Müllerian hormone. Non-mosaic Klinefelter syndrome (47, XXY) was diagnosed in 65% of cases (13/20) by the geneticist (including 7 cases prenatally), in 25% (5/20) by the endocrinologist and in 10% (2/20) by the hematologist. Ultrasound assessment revealed bilateral testicular microlithiasis (TM) in all patients. The youngest KS patient with TM was 3 months old. TM patterns have not changed during follow-ups of up to 6 years in any of the patients. In all KS patients markedly reduced echogenicity and in pubertal KS patients, also irregular echostructure of the testes was observed. The hormonal patterns observed in the study group were typical for those already described in KS. Sertoli and Leydig cell function was intact in prepubertal patients and deteriorated after the start of puberty. CONCLUSION: Although the degenerative process in the testicular tissue starts very early in the testes in KS and is reflected in morphological changes seen in ultrasonography, Sertoli and Leydig cell hormonal function is normal in prepubertal KS patients. WHAT IS KNOWN: • So far, normal Leydig and Sertoli cell function was observed in infants and prepubertal KS patients. WHAT IS NEW: • The morphological changes in the testes in KS may already be seen in early infancy.

MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

Effective and economical column-based method for RNA isolation from animal cells.

OBJECTIVES: To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. RESULTS: Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 106 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. CONCLUSIONS: The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.

Characterization, recellularization, and transplantation of rat decellularized testis scaffold with bone marrow-derived mesenchymal stem cells.

BACKGROUND: Regenerative medicine potentially offers the opportunity for curing male infertility. Native extracellular matrix (ECM) creates a reconstruction platform to replace the organs. In this study, we aimed to evaluate the efficiency of the testis decellularized scaffold as a proper niche for stem cell differentiation toward testis-specific cell lineages. METHODS: Rats' testes were decellularized by freeze-thaw cycle followed by immersion in deionized distilled water for 2 h, perfused with 1% Triton X-100 through ductus deferens for 4 h, 1% SDS for 48 h and 1% DNase for 2 h. The decellularized samples were prepared for further in vitro and in vivo analyses. RESULT: Histochemical and immunohistochemistry studies revealed that ECM components such as Glycosaminoglycans (GAGs), neutral carbohydrate, elastic fibers, collagen I & IV, laminin, and fibronectin were well preserved, and the cells were completely removed after decellularization. Scanning electron microscopy (SEM) showed that 3D ultrastructure of the testis remained intact. In vivo and in vitro studies point out that decellularized scaffold was non-toxic and performed a good platform for cell division. In vivo implant of the scaffolds with or without mesenchymal stem cells (MSCs) showed that appropriate positions for transplantation were the mesentery and liver and the scaffolds could induce donor-loaded MSCs or host migrating cells to differentiate to the cells with phenotype of the sertoli- and leydig-like cells. The scaffolds also provide a good niche for migrating DAZL-positive cells; however, they could not differentiate into post meiotic-cell lineages. CONCLUSION: The decellularized testis can be considered as a promising vehicle to support cell transplantation and may provide an appropriate niche for testicular cell differentiation.

Localization of EFA6 (exchange factor for ARF6) isoform D in steroidogenic testicular Leydig cells of adult mice.

EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP and the resulting activated form of Arf6 is involved in the membrane trafficking and actin remodeling of cells. Our previous study has shown the selective expression/localization of EFA6D in steroidogenic adrenocortical cells in situ of adult mice. In view of the previous finding, the present study was undertaken to examine its localization in mouse Leydig cells representing another steroidogenic cell species in order to further support the possible involvement of the EFA6/Arf6 cascade via membrane trafficking in the regulation of steroidogenesis and/or secretion. A distinct band for EFA6D with the same size as that of the brain was detected in the testis of adult mice. In immuno-light microscopy, immunoreactivity for EFA6D was seen throughout the cytoplasm in most Leydig cells without any distinct accumulation along the plasmalemma. Lack of immunoreactivity for EFA6D was seen in the seminiferous tubular epithelium. In immuno-electron microscopy, the immune-labeling was seen in sporadic/focal patterns on plasma membranes and some vesicles and vacuoles subjacent to the plasma membranes. More constant and rather predominant is the labeling on numerous mitochondria. No immuno-labeling was seen in lipid droplets. The present study suggests that EFA6D is somehow involved in regulation of the synthesis and/or secretion of testosterone through the membrane-traffic by activation of Arf6. In addition, EFA6D is suggested to play in mitochondria some yet unidentified roles rather independent of Arf6-activation, which remains to be elucidated.

