Postnatal testicular development and actin appearance in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.     

Malfunction of spermatogenesis in experimental ischemic mice

An experimental ischemia (EI)-induced mouse model was used to analyze pathological and biochemical alterations in testes. Initial morphological changes were observed in Sertoli cells of EI testes at the light microscopic level. Examination of the ultrastructure using transmission electron microscopy confirmed that Sertoli cells were partially detached from the basement membrane of the seminiferous epithelium and that the cell membranes of adjacent Sertoli cells were not joined. The functional integrity of the blood-testis barrier (BTB) was assessed using the lanthanum tracer technique. Lanthanum had penetrated into the spaces between adjacent Sertoli cells in the adluminal compartment up to the lumen of the seminiferous epithelium in EI testes. Proteome analysis showed that the expression of heat shock protein (HSP) 70 was significantly upregulated in EI testes. Western blot analysis confirmed that the expression of HSP70 increased in a time-dependent manner after the EI procedure. HSP70 immunostaining was observed in spermatocytes and in round and elongated spermatids in EI testes. Our results suggest that a change in the junctions between adjacent Sertoli cells on the basal compartment is involved in the BTB disruption in EI testes. Therefore, male infertility caused by the BTB disruption could be associated with heat stress induced by ischemia.     

Ultrastructure of the Hinny (Equus asinus × Equus caballus) Seminiferous Epithelium

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.     

Continual maintenance of the blood-testis barrier during spermatogenesis: the intermediate compartment theory revisited

Tight junctions occur between the lateral processes of neighboring Sertoli cells that divide the seminiferous epithelium into two compartments: basal and adluminal compartments. These tight junctions constitute the blood-testis barrier (BTB). The established theory that the BTB must open when spermatocytes translocate from the basal compartment to the adluminal compartment is marked by one contradiction, that is, normal spermatogenesis occurs in the testis because the BTB is expected to constantly seclude the adluminal compartment from the basal compartment in order to protect haploid germ cells from the autoimmune system. Subsequently, another concept was proposed in which two BTBs divide the seminiferous epithelium into three compartments: basal, intermediate and adluminal compartments. It has been suggested that the transition from the basal region to the adluminal region without the BTB open occurs through the agency of a short-lived intermediate compartment embodying some primary spermatocytes. In contrast, the results of recent findings in the molecular architecture of the BTB suggest that the BTB in the seminiferous epithelium must "open". In this paper, I re-examine the BTBs of boar and experimental cryptorchid mouse testes by transmission electron microscope (TEM). TEM analysis showed that an atypical basal compartment existed in the thin seminiferous epithelium of 14-day post-cryptorchid mice testes. In developmental boar testes, ectoplasmic specialization (ES) of the seminiferous epithelium showed dynamic behavior. The intermediate compartment was clearly observed between the basal and adluminal compartments of the mature boar seminiferous epithelium. ESs were observed between Sertoli cells and spermatids at all developmental stages, including early, late and mature. Furthermore, ESs were situated on the apical surface of the seminiferous epithelium. From these results, I propose that the BTB is continually maintained during spermatogenesis and suggest a model of ES circulation in the seminiferous epithelium.     

Dissertationen

Versuche zur Tiefgefrierkonservierungvonungeteiltenund geteilten Rinderembryonen unter Venvendung von Glycerin und Gelee Royal
Embryologie, Anatomie und Histologie der akzessorischen Geschlechtsdrüsen von Meerschweinchen und Hamsterartigen
Landw. Fakultät der Georg-August-Universität, Göttingen, 1987 (Institut für Tierzucht und Haustiergenetik)
(Ambulatorische und Geburtshilfliche Veterinärklinik)
Die medikamentelle Behandlung der Wehenschwäche mit dem β-Blocker Carazolol in einer Mutterkuhherde (The treatment of bovine uterine inertia with the beta-blocker carazolol in a cow-calfherd).
Elektronenmikroskopische Untersuchungen an der Sertolizelle während des Keimepithelzyklus beim Bullen (The examination of Sertoli cells during the cycle of the seminiferous epithelium by means of electron microscopy).
Elektronenmikroskopische Untersuchungen an Sertolizellen beim Hengst während des Keimepithelzyklus (Electron microscopic examination of Sertoli cells in the stallion during the cycle of the seminiferous epithelium).     

