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1.
Expression of the protein DDX4/MVH, or VASA, has been reported in germ cells of several species. The main objectives of this study were to (i) investigate VASA expression patterns in testicular cells of stallions at two different reproductive stages (pre‐pubertal and post‐pubertal) and (ii) evaluate the use of VASA antibody as a molecular marker for single germ cells from stallions. Testicular tissues were obtained from stallions and categorized as pre‐pubertal and post‐pubertal based on the formation of lumen and status of spermatogenesis on the cross section of seminiferous tubules. The results of Western blot showed a VASA protein band located at 76 kDa, indicating that the rabbit antibody has a cross‐reactivity with horse testicular tissues. The result of immunolabelling showed that VASA was expressed in the cytoplasm of spermatogonia at both reproductive stages and in spermatocytes and round spermatids at the post‐pubertal stage. GATA4‐positive Sertoli cells and Leydig cells located in the interstitial space were not immunolabelled with VASA. These results suggest that VASA can be utilized as a molecular marker for germ cells of stallions at pre‐pubertal and post‐pubertal stages. Interestingly, immunolabelling intensity was significantly higher in pachytene spermatocytes compared to spermatogonia and round spermatid. VASA antibody was also effective for staining of single germ cell preparations. In conclusion, VASA protein expression can be used as a marker for identification of spermatogonia, spermatocytes and round spermatids in testicular tissues of stallions.  相似文献   

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3.
Reasons for performing study: Specific patterns of cytoskeletal filaments reflect a functional state of the cell. In testicular cells intermediate filaments (IFs) are of the vimentin type. Since it is known that Sertoli cells regulate the spermatogenic function in the male gonad, it became important to propose a system that could quantify the state of seminiferous tubular quality. To date, a Johnsen score system has never been used to equine testes. Objectives: To demonstrate the expression pattern of vimentin in testes of mature Arabian stallions and correlate its distribution with grade of seminiferous tubule impairment as indicated by a Johnsen score. Methods: For histological examination by the Johnsen method, routine haematoxylin‐eosin staining was used. Vimentin expression and its presence in testicular sections and testicular homogenates were detected by immunohistochemistry and western blot, respectively. Both analyses were performed qualitatively and quantitatively and further validated by ANOVA tests. Results: Distinct morphology of seminiferous tubules was found in testes harvested from 3 stallions. Vimentin in IFs was immunolocalised to the cytoplasm of Sertoli, Leydig and peritubular‐myoid cells. The intensity and pattern of the IFs staining was different in individual seminiferous tubules suggesting a correlation between vimentin expression and the severity of tubule degeneration. Qualitative results by immunohistochemistry and western blot were confirmed by further quantitative analyses. Conclusions: In equine testes, differential expression of vimentin was found to be correlated with the impairment of seminiferous tubules indicated by a decrease in Johnsen score. Potential relevance: The Johnsen score system may be a useful method to facilitate the identification of tubular alterations in the stallion testes. Combined histological and immunohistochemical approach may provide a detailed phenotypic classification of stallions with decreased fertility.  相似文献   

4.
Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3 betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.  相似文献   

5.
Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β‐hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid‐metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre‐pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real‐time PCR (qRT‐PCR) were used. Pre‐pubertal horses showed higher testicular gene expression of 11βHSD1, 11βHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11βHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)‐dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11βHSD enzymes and the oxidative 11βHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.  相似文献   

6.
Thirty-four pairs of testes from wild adult raccoon dogs (Nyctereutes procyonoides) were obtained between September 2000 and May 2003. The cellular localization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits in wild raccoon dog testes was investigated. The testicular weight and size and seminiferous tubule diameters were measured. There were marked seasonal variations in testicular weight and size and seminiferous tubule diameters, with values relatively low in September and high in March. Spermatogonia and primary spermatocytes were observed in September, and spermatogonia, spermatocytes, and round spermatids were present in January. All types of spermatogenic cells, including mature spermatozoa, were found in March, indicating that the breeding season is around March in Japan. Thereafter, spermatogonia and degenerating spermatocytes were observed in April. The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB. The inhibin alpha and inhibin/activin (betaA and betaB) subunits were only expressed in Leydig cells in September. On the other hand, the inhibin alpha, betaA, and betaB subunits were observed in Leydig cells and Sertoli cells, but not in germ cells, in March. These results suggest that the testes of wild raccoon dogs have the ability to synthesize inhibins, and the cellular localization of inhibin/activin subunits showed season-related changes in the breeding and non-breeding seasons.  相似文献   