Expression patterns and changes of the LCN2 gene in the testes of induced cryptorchidism and busulfan-treated mice.

Lipocalin-2 (LCN2) was known to play various roles in different type cells; however, little was known about the effect of LCN2 in male fertility. In this study, we aimed to explore the expression pattern of LCN2 with increasing age in mice, and to obtain insight into the role of LCN2 in mice testes by induced cryptorchidism and busulfan-treated infertility. In situ hybridization showed that LCN2 was localized primarily in Leydig cells, but was absent in Sertoli and germ cells. Its expression in testes exhibited an age-related increase from day 1 to 8 months, then reduced by the twelth month. The mRNA and protein levels of LCN2 in the testes of both infertile models increased as measured by real-time PCR and western blotting, respectively. LCN2 mRNA and protein levels were higher (p<0.05) in busulfan treated mice than that of cryptorchidism. These observations have shown that LCN2 is developmentally regulated and highly expressed in the Leydig cells of mouse testes.

Valve interstitial cell culture: Production of mature type I collagen and precise detection.

Collagen often acts as an extracellular and intracellular marker for in vitro experiments, and its quality defines tissue constructs. To validate collagen detection techniques, cardiac valve interstitial cells were isolated from pigs and cultured under two different conditions; with and without ascorbic acid. The culture with ascorbic acid reached higher cell growth and collagen deposition, although the expression levels of collagen gene stayed similar to the culture without ascorbic acid. The fluorescent microscopy was positive for collagen fibers in both the cultures. Visualization of only extracellular collagen returned a higher correlation coefficient when comparing the immunolabeling and second harmonic generation microscopy images in the culture with ascorbic acid. Lastly, it was proved that the hydroxyproline strongly contributes to the second-order susceptibility tensor of collagen molecules, and therefore the second harmonic generation signal is impaired in the culture without ascorbic acid.

Different expression of leptin and IGF1 in the adult and prepubertal testis in dogs.

Leptin (Lep) and insulin-like growth factor 1 (IGF1) are implicated in the regulation of testicular function, but in dogs, our knowledge is limited to the possible role of the IGF1 system in testicular tumours. In this study, we aimed to describe and compare gene expression and protein localization of Lep, IGF1 and their receptors (LepR and IGF1R, respectively) in the testis of healthy adult and prepubertal dogs. Testes were collected from sexually healthy mature (n = 7) and from 8-week-old dogs (n = 7). Relative gene expression of Lep, LepR, IGF1 and IGF1R was determined by semi-quantitative real-time (TaqMan) PCR and cellular distribution in the testis by immunohistochemistry. Statistical analysis was carried out with Student's t test. Lep and LepR mRNA concentration was similar between the two groups, but IGF1 and IGF1R gene expression was significantly higher in the 8-week-old pups. Protein localization and the intensity of signals differed by age. In adults, Lep and LepR immunoreactivity was detected in spermatocytes and spermatids. Leydig cells showed sporadic, weak Lep staining. In prepubertal animals, intense Lep signals were present in Leydig and Sertoli cells, and LepR was found in Leydig cells. IGF1 and IGF1R protein was expressed in spermatogonia of the mature testis; IGF1 signals in Leydig cells seemed stronger than IGF1R. In the pups, IGF1 and IGF1R staining was detected in Leydig cells and in gonocytes. Sertoli cells showed weak IGF1 and sporadic, weak IGF1R signals. In conclusion, Lep and IGF1 may support spermatogenesis in adult dogs and mediate Leydig cell function. In the immature testis, they may promote development of Sertoli and Leydig cells and gonocytes.

