Internalization of Intact Intercellular Junctions in the Testis by Clathrin/Actin-Mediated Endocytic Structures: Tubulobulbar Complexes.

Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin-related structures at intercellular junctions. The appearance of these so called "tubulobulbar complexes" (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin-coated pits with extremely long necks. As they mature, the actin filament networks around the "necks" of the structures progressively disassemble and the membrane cores expand or swell into distinct "bulbs". These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin-mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and "intact" intercellular junctions that occurs during spermatogenesis. Anat Rec, 301:2080-2085, 2018. © 2018 Wiley Periodicals, Inc.

Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium.

TAR DNA-binding protein of 43 kDa (TDP-43) is an evolutionarily conserved, ubiquitously expressed, multi-functional DNA/RNA-binding protein with roles in gene transcription, mRNA splicing, stability, transport, micro RNA biogenesis, and suppression of transposons. Aberrant expression of TDP-43 in testis and sperm was recently shown to be associated with male infertility, which highlights the need to understand better the expression of TDP-43 in the testis. We previously cloned TDP-43 from a mouse testis cDNA library, and showed that it functions as a transcriptional repressor and regulates the precise spatiotemporal expression of the Acrv1 gene, which encodes the acrosomal protein SP-10, during spermatogenesis. Here, we performed immunoblotting and immunohistochemistry of the mouse testis using four separate antibodies recognizing the amino and carboxyl termini of TDP-43. TDP-43 is present in the nuclei of germ cells as well as Sertoli cells. TDP-43 expression begins in type B/intermediate spermatogonia, peaks in preleptotene spermatocytes, and becomes undetectable in leptotene and zygotene spermatocytes. Pachytene spermatocytes and early round spermatids again express TDP-43, but its abundance diminishes later in spermatids (at steps 5-8). Interestingly, two of the four antibodies showed TDP-43 expression in spermatids at steps 9-10, which coincides with the initial phase of the histone-to-protamine transition. Immunoreactivity patterns observed in the study suggest that TDP-43 assumes different conformational states at different stages of spermatogenesis. TDP-43 pathology has been extensively studied in the context of neurodegenerative diseases; its role in spermatogenesis warrants further detailed investigation of the involvement of TDP-43 in male infertility.

Ductuli efferentes of the male Golden Syrian hamster reproductive tract.

Efferent ductules are responsible for the transportation of spermatozoa from the testis to the epididymis and their epithelium is responsible for the reabsorption of over 90% of the luminal fluid. The purpose of this research was to characterize the gross morphology and histology of efferent ductules in the male Golden Syrian hamster. The efferent ductules emerge from rete testis with a unique polarity at the apex or cephalic pole of the testis. The number of efferent ductules varied from 3 to 10 with an average of 6.0 and blind ending ducts were observed in approximately 56% of the males. The ductules merged into a single common duct prior to entering the caput epididymidis. The proximal efferent ductule lumen was wider than the distal (conus and common ducts), consistent with reabsorption of most of the luminal fluid, as was morphology of the ductal epithelium. Non-ciliated cells in the proximal region had prominent endocytic apparatuses, showing both coated pits and apical tubules in the apical cytoplasm. Large basolateral, intercellular spaces were also present in the epithelium of the proximal region. Distal non-ciliated cells had an abundance of large endosomes and lysosomal granules. Localisation of sodium/hydrogen exchanger-3 (NHE3; SLC9A3) and aquaporins 1 and 9 (AQP1, AQP9) along the microvillus border was also consistent with ion transport and fluid reabsorption by this epithelium. In comparison, the caput epididymidis epithelium expressed only AQP9 immunostaining. Another unusual feature of the hamster efferent ductules was the presence of glycogen aggregates in the basal cytoplasm of small groups of epithelial cells, but only in the proximal ducts near the rete testis. Androgen (AR), estrogen (ESR1 and ESR2) and vitamin D receptors (VDR) were also abundant in epithelial nuclei of proximal and distal efferent ductules. In comparison, caput epididymidis showed very little immunostaining for ESR1.

Late morfofunctional alterations of the Sertoli cell caused by doxorubicin administered to prepubertal rats.

