In vitro regulation of extracellular superoxide dismutase in sertoli cells

Rat Sertoli and germ cells express extracellular superoxide dismutase (SOD(EX)), however, the relative level of SOD(EX) expressed by these cells was not known. We report herein germ cells consisting largely of spermatogonia, spermatocytes, and round spermatids expressed only one-third SOD(EX) as that of Sertoli cells when examined by semi-quantitative RT-PCR. While cocultures of germ cells with Sertoli cells failed to induce any changes in SOD(EX) expression possibly due to the limited number of cells that can be supported by the in vitro culture system dissimilar to the in vivo condition, incubation of total germ cell-conditioned medium with Sertoli cells was able to significantly inhibit Sertoli cell SOD(EX) expression dose-dependently suggesting a germ cell-derived soluble factor(s) may regulate SOD(EX) in the testis. On the other hand, cytokines such as TGF-beta1, beta-NGF, or FGF and steroid hormones such as estradiol-17beta, progesterone, testosterone, and DHT were unable to effect the expression of Sertoli cell SOD(EX). However, FSH at 100 ng/dish was able to induce a significant increase in Sertoli cell SOD(EX) expression. While cytokines, the known mediators of the inflammatory response, were unable to affect Sertoli cell SOD(EX) expression, the induction of generalized inflammation in vivo was able to cause a 2- to 2.5-fold increase in testicular SOD(EX) expression concomitant with a transient increase in the liver but not in the brain. Taken collectively, these results demonstrate that while SOD(EX) is an important antioxidant enzyme protecting the testis from reactive oxygen species, the mechanism(s) regulating its expression may involve an array of molecules and is a complicated cellular event.     

IL17A impairs blood–testis barrier integrity and induces testicular inflammation

Experimental autoimmune orchitis is a useful model for studying testicular inflammation and germ/immune cell interactions. Th17 cells and their hallmark cytokine IL17A were reported to be involved in the development of autoimmune orchitis. The aim of the present work is to investigate the pathogenic role of IL17A in rat testis. In vitro experiments were performed in order to analyze effects of IL17A on Sertoli cell tight junctions. The addition of IL17A to normal rat Sertoli cell cultures induced a significant decline in transepithelial electrical resistance and a reduction of occludin expression and redistribution of occludin and claudin 11, altering the Sertoli cell tight junction barrier. Intratesticular injection of 1 μg of recombinant rat IL17A to Sprague–Dawley rats induced increased blood–testis barrier permeability, as shown by the presence of biotin tracer in the seminiferous tubule adluminal compartment, and delocalization of occludin and claudin 11. Results showed that IL17A induced focal inflammatory cell infiltration in the interstitium and germ cell sloughing in adjacent seminiferous tubules. Moreover, an increase in TUNEL+ apoptotic germ cells was also observed. Inflammatory ED1+ macrophages were the main population infiltrating the interstitium following IL17A injection. This correlated with an increase in mRNA expression of the monocyte chemoattractant protein Ccl2, its receptor Ccr2 and the vascular cell adhesion molecule Vcam1. Overall results suggest a relevant role of IL17A in the development of testicular inflammation, facilitating the recruitment of immune cells to the testicular interstitium and inducing impairment of blood–testis barrier function.     

Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells.

The expression of the mRNA for SLF (the c-kit ligand), a product of the "steel" locus, has been investigated in postnatal mouse testis and homogeneous populations of testicular cells. The message was found expressed in postnatal mouse testis but not in germ cells. Studies on primary mouse Sertoli cell cultures from 18 day old mice show that Sertoli cells are the site of SLF mRNA expression in the seminiferous tubules. Treatment of Sertoli cell cultures with cAMP analogs led to a significant increase in the SLF mRNA levels.     

A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.