7.
The expression of cyclins A, D1, D2 and E were examined immunohistochemically in 5 canine normal testes and 31 testicular tumors, including 14 seminomas, 11 Sertoli cell tumors and 6 Leydig cell tumors. In canine normal testes, cyclin A expression was detected in spermatogonia and primary spermatocytes. This suggests that A-type cyclins may play some role in canine spermatogenesis. Cyclin A expression was also observed in 13/14 (92.9%) seminomas and 2/11 (18.2%) Sertoli cell tumors, but no positive reaction was observed in Leydig cell tumors. Parallel examinations for cyclins D1, D2 and E gave negative results in canine normal testes and testicular tumors. High levels of cyclin A expression in canine seminomas indicate that the neoplastic germ cells may be arrested at the spermatogonia and primary spermatocyte stages of differentiation.  相似文献   

8.
The Akt/protein kinase B (PKB) and extracellular signal-regulated kinase (ERK) pathways are involved in cell survival. This study examined the temporal profiles and localization of Akt/PKB and ERK1/2 activation in rat testis after ischemia/reperfusion (I/R). Testicular tissue was collected from normal control rats and rats exposed to reperfusion for 6, 24, and 48 hr after ischemic injury; the tissues were analyzed via Western blotting and immunohistochemistry. Western blot analysis showed that the levels of phosphorylated Akt/PKB (pAkt/PKB) and ERK1/2 (pERK1/2) increased significantly during the first 6-24 hr of reperfusion after ischemia. However, both of these activated proteins were decreased slightly at 48 hr after reperfusion. Immunohistochemically, low levels of pAkt/PKB expression were observed in Sertoli cells from the normal control. After I/R, pAkt/PKB expression increased mainly in the adluminal portion of the Sertoli cells, as well as in spermatogenic cells. In addition, pERK1/2 expression was observed in Sertoli and Leydig cells in the normal control. After I/R, pERK1/2 expression increased in some surviving spermatogenic cells (mainly spermatocytes), as well as in the adluminal portion of Sertoli cells. These results suggest that both Akt/PKB and ERK1/2 are involved in the survival of testicular cells during the early phase of testicular I/R. These pathways may represent important targets for increasing cell survival in testicular injury, including testicular torsion.  相似文献   

9.
The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d ‐galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N‐acetyl‐d ‐galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N‐acetyl‐d ‐glucosamine and sialic acid (wheat germ agglutinin WGA, s‐WGA), d ‐mannose and d ‐glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), l ‐fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA‐E) sugar residues. In Golgi‐, cap‐, and acrosome‐phase spermatids, lectin‐bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s‐WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA‐E). s‐WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA‐E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA‐E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA‐E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin‐bindings noted in the testis of lesser mouse deer included the limited distribution of s‐WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.  相似文献   

10.
Dietary cobalt (265 ppm Co) induced polycythemia and consistent degenerative and necrotic lesions in the seminiferous tubules of rats. Cyanosis and engorgement of testicular vasculature on day 35 and thereafter was followed on day 70 by degenerative and necrotic changes in the germinal epithelium and Sertoli cells. Spermatogonia, primary spermatocytes and round spermatids were markedly affected, while elongated spermatids, spermatozoa, and sertoli cells were more resistant. Damaged tubules, often present side by side with normal tubules, contained multinucleated giant cells composed of degenerated and necrotic spermatocytes and/or spermatids, sloughed germinal and Sertoli cells, and calcified necrotic debris. Necrotic tubules were frequently collapsed and devoid of epithelium except for occasional spermatogonia and surviving Sertoli cells. Lesions were not observed in the Leydig cells, cauda epididymis or seminal vesicles.  相似文献   

