Position and histological structure of the testes in the brown hare (Lepus europaeus) during seasonal regression and recrudescence

The position and histological structure of the testes of 33 brown hares (Lepus europaeus) were studied from July to December. From July to September, the testes were located in the scrotum; in October and November, in some animals, the testes were positioned more or less in the inguinal canal towards the abdominal cavity, and in December none of the investigated animals had testes located in the scrotum. Testes were weighed and a quantitative analysis of tissue components was performed: the diameter of the seminiferous tubules, the depth of the seminiferous epithelium, the thickness of the tunica albuginea, the thickness of the peritubular tissue and the relative proportion of seminiferous tubules were determined. The tunica albuginea and peritubular tissue were thickest in September, October and at the beginning of November. In the same months the testis weight was low, and the diameter of the seminiferous tubules, the depth of the seminiferous epithelium and the relative proportion of seminiferous tubules in the testis tissue were significantly lower than in other months. We did not find any correlation between testicular regression or testis weight reduction and the change in the position of the testes. During recrudescence of spermatogenesis in November and December the testes were located in the inguinal canal.     

Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black‐Backed Jackal (Canis mesomelas)

Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black‐backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.     

A polyorchid dog

The case of a polyorchid Irish Setter is presented here. Castration and intra-abdominal testis removal were performed one year of age when one scrotal and one cryptorchid testis near the right inguinal canal were removed. Later it became apparent that there was still testosterone production. A third testis, abdominal cryptorchid, was found on the right side cranially and right to the bladder. The third testis had a strong cranial suspensory ligament and the tail of the epididymis was elongated. The ductus deferens did not enter the prostate but followed the gubernaculum to the inguinal canal near the stump of the previous operation on the caudal right testis. This suggests that two right cryptorchid testes had common ductus deferens.     

The Case of an Sry‐Negative XX Male Pug With an Inguinal Gonad

A case of intersexuality in a Pug that was bought as a male in a pet shop is described. The dog was presented at the Veterinary Teaching Hospital, University of Turin, for a reddish mass protruding from the prepuce. The mass had the aspect of an enlarged clitoris, with a caudoventral direction and a dorsal urethral ostium. A gonad was palpable in the left inguinal region. Laparotomy confirmed ultrasound detection of an abdominal uterine structure together with the right gonad. The histology of both gonads was similar, showing an exclusively masculine character, with seminiferous tubules lined only by Sertoli cells; the uterus showed a normal histological structure. Karyological analysis revealed a female karyotype (78,XX), and polymerase chain reaction showed the absence of Sry. The diagnosis was an XX male. The pathogenesis of the XX sex reversal syndrome in dogs is not completely understood, as Sry, the master gene regulating testis differentiation, is not present; to date, no genetic cause has been identified for this phenotypic condition in dogs. This case is unusual because the dog showed an inguinal testis, implying a partial activity of the mechanisms leading to abdominal testis translocation along a gubernaculum and transinguinal migration.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

Relationship between the development of the gubernaculum and the testicular descent in the rat fetus: macroscopic and light and electron microscopic observation

The gubernaculum consists of the gubernacular cord connected with the vas deferens and the gubernacular bulb connected with the retroabdominal wall. The cord was first distinguished on day 15 of gestation. Its length reached the peak on day 16, followed by a reduction until day 18 to be almost constant thereafter. The bulb was first distinguished on day 16, and its length was steadily increased toward term. The testis began to descend with a reduced length of the cord. By day 19, the testis descended close to the caudal part of the bulb by the sharp bending of the cord. Microscopically, the bulb consisted at first of a centrally located mesenchymal mass and of a covering layer of myoblasts. Thereafter, the mesenchymal mass was gradually shifted toward the cranial part of the bulb, trailing loose mesenchymal tissue in the caudal part of the bulb. On day 19, the mass was decreased in cell population but increased in amount of collagen fibers. It is suggested that the testicular descent starts on day 16 together with the commencement of shortening of the gubernacular cord and enlargement of the gubernacular bulb.     

Zur Entwicklung des Pferdehodens

The aim of the study was to answer the open questions concerning the development of the horse's testis. This study revealed that the seminiferous tubules originate from the sex cords of the coelomic epithelium and Leydig cells from the proximal part of mesonephric nephrones, whereas the rete and the ductuli efferentes derive from intermediate and distal parts of the mesonephric tubules. During the development the Leydig cells undergo an enormous proliferation due to the PMSG secretion in the mare. The proliferation of these cells prevent the deep penetration of the rete into the medulla and is therefore the reason for the reduced extension of the rete and mediastinum testis in the stallion, although 80% of these cells degenerate in the last third of pregnancy. The growth of the seminiferous tubules during sexual maturity reduces the rete to the extremitas capitata of the testis.     