An ultrastructural study on the Leydig and Sertoli cells in the immature lesser mouse deer (Tragulus javanicus)

Leydig and Sertoli cells of the immature lesser mouse deer testes, obtained in East Malaysia, were observed using light and transmission electron microscopy (TEM). The testes were fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethanol, and embedded in Araldite M. Serial semi-thin sections were cut, stained with toluidine blue and observed using light microscopy. Serial ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and examined using TEM. As a result, ultrastructurally, two types of underdeveloped filament bundles were infrequently recognized in Leydig cells, but not in other testicular cells. One type was the underdeveloped bundles of actin filaments (approximately 5 nm in diameter), which were found in the nucleus of Leydig cells. The other type was the underdeveloped bundles of intermediate filaments (approximately 10 nm in diameter), which were found in the cytoplasm of Leydig cells. A multivesicular nuclear body (MNB)--specifically present in the Sertoli cell nucleus of ruminant testes--was infrequently observed. The MNB is situated in the vicinity of nuclear membrane, still in an underdeveloped stage.     

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.     

Age-related changes in the testes of horses

Atrophy of seminiferous tubules and interstitial fibrosis are frequently observed in aged horses. Samples from 8 male Thoroughbreds, age 4-24 years, were subjected to histological, electron microscopical and immunohistochemical examination and statistical analysis. There were statistically significant increases in collagen fibres in the lamina propria of seminiferous tubules and testicular interstitium in 3 horses age 23 and 24 years compared with 5 horses age 4-20 years (P<0.001). Lamina propria surrounding atrophic tubules was thickened by an increase in collagen type IV and elastic fibres and by proliferation of bizarre myoid cells. Basal lamina was also thickened but had decreased reactivity for collagen type IV. Some myoid cells changed morphologically to a swollen and irregular shape and contained abundant cytoplasmic organelles. Laser scanning microscopy revealed that cytoplasmic actin filaments were decreased; the remaining filaments were positive for alpha-smooth muscle actin and vimentin, and matrix metalloproteinase-2 was secreted. These myoid cells transformed into myofibroblasts. The changes are interpreted as evidence of injured structure and function of the lamina propria and basal lamina and may explain the functional decline of the blood-testis barrier. Myoid cells may play an important role in the progression of testicular fibrosis.     

The Sertoli cell of the water buffalo--an electron microscopic study.

The ultrastructure of Sertoli cells in the seminiferous tubules of water buffaloes before and during sexual maturity was studied by transmission electron microscopy, with emphasis on the intranucleolar vesicular elements. Sertoli cells of animals under 12 months of age were distinguished from the germ cells by the presence of electron dense membrane bound bodies within their cytoplasm. These cells, referred to as basal indifferent supporting cells, were probably involved in the phagocytosis and elimination of degenerating spermatocytes, which failed to differentiate into spermatids and spermatozoa in animals under one year of age. In 12 month old animals, a few Sertoli cells exhibiting the vesicular elements appeared in the nucleolar region while in animals over 15 months of age Sertoli cells could be positively identified by the characteristic cytoplasm containing microtubules, elongated and electron dense mitochondria, extensive granular endoplasmic reticulum and the presence of spermatids in various stages of spermiogenesis. The vesicular elements in the nucleolar region of the Sertoli cells were most prominent at this stage. Ultrastructural features of the Sertoli cells revealed an abundance of ribosome-like particles surrounding the vesicles of varying size. Some of these vesicular elements contained amorphous material suggesting that they represent the products sequestered in the nuclear region for transport to the cytoplasm and that the process of spermiogenesis may be dependent on the ability of Sertoli cells to generate these products at sexual maturity.     

Sertoli Cell - Spermatid Tubulobulbar Complexes in the Ram

The fine structure of the tubulobulbar complex (TBC) of the ram seminiferous epithelium was studied in immersion-fixed testicular samples obtained in autumn (sheep's mating period). The TBC was visible during the last two stages of the seminiferous epithelium cycle. It originated as a tubular invagination of the Sertoli cytoplasm harbouring a complementary evagination from the spermatid head. Later, it expanded into a bulbous structure, finally becoming detached from the spermatid and apparently phagocytosed by the Sertoli cell. The significance of this transient structure is discussed and compared with previous reports for other eutherian mammals.     

Testicular Structure and Seminal Pathway in the Yellowtail Tetra Astyanax altiparanae (Characiforms: Characidae)

This study aimed to describe testicular and its main ducts structure in the yellowtail tetra Astyanax altiparanae, contributing to the knowledge of the region in which semen is produced, storage and released, focusing mainly on the dynamic of germinal epithelium and Sertoli cells during germ cell maturation. Ten sexually mature male A. altiparanae had their testes processed according to the routine protocols to optical microscopy. Moreover, spermatic ducts and tubular compartment of the testes of three specimens were perfused with vinyl resin for gross anatomy and scanning electron microscopy. Astyanax altiparanae testes are paired organs, separated for most of their extension, joining posteriorly in a spermatic duct formed by a squamous simple epithelium. Seminiferous compartment presents anastomosing tubular type organisation, and spermatogonia spread along its extent. Spermatogenesis is of cystic type, and there is no main testicular duct. Spermatogenesis develops in ‘waves’, from posterior to anterior part of the gonad. Thus, while sperm is storage posteriorly, spermatogenesis keeps maturing germ cells anteriorly, making the germinal epithelium very dynamic, holding Sertoli cells that change their function as a cystic envelope to produce secretions of the seminal fluid and store sperm. Such kind of development is thought to be responsible by the high prolificacy of this species.     