11.
In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.  相似文献   

12.
The crude testicular cells (CTCs) contain many cell types, such as Sertoli cells, leydig cells, spermatogonial stem cells (SSCs), spermatocytes, and other somatic testicular cells, that secrete various growth factors needed in spermatogenesis. The objective of this study was to characterize development of 5‐day‐old mice testicular cells cultured. Crude testicular cells prepared from the testes of 5‐day‐old male mice were cultured in Dulbecco's Modified Eagle Medium and incubated at 37°C in a 5% CO2 atmosphere for 6 days. The results demonstrated that the testicular cells developed rapidly with a population doubling time (PDT) of 0.63 days and more than 90% of cells were viable after being cultured for 3 days. The number of Sertoli‐like cells increased significantly over days 1, 3, and 6 to 22.1%, 34.6%, and 50.1%, respectively. A significant increase was also observed in fibroblast‐like cells (15.5% on day 1 to 28.8% on day 3 and to 26.6% on day 6). In contrast, the number of spermatogonia‐like cells decreased significantly (54.3%, 30.4%, and 18.7%, on days 1, 3, and 6, respectively). These data indicated that the developmental pattern of the testicular cell in this study might be affected by the niche provided by the cultured testicular cells.  相似文献   

13.
The gonads and the germinative cells of 3 male hinnies were studied with light and transmission electron microscopy with the aim to observe the development of germ cells and verify the morphological modifications due to the hybridization. The hinny seminiferous epithelium presented Sertoli cells and spermatogonia with normal features and anomalous spermatocytes I. The other cells from the spermatogenic sequence were not seen. Most of the alterations began to occur in the cytes I, which presented nuclear vacuolization and deposits of amorphous material between the carioteca and the nuclear lamina, forming vesicles, or exaggerated chromatin condensation, resulting in pyknosis. In the cytoplasm vacuolization was also observed, besides organelle destruction.
The arrest of meiosis due to lock of chromosome homologies leads to germinative cell degeneration and, therefore, the spermatogenesis arrest. This fact causes a profound alteration in the seminiferous epithelium morphology in comparison with the parental species.  相似文献   

14.
The purpose of this study was to explore the variations in the circulating leptin concentrations of the wild ground squirrels in relation to seasonal changes in testicular activities. Hematoxylin-eosin staining showed all types of elongated spermatids and spermatogenic cells existed in the testis in April, while the primary spermatocytes and spermatogonia were most advanced stages of germ cells in June. In addition, the primary spermatocytes, secondary spermatocytes, and spermatogonia were most advanced stages of germ cells in September. The highest circulating leptin concentration was consistent with the maximum body weight results from accumulation of adipose tissue in September. The mRNA expression level of leptin receptor (Ob-R) and STAT3 was lowest in June, raised in September, and remained increased in April. Ob-R and STAT3 were stronger staining in the Leydig cells in July. Moreover, the concentrations of testosterone (T) showed the maximum values in April, the minimum values in June, and significant increases in September. Furthermore, it is worth noting that the levels of T increased with the mRNA levels of Ob-R, STAT3, StAR, and testicular steroidogenic enzymes (3β-HSD, P450c17, and P450scc). Moreover, RNA-seq analyses of testis during the different periods showed that a total of 4209 genes were differentially expressed genes (DEGs); further analysis revealed that DEGs related with the Jak/STAT pathways and reproduction were altered. Taken together, the results suggested that the leptin regulated testicular function through the Jak/STAT pathways and testicular steroidogenic factor expressions.  相似文献   