Differential localization of immunoreactive alpha- and beta-subunits of S-100 protein in feline testis

This study investigates the differential localization of the alpha-subunit (S100-alpha) and the beta-subunit (S100-beta) of the S-100 protein in the feline testis, using immunohistochemistry with polyclonal antibodies to bovine S-100 protein (S-100) and monoclonal antibodies to bovine S100-alpha and S100-beta. Appreciable differences were observed in the cellular localization of the immunoreactivity of each subunit. S-100 was observed in the Sertoli cells, the epithelial cells of the transitional segment of the seminiferous tubules, Leydig cells and the peritubular cells of the seminiferous tubules, but was not observed in the epithelial cells of straight tubules and the rete testis or in the endothelial cells of blood and lymph vessels. S100-alpha immunoreactivity was localized in Sertoli cells, peritubular cells and the epithelial cells of the terminal segment of the tubules, whereas S100-beta immunoreactivity was localized in Leydig cells. The differential localization of the alpha- and beta-subunits of the S-100 protein in the feline testis suggests that this protein is multifunctional and be useful as an investigative tool in studying feline testis function.     

Expression of constitutive endothelial, neuronal and inducible nitric oxide synthase in the testis and epididymis of horse

The expression of three isoforms of nitric oxide synthase (NOS) were examined in the testis and epididymis of a thoroughbred horse. Immunohistochemical studies demonstrated the presence of eNOS immunostaining in some germ cells in the seminiferous tubules and in vascular endothelial cells in the interstitial tissues. Interstitial cells, most likely Leydig cells, were also intensely immunopositive for eNOS. The pattern of immunostaining for nNOS was similar to that for eNOS in the testis. Weak expression of iNOS was detected in the seminiferous tubules of the testis, but intense expression was found in interstitial cells. Inducible NOS was also strongly detected in stereocilia, sperm, epithelium and connective tissue of the epididymis of normal horses. These findings suggest that three isoforms of NOS are expressed in the testis and epididymis of horse and that they play important roles in the biology of interstitial cells that produce testosterone, as well as in spermatogenesis in the seminiferous tubules.     

Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.     

Stereological analysis of Wistar rat testis during early post-natal development

Quantitative analysis of testis structure was performed during early post-natal development in Wistar rats. For this purpose, at 1, 7, 14, 21, 28, 35, 42, 60 and 90 days after birth, weights and volumes of testes were recorded and fixed in Bouin's solution. For stereological studies, 5-μm paraffin sections were stained with haematoxylin-eosin. Relative and then absolute volumes of seminiferous tubules, germinal epithelium, seminiferous tubuli lumina and interstitial tissue were estimated by point-counting method. The results showed that weight and volume of testis in rats increase 75- and 86.28-fold during post-natal development, respectively. The greatest growth rate (2.96-fold) of testis was observed between days 35 and 42. Also, diameter of seminiferous tubules increased significantly (P<0.001) between different ages and represented 5.55-fold increase during post-natal development. The percentage volume of tubular tissue increased 1.4-fold between birth and 90 days of age. Interstitial tissue formed 36.68±0.90% of testicular parenchyma at birth. This percentage decreased progressively during post-natal development until 90 (9.00±0.55%) days of age. Lumen of seminiferous tubules was recognized at day 28, and its relative volume increased with age. This study provides systematic data on the stereological characteristics of developing Wistar rat testis.     

Reducing endogenous estrogen during development alters hormone production by porcine Leydig cells and seminiferous tubules

High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1 mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5-4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24h with and without porcine LH. Media were assayed for estradiol (E(2)) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4h with and without porcine FSH; media were assayed for E(2) and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E(2) and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E(2) but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E(2) production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins.     