Peculiar bundles of filaments in Leydig cells of the lesser mouse deer (Tragulus javanicus): an ultrastructural study

Leydig cells of lesser mouse deer (Tragulus javanicus) testes were observed using light and transmission electron microscopies. Sexually mature lesser mouse deer were obtained in East Malaysia. The testes were perfused with 5% glutaraldehyde, postfixed with 1% OsO4, dehydrated in ethanol and embedded in Araldite. The semithin sections were cut, stained with toluidine blue and observed under light microscopy. The ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using a JEM-1200 transmission electron microscope. As a result, two types of filament bundles were frequently recognized in Leydig cells, but not in other testicular cells. These bundles were clearly seen at even a light microscopic level. One type was bundles of actin filaments (approximately 5 nm in diameter). These structures were found not only in the cytoplasm but also in the nucleus. The other type was bundles of intermediate filaments (approximately 10 nm in diameter). These structures were found only in the cytoplasm. The existence of filament bundles has never been reported in the testicular cells of another mammalian species. Thus, while bundles of actin and intermediate filaments are specifically present in the Leydig cells of the lesser mouse deer, their functions are still unclear.     

Immunohistochemical Demonstration of Cytoskeletal Proteins in the Testis of the Japanese Black Bear, Ursus thibetanus japonicus

The seasonal changes of the cytoskeletal protein expressions were immunohistochemically investigated in the testes of Japanese black bear, Ursus thibetanus japonicus. A strong immunoreaction for α-smooth muscle actin is restricted to the vascular smooth muscle cells and the peritubular cells which surround the seminiferous tubules by several layers throughout the year. Weak immunoreactions for B4 antigen and desmin were observed in the vascular smooth muscle cells and in a part of peritubular cells throughout the year. A strong immunoreaction for vimentin was also detected in the fibroblasts and Leydig cells, in addition to the vascular smooth muscle and epithelial cells and the peritubular cells throughout the year. A strong α-tubulin immunoreaction was detected in the elongating spermatids during the acrosome phase of spermiogenesis in May and June. The cytoplasm of several Sertoli cells was faintly immunoreacted for vimentin in the basal and lateral region, while an intense α-tubulin reaction was seen in the entire cytoplasm in May, April and June. In November, January and March, the immunoreactions for vimentin and α-tubulin strongly accumulate in a perinuclear region of Sertoli cells when developmental spermatids are not seen in the seminiferous tubules. These accumulations in the immunoreactions for vimentin and α-tubulin seem to be caused by the reduction in size of Sertoli cells cytoplasm with season. However, the seasonal changes of distributions in the cytoskeletal proteins are obscure in the bear testes. These results suggest that the contents of cytoskeletal proteins may not change in relation to the morphological differences with season in the testes of the seasonal breeders.     

AR regulates porcine immature Sertoli cell growth via binding to RNF4 and miR-124a

Sertoli cells are the only somatic cells in the seminiferous epithelium which directly contact with germ cells. Sertoli cells exhibit polarized alignment at the basal membrane of seminiferous tubules to maintain the microenvironment for growth and development of germ cells, and therefore play a crucial role in spermatogenesis. Androgens exert their action through androgen receptor (AR) and AR signalling in the testis is essential for maintenance of spermatogonial numbers, blood–testis barrier integrity, completion of meiosis, adhesion of spermatids and spermiation. In the present study, we demonstrated that AR gene could promote the proliferation of immature porcine Sertoli cells (ST cells) and the cell cycle procession, and accelerate the transition from G1 phase into S phase in ST cells. Meanwhile, miR-124a could affect the proliferation and cell cycle procession of ST cells by targeting 3′-UTR of AR gene. Furthermore, AR bound to the RNF4 via AR DNA-binding domain (DBD) and we verified that RNF4 was necessary for AR to regulate the growth of ST cells. Above all, this study suggests that AR regulates ST cell growth via binding to RNF4 and miR-124a, which may help us to further understand the function of AR in spermatogenesis.     