15.
Fifteen stallions of different breeds, age 3-11 years, had their right testicles evaluated by fine needle aspiration cytology (FNAC). Cytological analysis showed the following spermatogenic cell types: spermatogonia (1.6% +/- 1.1); spermatocyte I (3.4% +/- 2.2); spermatocyte II (0.8% +/- 0.7); early spermatids (25.5% +/- 9.5); late spermatids (37.0% +/- 9.3). Spermatozoal numbers were expressed as the spermatic index (SI = 31.5% +/- 8.5) and Sertoli cells were expressed as the Sertoli cell index (SEI = 20.9% +/- 17.0) (means +/- s.d). Identification of cell types was relatively easy and no immediate adverse effects of aspiration were noted. The results suggest that FNAC of testis may assist clinical diagnosis in the study of male equine infertility.  相似文献   

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18.
Ziwuling black goats are typically found in loess plateaus regions and the Ziwuling Nature Reserve. Cryptorchidism is a common disease in this inbred goat, and its pathogenesis has been linked with the expression of insulin-like factor 3 (INSL-3). Therefore, this study aimed to investigate anatomical alterations caused by cryptorchism and the expression and distribution of INSL-3 in normal and cryptorchid testicular tissues. The testicular tissues of 6-month-old Ziwuling black goats were collected for microscopic analyses using histochemical, immunohistochemical, immunofluorescence and biometrical methods, as well as Western blotting to compare the expression and distribution of INSL-3. A lower expression of INSL-3 was observed in cryptorchid compared with normal testicular tissues (p < .01). Cryptorchidism caused a significant reduction in layers of spermatogenic epithelium and tubule areas in Ziwuling black goat (p < .01). The interstitial to seminiferous tubule area ratio was larger in cryptorchid than in normal group. Periodic Acid-Schiff (PAS) staining revealed pronounced positive bands in the interstitial tissue, while positive Alcian blue (AB) staining was not clear, and AB-PAS staining revealed a positive red band in the basement membrane of cryptorchid group. Immunofluorescence revealed a strong signal of INSL-3 expression in Sertoli and peritubular myoid cells, and moderate signal in Leydig and spermatogenic cells in the normal group. However, in cryptorchid testicular tissues, the signal of INSL-3 expression was strong in primary spermatocytes, occasional in Sertoli cells, limited in Leydig cells and absent in peritubular myoid cells. Furthermore, immunohistochemistry showed that INSL-3 expression was higher in normal testes compared with cryptorchid testicular tissues (p < .05), especially in primary spermatocytes and Sertoli cells. Collectively, our results indicate that cryptorchidism is closely related to the disorder of acid glycoprotein metabolism and the reduction in release of INSL-3 from Leydig cells. Moreover, Sertoli and peritubular myoid cells are crucial for INSL signalling and could underpin further research on the mechanism of cryptorchidism in animal.  相似文献   

19.
Decorin, a small leucine‐rich proteoglycan, plays an important role in cellular activities through modification of growth factors. It also acts as a signaling molecule to non‐muscle cells through epidermal growth factor receptor or insulin‐like growth factor I receptor (IGF‐IR). However, it is unclear if decorin acts as a signaling molecule to myogenic cells. In this study, we investigated the effect of decorin on the differentiation of myoblasts and the signaling via IGF‐IR to myogenic cells. C2C12 myoblasts cultured in media containing decorin for 72 h showed more extensive formation of multinucleated myotubes than control cells cultured in the same media without decorin. The protein expressions of myogenin and myosin heavy chian were higher in decorn‐treated cells than in control cells. These results suggest that decorin enhances the differentiation of myoblasts. Western blot analysis and immunocytochemistry showed that IGF‐IR was expressed in myoblasts and myotubes. Furthermore, Akt, which is downstream of IGF‐IR, was more phosphorylated in myoblasts cultured in media containing decorin than those in media without decorin. These results suggest that decorin activates Akt downstream of IGF‐IR and enhances the differentiation of myogenic cells.  相似文献   

20.
The cellular effects of tall fescue grass–associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel‐free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5°C was to make the sperm cells less likely to undergo a calcium ionophore–induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts.  相似文献   

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