Changes in Testicular Interstitial Connective Tissue of Hamsters (Mesocricetus auratus) During Ageing and After Exposure to Short Photoperiod.

The testicular interstitium of Syrian hamster (Mesocricetus auratus) was studied during ageing and in testicular regression after exposure to a short photoperiod, in relation to the interstitial cells and their connective tissue. This tissue was assessed histochemically using Masson's trichrome technique and the expression of Heat Shock Protein 47 (HSP-47) and collagen IV (α5) was assessed in Leydig cells. Finally, an ultrastructural analysis of some cells of the testicular interstitium was made. Leydig cells were positive for HSP-47 and collagen IV (α5). Ageing did not change the parameters studied while the short photoperiod altered the synthetic activity of Leydig cells. The positivity index of these cells for HSP-47 was significantly higher in the regressed testis, but was lower for collagen IV (α5). During ageing no change were observed. Ultrastructural Leydig cells showed a discontinuous basal lamina that did not change during ageing. The basal lamina was not identified in Leydig cells regressed by exposure to a short photoperiod. In conclusion; the intertubular connective tissue suffers little change with age. By contrast, in the testis regressed after exposure to a short photoperiod the studied parameters related to the intertubular connective tissue were altered. These changes are probably related with the low synthetic activity of regressed Leydig cell.

Expression analyses of insulin-like peptide 3, RXFP2, LH receptor, and 3ß-hydroxysteroid dehydrogenase in testes of normal and cryptorchid dogs.

Insulin-like peptide 3 (INSL3) plays a key role in testicular descent in rodents, whereas in domestic animals, many aspects of the roles of INSL3 in reproductive organs after puberty are still unknown. This study was undertaken to (1) determine the quantitative changes of gene expression of testicular INSL3, its receptor (RXFP2), LH receptor, and 3ß-hydroxysteroid dehydrogenase during and after puberty in normal male dogs; (2) compare the expressions of these substances in normal and cryptorchid dogs; and (3) localize the cells expressing INSL3 in normal and retained canine testes. Testes were obtained from small-breed normal male dogs (n = 56) and cryptorchid dogs (n = 22). Normal scrotal testes from the normal dogs (normal testes), retained testes from both the unilateral and bilateral cryptorchid dogs (retained testes), and scrotal testes of the unilateral cryptorchid dogs (cryptorchid scrotal testes) were used. We measured the concentrations of these testicular messenger RNAs (mRNAs) by quantitative real-time reverse transcription polymerase chain reaction, and an enzyme immunoassay was used for measuring INSL3 peptide. Immunohistochemistry for INSL3 peptide was done in paraformaldehyde-fixed frozen testicular tissue. In the normal dogs, total amount of INSL3 mRNA per testis tended to decrease (P = 0.05) from pubertal (6-12 months) to postpubertal (1-5 years) and decreased (P < 0.01) to middle age (5-10 years), but total amount of INSL3 peptide per testis did not change among age groups. Concentrations of INSL3 mRNA were higher (P < 0.01) in retained testes than those in the normal testes and cryptorchid scrotal testes, and similar differences were observed for INSL3 peptide. Reversely, total amounts of INSL3 mRNA and peptide per retained testis were lower (P < 0.01) than those per normal testis because of smaller weight of retained testes. Concentrations and total amount of RXFP2 mRNA in the retained testes were almost nil and lower (P < 0.01) than those in the normal testes and in the cryptorchid scrotal testes. Total amount of LH receptor mRNA per retained testis was lower (P < 0.01) than that per normal testis. The immunohistochemical analysis revealed that INSL3 was expressed only in Leydig cells of both the normal and retained canine testes. These results suggest that INSL3 in retained testes of cryptorchid dogs is substantially expressed per unit-weight basis but may be produced with lower amount as a whole testis. Also, this study provides findings that RXFP2 gene is expressed scarcely in the retained testes but normally in cryptorchid scrotal testes.