BACKGROUND: Doxorubicin is a potent chemotherapeutic drug used against a variety of cancers. It acts through interaction with polymerases and topoisomerase II and free radical production. Doxorubicin activity is not specific to cancer cells and can also damage healthy cells, especially those undergoing rapid proliferation, such as spermatogonia. In previous studies our group showed that etoposide, another topoisomarese II poison, causes irreversible damage to Sertoli cells. Thus, the aim of this study was to address the effects of doxorubicin on Sertoli cell morphology and function and on the seminiferous epithelium cycle when administered to prepubertal rats. METHODS: Prepubertal rats received the dose of 5 mg/Kg of doxorubicin, which was fractioned in two doses: 3 mg/Kg at 15dpp and 2 mg/Kg at 22 dpp. The testes were collected at 40, 64 and 127 dpp, fixed in Bouin's liquid and submitted to transferrin immunolabeling for Sertoli cell function analysis. Sertoli cell morphology and the frequency of the stages of the seminiferous epithelium cycle were analyzed in PAS + H-stained sections. RESULTS: The rats treated with doxorubicin showed reduction of transferrin labeling in the seminiferous epithelium at 40 and 64 dpp, suggesting that Sertoli cell function is altered in these rats. All doxorubicin-treated rats showed sloughing and morphological alterations of Sertoli cells. The frequency of the stages of the seminiferous epithelium cycle was also affected in all doxorubicin-treated rats. CONCLUSIONS AND DISCUSSION: These data show that doxorubicin administration during prepuberty causes functional and morphological late damage to Sertoli cells; such damage is secondary to the germ cell primary injury and contributed to enhance the spermatogenic harm caused by this drug. However, additional studies are required to clarify if there is also a direct effect of doxorubicin on Sertoli cells producing a primary damage on these cells.

Glycomolecule modifications in the seminiferous epithelial cells and in the acrosome of post-testicular spermatozoa in the alpaca.

A lectin histochemical investigation of the seminiferous epithelium and acrosomes of spermatozoa present in the efferent ductules and epididymal regions was carried out in the alpaca. The histochemical characterization was performed using a battery of different lectins: Con-A, UEA-I, LTA, WGA, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosilation pre-treatments were also employed. The cytoplasm of the Sertoli cells contained N-linked oligosaccharides with α-D-Man/α-D-Glc and GlcNAc and O-linked glycans with α-L-Fuc, ß-GalNAc, ß-D-Gal-(1-4)-D-GlcNAc, α-Gal and Neu5Acα2,6α-GalNAc moieties whereas ß-D-Gal-(1-3)-D-GalNAc residues were included in both O- and N-glycoproteins. Spermatogonia expressed α-D-Man/α-D-Glc residues included in N-glycoproteins and α-Fuc in O-glycoproteins. Spermatocytes contained the N-glycoproteins residues α-D-Man/α-D-Glc and GlcNAc and the O-glycoproteins residues α-L-Fuc, ß-D-Gal-(1-4)-D-GlcNAc, α-Gal, ß-GalNAc, Neu5Acα2,6α-GalNAc and Neu5Acα2,6ß-D-Gal-(1-3)-D-GalNAc. The results of the present study show differences in the presence and distribution of lectin reactive sites throughout the acrosomal development in the alpaca. In particular, Fuc moieties were found only during the Golgi-phase of spermatids, α-Gal were found in the acrosome of Golgi- and cap-phase spermatids, sialic-acid/α-GalNAc sequence was revealed during the cap-phase and elongated spermatids, and α-D-Man/α-D-Glc and GlcNAc were detected only in the acrosomes of elongated spermatids. Finally, ß-GalNAc, ß-D-Gal-(1-3)-D-GalNAc and ß-D-Gal-(1-4)-D-GlcNAc were added to acrosomal glycoproteins in the early stages of spermatogenesis and remained unchanged during the later phases. Differences in the carbohydrate expression were also demonstrated on the sperm acrosomes during passage through the post-testicular ducts.

Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes.

The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis.

An intracellular trafficking pathway in the seminiferous epithelium regulating spermatogenesis: a biochemical and molecular perspective.