When Sertoli cells were cultured in vitro on Matrigel-coated bicameral units, the assembly of the inter-Sertoli tight junction (TJ) permeability barrier correlated with an induction of occludin expression. Inclusion of a 22-amino acid peptide, NH(2)-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to residues 209-230 in the second extracellular loop of rat occludin, at 0.2-4 microM into Sertoli cell cultures could perturb the assembly of Sertoli TJs dose-dependently and reversibly. This peptide apparently exerts its effects by interfering with the homotypic interactions of two occludin molecules between adjacent Sertoli cells at the sites of TJs, thereby disrupting TJs, which, in turn, causes a decline in transepithelial electrical resistance across the Sertoli cell epithelium. When similar experiments were performed using a 22-amino acid myotubularin peptide, NH(2)-TKVNERYELCDTYPALLAVPAN-COOH (residues 156-177), no effects on the assembly of inter-Sertoli TJs in vitro were noted. When a single dose of this synthetic occludin peptide was administered to adult rats intratesticularly at 1.5-10 mg/testis, germ cells began to deplete from the seminiferous epithelium within 8-16 days. By 27 days, virtually all tubules were devoid of germ cells. This antispermatogenic effect was reversible, because germ cells progressively repopulated the epithelium thereafter. Treated testes were indistinguishable from normal or control testes by 68 days post-occludin peptide treatment when assessed using histological analysis. In contrast, control rats receiving either no treatment, vehicle alone, or a 22-amino acid synthetic peptide of myotubularin displayed no changes in the testicular morphology at all time points. The occludin peptide-induced germ cell depletion was also accompanied by a disruption of the blood-testis barrier (BTB) when assessed by micropuncture techniques quantifying [(125)I]-BSA in rete testis fluid and seminiferous tubular fluid following i.v. administration of [(125)I]-BSA through the jugular vein. These results illustrate that the occludin peptide-induced disruption of the BTB may possibly affect the underlying adherens junctions, which causes premature release of germ cells from the epithelium and reversible infertility.     

The role of tumor necrosis factor-alpha and interleukin-1 in the mammalian testis and their involvement in testicular torsion and autoimmune orchitis

This review will focus the roles of TNF-alpha, IL-1 alpha, and IL-1 beta in the mammalian testis and in two testicular pathologies, testicular torsion and orchitis. TNF alpha in the testis is produced by round spermatids, pachytene spermatocytes, and testicular macrophages. The type 1 TNF receptor has been found on Sertoli and Leydig cells and numerous studies suggest a paracrine mode of action for TNF alpha in the normal testis. IL-1 alpha has been reported to be produced by Sertoli cells, testicular macrophages, and possibly postmeiotic germ cells. IL-1 receptors have been reported on Sertoli cells, Leydig cells, testicular macrophages, and germ cells suggesting both autocrine and paracrine functions. While these proinflammatory cytokines have important roles in normal testicular homeostasis, an elevation of their expression can lead to testicular dysfunctions. Testicular torsion is a clinical pathology with results in testicular ischemia and surgical intervention is often required for reperfusion. A pivotal role for IL-1beta in the pathology of testicular torsion has been recently described whereby an increase in IL-1beta production after reperfusion of the testis is correlated with the activation of the stress-related kinase, c-jun N-terminal kinase, and ultimately resulting in neutrophil recruitment to the testis and germ cell apoptosis. In autoimmune orchitis, on the other hand, TNF alpha produced by T-lymphocytes and macrophages of the testis has been implicated in the development and progression of the disease. Thus, both proinflammatory cytokines, TNF alpha and IL-1, have significant roles in normal testicular functions as well as in certain testicular pathologies.     

Effect of neonatal gonadotropin-releasing hormone antagonist administration on sertoli cell number and testicular development in the marmoset: comparison with the rat

The primary purpose of this study was to establish whether Sertoli cells proliferate in the neonatal period in the marmoset monkey (Callithrix jacchus) and whether administration of a long-acting GnRH antagonist (GnRHa) during this phase induced any transient or permanent effects on Sertoli cell number or on any other aspect of testicular development. Male marmoset co-twins (n = 9) were treated during Weeks 1-14 with either vehicle or GnRHa. Four sets of co-twins were examined at Weeks 18-22 (start of infancy) and 5 sets in adulthood (92+ wk), and Sertoli cell number was determined using either the nucleator or optical disector methods; other testicular morphometric analyses (e.g., germ cell volume, Leydig cell volume) used standard point-counting. Data for the marmoset were compared with that obtained in similarly treated rats. Sertoli cell number in marmosets treated neonatally with GnRHa was reduced by 35% compared with that of controls at Weeks 18-22 but was comparable to control values in adulthood. However, seminiferous epithelium volume was reduced significantly in adult marmosets treated neonatally with GnRHa, and there was a tendency for reduced germ cell volume per Sertoli cell. In the same animals, there was significant expansion of the interstitium and an increase in Leydig cell volume per testis when compared with co-twin controls; a similar increase in Leydig cell volume was evident in adult rats treated neonatally with GnRHa. Comparison of Sertoli cell numbers in 6 infantile (18-24 wk) and 10 adult marmosets showed that adult numbers of Sertoli cells were present by the start of infancy but, unlike rats, marmosets were still able to replicate Sertoli cells beyond this period. However, marmoset Sertoli cells supported only approximately 20% of the germ cell volume supported by rat Sertoli cells, indicative of poor efficiency of spermatogenesis, as shown previously in the human. This finding, together with the demonstration of a temporal pattern of Sertoli cell replication similar to that in the human, supports the use of marmosets as a model for human male testicular development and function.     