Morphological study of the effects of the GnRH superagonist deslorelin on the canine testis and prostate gland

The present study is part of a programme of research designed to evaluate the efficacy of the GnRH superagonist,deslorelin (D-Trp6-Pro9-des-Gly10-LHRH ethylamide), as a contraceptive for male dogs. Adult dogs were assigned to a completely randomized design comprising six groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other five groups received a 6 mg deslorelin implant. One group of deslorelin treated dogs was sacrificed on each of days 16, 26, 41, 101 and 620, and testicular and prostate tissues were collected for study by light and electron microscopy. On days 16 and 26 after implantation, we observed partial disruption of the seminiferous tubules, with early spermatids shed into the lumen. On days 41 and 101 after implantation, 90–100% of the seminiferous tubules were atrophic and aspermatogenic.On day 101 after implantation, 99% of all sections showed atrophy of the epithelium and shrinkage of epithelial height in the ductus epididymides. On days 41 and 101 after implantation, prostate tissue showed complete atrophy of the glandular epithelium (100% of sections) and an apparent increase in the relative proportion of connective tissue. At the electron microscopic level, in dogs treated with deslorelin for 41 and 101 days, the Sertoli cells were smaller and their nucleoli appeared smaller than in the control dogs. The nucleoli of the Leydig cells were atrophied and prostate glandular epithelium showed reduced epithelial height, a trophy of the nucleolus and an absence of secretory granules.Tissues collected during the recovery phase revealed a complete recovery of spermatogenesis. In conclusion, slow release implants containing deslorelin induce a striking a trophy of the testes and prostate gland by 26 days after implantation, explaining the previously reported loss of ejaculate and arrest of sperm output. At histological level,the entire process appears to be completely reversible, in accordance with data on endocrine variables and semen production.     

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.     

Testicular histomorphometry and the proliferative and apoptotic activities of the seminiferous epithelium in Syrian hamster during spontaneous recrudescence after exposure to short photoperiod

Syrian hamsters are photoperiodic rodents in which reproduction, including testicular function, is stimulated by long photoperiod exposure and curtailed by exposure to a short photoperiod. The objectives of this study were to characterize the testis histomorphometrically and to determine the role of the proliferation and apoptosis phenomena in the recovery of the seminiferous epithelium during spontaneous recrudescence after exposure to short photoperiod. The study was performed using conventional light microscopy, proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP in situ nick end labelling staining, image analysis software, and transmission electron microscopy in three recrudescence groups: initial recrudescence (IR), advanced recrudescence (AR) and total recrudescence (TR). The results morphometrically pointed to the gradual recovery of the testicular and tubular volumes, as well as of the seminiferous epithelium. Among the IR and AR groups, the increase in testicular and tubular volumes was accompanied by an increase in tubular diameter and length, with an increase in interstitial volume. From AR to TR, there was an increase in the tubular and total volumes, but, in this case, with a gradual increase in tubular diameter. Recovery of the seminiferous epithelium was accompanied by changes in apoptosis and proliferation activities. The first decreased halfway through the process, and the second remained higher than the control levels throughout the recrudescence stage. Alterations in the spermatozoa were ultrastructurally observed, which indicated that spermiogenesis was not yet completely normal. In conclusion, spontaneous testicular recrudescence in Syrian hamster comprises two histomorphometrical phases: the first related to an increase in tubular length and diameter and interstitial volume and the second depending principally on the gradual increase in tubular diameter. The restoration of the seminiferous epithelium is due to apoptosis reaching normal values in the AR group accompanied by higher proliferative activity than that observed in the Control group.     

Actinfilaments in the Ruminal Epithelium of Cattle

Phalloidin and immunogold labeling were used to localize actinfilaments in the ruminal pilar epithelium of adult catde on the light and electron microscopic level. Incubation with rhodaminyl-phalloidin permitted an overview of general actin distribution pattern. Distinct subcellular localization of actin was revealed with the biotin-streptavidin bridge technique in a postembedding procedure. Superior fixation of membranes and filaments was achieved with tannic acid-glutaraldehyde. Surface-etching prior to immunoincubation ensured restoration of actin immunoreactivity. Patterns of distribution in non-keratinized and keratinized epithelial cells as well as in pericytes and endothelial cells point to cell-specific cytoskeletal and motile functions of actinfilaments.     

支持细胞骨架与精子发生研究进展

精子发生过程是支持细胞参与调控的一个精密的过程,支持细胞结构的完整性保证了精子发生的正常进行.支持细胞骨架包括微丝、微管和中间纤维,它们参与构成血睾屏障,为精子发生提供了物理支撑和稳定的微环境,对于维持生精上皮的完整性至关重要,影响支持细胞蛋白质的分泌,使分泌物定向转运.支持细胞骨架的损坏引起生殖细胞的凋亡和精细胞的变形及迁移异常.文章主要围绕影响精子发生的诸多因素对支持细胞骨架的作用及机制加以阐述.