Intratesticular morphometric, cellular and endocrine changes in dromedary bulls exhibiting azoospermia

Twenty bulls, aged 7-12 years and selected from six dromedary farms were used in this study. Fifteen previously fertile animals were divided into fertile (controls) and infertile groups on the basis of abnormal scrotal contents following palpation and azoospermic ejaculates collected by electroejaculation. An examination of the clinical and histological findings as well as the testicular patterns of oestradiol-17beta, testosterone and histamine indicated that three bulls displayed normal ranges comparable to the controls but with bilateral spermatocoeles in the caput epididymides in conjunction with the soft texture of the testicles. Seven bulls showed moderate testicular firmness and springiness, a marked increase in testicular oestradiol-17beta and histamine concentrations, and increases in surface area, density of mast cells and percentages of seminiferous tubules containing premeiotic spermatogenic cells as well as decreases in testicular testosterone concentrations, surface area of Leydig cells and diameter of the seminiferous tubules. The remaining five infertile animals had small hard testicles, supranormal testicular testosterone concentrations, baseline values of testicular oestradiol-17beta and histamine, decreased numbers of Sertoli and mast cells, with a predominance (98.2%) of seminiferous tubules containing spermatogonia resting on a thickened tubular basement membrane. The results provide information on the relationship between gonadotrophin, testicular oestrogen, androgens and histamine as well as spermatogenesis in normal and azoospermic dromedary bulls.     

Testicular Mineralization in KK-Ay Mice Treated with an Oxovanadium Complex

Vanadium has potential for use in diabetes therapy. Many investigators have reported toxic effects of inorganic vanadium salts; however, there are few reports on toxic effects of oxovanadium(VO²⁺) complexes. Therefore, we studied VO²⁺ toxicity by examining histological changes and measuring the vanadium concentration in the testis after repeated oral administration of bis(1-oxy-2-pyridine-thiolato)oxovanadium(VO²⁺) (VO(opt)₂) for 2 or 4 weeks in KK-A^y mice. Severe mineralization and degeneration/necrosis of the seminiferous tubules were detected after either 2 or 4 weeks of administration. Vacuolar changes in Sertoli cells and the seminiferous epithelia, and hyperplasia of Leydig cells were observed in the testes of some animals. Vanadium concentrations in the mineralized testis were much higher than those in the testis of untreated KK-A^y mice. These results represent the first report of the possibility for seminiferous tubules mineralization induced by VO(opt)₂ administration. Therefore, our research provides important information about the potentially toxic effects of VO²⁺ complexes.     

Testis Morphometry and Stages of the Seminiferous Epithelium Cycle in an Epididymal Sperm‐storing Neotropical Vespertilionid,Myotis levis (Chiroptera)

Yellowish myotis, Myotis levis, is a seasonal, epididymal sperm‐storing Neotropical vespertilionid. In the dry season, males show simultaneous testis regression and sperm storage in cauda epididymis, enabling them to mate during this season. In this study, we investigated seasonal variations in body mass, diameter and height of seminiferous tubules and nuclei of Leydig cells in a population of southeastern Brazil. We also determined the frequencies of the stages of the seminiferous epithelium cycle (SEC) of mature individuals of this population. Body mass and diameter of Leydig cell nuclei showed no significant differences between dry and rainy seasons and stages of annual reproductive cycle; however, all other morphometric parameters varied significantly. The relative cumulative frequency of pre‐meiotic stages of the SEC (1–3) was 51%, of meiotic stage (4) was 2% and of post‐meiotic stages (5–8) was 47%. We confirmed that the yellowish myotis presents seasonal sperm production as revealed by testis regression and epididymal sperm storage during the dry season.     

A Stereological Study of the Different Cell Populations in Chicken Testes Treated with Follicle-Stimulating Hormone during Embryonic Development

Quantitative morphological methods were used to analyse the histomorphometric changes and variations in the number and size of cells from diverse cellular populations in testes of newly hatched chicks treated with follicle stimulating hormone (FSH) during embryonic development. The tissue was fixed and embedded in Epon and sections were morphometrically measured under light microscopy, using point counting for volume densities and the Floderus equation to determine numerical density. The average volume of the individual cell was determined by dividing the volume density by the numerical density. Results indicate that FSH administration causes an increase in the number of Sertoli cells and spermatogonia, as well as enlargement of the individual Sertoli cells leading consequently to an increase in the diameter and volume density of the testicular seminiferous tubules. Results also reveal an increase in the volume density of the interstitial cords of the Leydig cells, this expansion is due to the enlargement of the individual Leydig cells and not to an increase in their number, which remains constant. We conclude that testes of chick embryos are able to respond to FSH treatment, as revealed by the changes in the number and size of the cells conforming the diverse cellular populations of the testis. FSH treatment during embryonic development induces histomorphometric changes in both the interstitial tissue and seminiferous tubules, accelerating their growth and differentiation.     