硝基还原酶结构域蛋白1在大鼠睾丸组织中特异性高表达在人类睾丸癌中表达下调

通过克隆分离鉴定得到大鼠硝基还原酶结构域蛋白1(rNOR1),发现rNOR1 cDNA含有1 418 个碱基,编码含379个氨基酸残基的rNOR1蛋白．rNOR1与人类NOR1 (hNOR1)和小鼠NOR1 (mNOR1)的同源性分别为89% 和93%,这三种同源蛋白都含有OSCP1家族的保守结构域．rNOR1基因在大鼠睾丸中选择性高表达,而且与之同源的人类hNOR1也选择性高表达于睾丸中．通过免疫组化检测人类不同睾丸癌中的hNOR1蛋白表达,发现hNOR1蛋白在非癌变睾丸组织和胚胎性癌组织中高表达,而在精原细胞癌和分化型非精原细胞癌(畸胎瘤,卵黄囊瘤)中低表达．这些数据表明,hNOR1可能是一种睾丸选择性表达基因,睾丸癌hNOR1表达的改变或许可以帮助我们阐明hNOR1蛋白在生殖细胞系肿瘤发生中的功能．     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Quantitative studies of compensatory testicular hypertrophy following unilateral castration in the boar

Unilateral castration of Large White X Landrace boars at monthly intervals up to 5 months of age, with the remaining testis being removed 2 months later, resulted in compensatory hypertrophy of the testis which decreased with age. In pigs 3 and 4 months old there was significant hypertrophy of the testis but at 5 and 7 months of age testicular weight of the hemicastrates did not differ significantly from control values. The increase in the testicular weight of unilaterally castrated pigs was correlated with an increase in the number of Sertoli and germ cells at 3 months of age and germ cells at 4 months of age occupying the seminiferous epithelium. This was correlated with increased total seminiferous tubule length and larger cross-sectional area of the tubule. Sertoli cell occupancy did not differ significantly between unilaterally castrated and intact boars.     

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood-testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150(Glued), EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.     

Impact of methoxyacetic acid on mouse Leydig cell gene expression

Background  

Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function.     

Role of β-catenin in post-meiotic male germ cell differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.     

Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-beta-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome ct and lactate dehydrogenase-C4 isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.     

Differential expression of Prx I and II in mouse testis and their up-regulation by radiation

Testis is one of the most sensitive organs to ionizing radiation. The present study was designed to unravel the possible role of antioxidant proteins, peroxiredoxin I and II (Prx I and II) in the testis. Our results show that Prx I and II are constitutively expressed in the testis and their expression levels are decreased to some extent as the testis develops. Interestingly, immunohistochemical analysis revealed a preferential expression of Prx I and II in Leydig and Sertoli cells, respectively. Neither Prx I nor Prx II expression was obvious in the testicular germ cells including spermatogonia and spermatocytes. Ionizing radiation exerted oxidative stress on the testis and induced apoptosis primarily in the germ cells. When the irradiated testis was examined, the Prx system was found to be transiently up-regulated. Taken together, we suggest that the relative radiation-resistance of Leydig and Sertoli cells could be attributed in part to the antioxidant function of the Prx system in these cells.     

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis

Summary. Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s K_m was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).     

mRNAs encoding a von Ebner's-like protein and the Huntington disease protein are induced in rat male germ cells by Sertoli cells

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.