Levels of liver X receptors in testicular biopsies of patients with azoospermia.

OBJECTIVE: To determine whether the transcription factors liver X receptors (LXRs) and their downstream genes, which are involved in the regulation of several testicular functions in mouse models, are differentially expressed in testes of men with nonobstructive azoospermia (NOA) or obstructive azoospermia (OA). DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Patients with various types of NOA (n=22) and with OA (n=5). INTERVENTION(S): Human testicular biopsies. MAIN OUTCOME MEASURE(S): Transcript levels were measured in testicular biopsies with the use of quantitative polymerase chain reaction. Correlations of LXR mRNA levels with the number of germ cells, the expression of proliferation and apoptosis markers, and the amount of intratesticular lipids and testosterone were evaluated. The localization of LXRα was analyzed by immunofluorescence. RESULT(S): LXR mRNA levels were decreased by 49%-98% in NOA specimens and positively correlated with germ cell number. Accumulations of IDOL and SREBP1c (LXR targets involved in lipid homeostasis) were 1.8-2.1 times lower in NOA samples and mRNA levels of the SREBP1c target gene ELOVL6 were increased 1.9-2.4-fold. Interestingly, the amount of triglycerides and free fatty acids were higher in NOA testes (3.4-12.2-fold). LXRα was present in Leydig cells. Accumulations of LXR downstream genes encoding the steroidogenic proteins StAR and 3ßHSD2 were higher in NOA testes (5.9-12.8-fold). CONCLUSION(S): Knowledge of changes in the transcript levels of LXRs and some of their downstream genes during altered spermatogenesis may help us to better understand the physiopathology of testicular failure in azoospermic patients.

Molecular cloning of VIP and distribution of VIP/VPACR system in the testis of Podarcis sicula.

Using molecular, biochemical, and cytological tools, we studied the nucleotide and the deduced amino acid sequence of PHI/VIP and the distribution of VIP/VPAC receptor system in the testis of the Italian wall lizard Podarcis sicula to evaluate the involvement of such a neuropeptide in the spermatogenesis control. We demonstrated that (1) Podarcis sicula VIP had a high identity with other vertebrate VIP sequences, (2) differently from mammals, VIP was synthesized directly in the testis, and (3) VIP and its receptor VPAC2 were widely distributed in germ and somatic cells, while the VPAC1 R had a distribution limited to Leydig cells. Our results demonstrated that in Podarcis sicula the VIP sequence is highly preserved and that this neuropeptide is involved in lizard spermatogenesis and steroidogenesis.

Immunolocalization of aquaporin water channels in the domestic cat male genital tract.

Four different aquaporins (AQP1, 2, 5 and 9), integral membrane water channels that facilitate rapid passive movement of water, were immuno-localized in the excurrent ducts collected from sexually mature cats during orchiectomy. Aquaporins 1, 2 and 9, were immuno-localized at distinct levels, whereas AQP5 was undetectable all along the feline genital tract. No immunoreactivity was present at the level of the rete testis with any of the antibodies tested. In the efferent ducts, AQP1-immunoreactivity was strongly evidenced at the apical surface of the non-ciliated cells, and AQP9-immunoreactivity was shown at the periphery of both ciliated and non-ciliated cells. Aquaporins 2 was absent in the caput epididymidis, either in the efferent ducts or in the epididymal duct. Otherwise, AQP2 was increasingly localized at the adluminal surface of principal cells from the corpus to the cauda epididymidis and more weakly in the vas deferens epithelium. The supranuclear zone of the epididymal principal cells was AQP9-immunoreactive throughout the duct, with the exclusion of the vacuolated sub-region of the caput and with higher reaction intensity in the cauda region. AQP1 was present in the blood vessels all along the genital tract. AQP1 was expressed also in the smooth muscle layer of the vas deferens. The tested AQP molecules showed a different expression pattern in comparison with laboratory mammals, primates and the dog, unique other carnivore species studied to date. The present information is possibly useful in regard to the regional morphology of the feline epididymis and correlated functions, which are still a matter of debate.