Alterations in the immunohistochemical localization patterns of alpha-smooth muscle actin (SMA) and vimentin in the postnatally developing bovine cryptorchid testis

Previously, we reported the normal postnatal developmental changes in immunohistochemical localization of alpha-smooth muscle actin (SMA) and vimentin in the bovine testis. In this study, we demonstrate the alterations of these cytoskeletal proteins in the bovine cryptorchid testis as compared to the contralateral scrotal testis during postnatal development. Seminiferous peritubular alpha-SMA did not appear in the cryptorchid testis until 8 months of age, except for very weak intermittent filaments in relatively larger seminiferous tubules. However, a similar peritubular pattern was observed in the 18-month-old cryptorchid and scrotal testes. Moreover, weak expression of alpha-SMA in the straight tubules and rete testes at 5 months of age did not improve until 18 months of age in the cryptorchid testes. The Sertoli cell vimentin in the cryptorchid testes revealed a highly immature pattern at 5 months of age, a pattern similar to a transforming pattern with infranuclear vimentin extensions at 8 months of age, and a pattern that was almost a transforming pattern, but with considerable weakening of the vimentin filaments, at 18 months of age. In conclusion, cryptorchidism may cause considerable delay in testicular myoid cell differentiation and in attainment of the transforming pattern of the Sertoli cell vimentin, which weakens and fails to attain the mature pattern in the cryptorchid testis. These alterations may be related to the structural immaturity and functional failure of postnatally developing bovine testes exposed continuously to body heat.     

Tubule length and Leydig cell volume in the normal bull testis.

Data are presented on the size, Leydig cell content and seminiferous tubule dimensions of the normal bull testis. A method of estimating total tubule length, requiring only the measurement of testis volume and the number of tubule cross-sections in a unit area is described, and its applicability to the bull established. The average bull testis contains about 5.2 km of tubules.     

老龄哈多利系博美犬睾丸组织结构分析

为比较哈多利系博美犬老龄与青年阶段睾丸的组织结构差异及相关蛋白分布特征，探索博美犬不同年龄阶段睾丸组织结构变化及其对生殖功能的影响，本试验应用特殊染色、免疫组织化学法结合免疫荧光观察比较青年与老龄博美犬睾丸组织化学特点，并用IPP图像分析软件进行定量分析。结果显示，与青年博美犬相比，老龄博美犬生精上皮层数与厚度降低，Leydig细胞数及Sertoli细胞数显著增加（P<0.05），生精小管基底膜及血管管壁层胶原纤维与网状纤维含量明显增加。免疫组织化学结果显示，老龄博美犬睾丸细胞外基质（exreacellular matrix，ECM）相关蛋白Ⅳ型胶原（collage，Col Ⅳ）、硫酸乙酰肝素蛋白多糖（heparan sulfate proteoglycan，HSPG）含量极显著低于青年博美犬（P<0.01），层黏连蛋白（laminin，LN）含量显著低于青年博美犬（P<0.05）。免疫荧光结果显示，Col Ⅳ、HSPG和LN主要在Leydig细胞及Sertoli细胞强表达。因此，老龄博美犬胶原纤维与网状纤维含量增加，Leydig细胞数及Sertoli细胞数增多可能与其生精功能的维持相关，其生精功能的下降与Col Ⅳ和HSPG的变化密切相关。     

Histologic and immunohistochemical characterization of a testicular mixed germ cell sex cord-stromal tumor and a leydig cell tumor in a dog

Mixed germ cell sex cord-stromal tumors (MGSCTs) of the testis are rare in dogs. We describe the histopathology and immunohistochemical characteristics of an MGSCT associated with a Leydig cell tumor in a cryptorchid testis. Histologically, MGSCT consisted of two nodules of seminiferous tubules lined by germ cells and Sertoli cells in variable proportions. Germ cells had variable size and nuclear features, with frequent giant cells. Germ cells were evenly mixed with Sertoli cells or located in the center of tubules. Markers that labeled mainly germ cells and few or no Sertoli or Leydig cells were calretinin, KIT, and PGP 9.5. E-cadherin, GATA-4, inhibin-alpha (INH-alpha), and neuron-specific enolase (NSE) were predominantly detected in Sertoli cells, whereas melan A was particularly expressed in Leydig cells and vimentin in all three cell types. OCT3/4 was not detected in any cell type. Although more cases of canine MGSCT need to be examined, our results suggest that an immunohistochemical panel of E-cadherin, GATA-4, INH-alpha, KIT, NSE, PGP 9.5, and melan A will help distinguish the three main cell types in canine testicular germ cell and sex cord-stromal tumors.