The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells.

Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

Region-specific expression of aquaporin subtypes in equine testis, epididymis, and ductus deferens.

The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, -4, -5, -8, -10, and -11); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, -3, -7, and -9). Overall, we inferred that fluid transport in the stallion testicle involved a collaboration of AQP subtypes (primarily AQP2, -5, -7, and -8). Based on immunohistochemistry, expression of AQP subtypes analyzed (i.e., AQP0, -2, -5, and -9) was localized to Leydig cells and elongated and round spermatids. Functional significance of AQP expression by Leydig cells remained uncertain. In elongated and round spermatids, AQP s likely contributed to the volume reduction observed during spermatogenesis. Subtypes AQP2 and AQP9 were the predominant forms expressed in epididymal tissue. Regulation of AQP2 expression, especially in the epididymal head, seemed to occur at the post-transcriptional level, as protein expression upon immunohistochemistry was pronounced, despite low transcript abundance. In epididymal tissue, AQPs likely contributed to fluid resorbtion, given their localization on the apical membrane of principal cells.

Myeloid cell leukemia-1 (Mc1-1) is a candidate target gene of hypoxia-inducible factor-1 (HIF-1) in the testis.

BACKGROUND: Spermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5'-RCGTG-3') identified myeloid cell leukemia-1 (Mcl-1) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1. METHODS: The testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis. RESULTS: The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter. CONCLUSIONS: The results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.

Comparative evaluation of nonylphenol isomers on steroidogenesis of rat Leydig Cells.

Nonylphenol (NP) has been proven to be one of the most investigated xenohormones interacting with the estrogen receptor. Technical nonylphenol (t-NP) contains at least 20 para-substituted isomers. It has been shown that NP isomers vary in their estrogenic potency. So the use of mixtures or impure substances can lead to misinterpretations and unsatisfying conclusions. In the present study, experiments were performed to examine effects of NP isomers on steroidogenesis of rat Leydig cells. Primary cultured Leydig cells were exposed to NP isomers (p33-NP, p262-NP, p353-NP, p363-NP) at the optimized inhibitory concentration 5µmol/L for 6h. NP isomers showed various degrees of inhibition of testosterone biosynthesis, with p363-NP leading to the most significant decrease and others sharing the similar efficacy. The expression of 3b-HSD, Cyp11a1, Star and the apoptosis of Leydig cells were further measured to investigate the underlying mechanisms. We demonstrated that NP isomers can affect the steroidogenesis of rat Leydig cells, at least in part, through their influence on gene expression and cell apoptosis, but varied in their individual degree. However, the final results were not completely coincident with their estrogenic potency tested in vitro, which implies that effects of NP isomers on steroidogenesis appear to be mediated through some other underlying mechanisms besides their various estrogenic potency.

Immunohistochemical localization of transforming growth factor ß1 and ß2 in mouse testes during postnatal development.

We examined age-related changes in the expression of transforming growth factor-ß(1) (TGF-ß(1)) and transforming growth factor-ß(2) in mouse testes. The mice were assigned to three age groups: 35, 50, and 75 days old. Paraffin embedded testis sections were processed for the standard streptavidin biotin peroxidase complex immunohistochemistry method. TGF-ß(1) expression increased in aging round spermatids over the time studied. There was no expression in 35-day-old Leydig cells, whereas strong expression of TGF-ß(1) was observed in 50-day-old Leydig cells. Expression decreased in 75-day-old Leydig cells. TGF-ß(2) expression was weak in 35- and 50-day-old mouse spermatids, but expression was greater in 75-day-old elongated spermatids. In Leydig cells, TGF-ß(2) expression was strong in both 35- and 50-day-old mice, whereas the expression of TGF-ß(2) was less in 75-day-old Leydig cells. Our results suggest that TGF-ß(1) and TGF-ß(2) may play significant roles in testicular functions and germ cell development in mice.