During spermatogenesis in adult rat testes, fully developed spermatids (i.e. spermatozoa) at the luminal edge of the seminiferous epithelium undergo "spermiation" at stage VIII of the seminiferous epithelial cycle. This is manifested by the disruption of the apical ectoplasmic specialization (apical ES) so that spermatozoa can enter the tubule lumen and to complete their maturation in the epididymis. At the same time, the blood-testis barrier (BTB) located near the basement membrane undergoes extensive restructuring to allow transit of preleptotene spermatocytes so that post-meiotic germ cells complete their development behind the BTB. While spermiation and BTB restructuring take place concurrently at opposite ends of the Sertoli cell epithelium, the biochemical mechanism(s) by which they are coordinated were not known until recently. Studies have shown that fragments of laminin chains are generated from the laminin/integrin protein complex at the apical ES via the action of MMP-2 (matrix metalloprotease-2) at spermiation. These peptides serve as the local autocrine factors to destabilize the BTB. These laminin peptides also exert their effects on hemidesmosome which, in turn, further potentiates BTB restructuring. Thus, a novel apical ES-BTB-hemidesmosome regulatory loop is operating in the seminiferous epithelium to coordinate these two crucial cellular events of spermatogenesis. This functional loop is further assisted by the Par3/Par6-based polarity protein complex in coordination with cytokines and testosterone at the BTB. Herein, we provide a critical review based on the latest findings in the field regarding the regulation of these cellular events. These recent findings also open up a new window for investigators studying blood-tissue barriers.

A complex containing alpha6beta1-integrin and phosphorylated focal adhesion kinase between Sertoli cells and elongated spermatids during spermatid release from the seminiferous epithelium.

Spermiation is the final step of spermatogenesis and culminates in the disengagement (release) of elongated spermatids from Sertoli cells into the seminiferous tubule lumen. Spermiation failure, wherein spermatids are retained by Sertoli cells instead of releasing, occurs after hormone suppression. The mechanisms involved in spermatid disengagement and retention are not well understood. We previously showed that beta(1)-integrin is associated with spermatids until the point of disengagement, but the ectoplasmic specialisation junction (ES) is not. The aims of this paper are to further characterise the complex that is present immediately prior to spermatid disengagement by identifying the alpha-integrin form dimerised with beta(1)-integrin, localising focal adhesion kinase (FAK) and determining if microtubules are involved. Adult Sprague-Dawley rats received testosterone and oestradiol implants and an FSH antibody for 7 days to suppress testicular testosterone and FSH and induce spermiation failure. Control rats were treated with saline. Immunohistochemical analysis showed that alpha(6)-integrin and a phosphorylated form of FAK (FAK-Tyr(397)) are present between late spermatids and Sertoli cells after ES removal, until the point of disengagement, and both proteins remain associated with retained spermatids after spermiation failure induced by hormone suppression. Using dual-label immunofluorescence, tubulins (and thus microtubules) were observed to co-localise with ES, but were neither associated with elongated spermatids just prior to release nor with retained spermatids following hormone suppression. These results suggest that microtubules are not involved in the final release of spermatids from Sertoli cells. We conclude that spermatid release during spermiation is mediated by a 'disengagement complex' containing alpha(6)beta(1)-integrin and phospho-FAK, the function of which can be affected by gonadotrophin suppression.

Regulated production of activin A and inhibin B throughout the cycle of the seminiferous epithelium in the rat.

Production and regulation of activin A and inhibin B during the cycle of the seminiferous epithelium were investigated in adult rats. Immunohistochemistry localised the activin beta(A)-subunit to the Sertoli cell cytoplasm, with much weaker expression in spermatocytes and spermatids. Both activin A and inhibin B, measured by ELISA were secreted by, seminiferous tubule fragments over 72 h in culture. Activin A was secreted in a cyclic manner with peak secretion from tubules isolated at stage VIII. Tubules collected during stage VI produced the least activin A. Inhibin B secretion was highest from stage IX-I tubules and lowest from stage VII tubules. Addition of interleukin-1beta (IL-1beta) had relatively little effect on activin A or inhibin B secretion in culture. In contrast, the peak secretion of activin A by stage VIII tubules was blocked by co-incubation with an excess of human recombinant IL-1 receptor antagonist, whereas inhibin B secretion increased slightly. Dibutyryl cAMP stimulated activin A secretion by late stage VII and VIII tubules and stimulated inhibin B across all stages. These data indicate that activin A and inhibin B are cyclically regulated within the seminiferous epithelium, with endogenous IL-1 (presumably IL-1alpha produced by the Sertoli cells), responsible for a peak of activin A production subsequent to sperm release at stage VIII. These data provide direct evidence that production of activin A and inhibin B by the Sertoli cell is locally modulated by IL-1alpha , in addition to FSH/cAMP, under the influence of the developing spermatogenic cells.

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis.

Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.

Chromogranin-A expression in the bovine testis.

Chromogranin-A (CgA) is the most distributed member of the granin family. Chromogranins are soluble anionic glycoproteins, found in the majority of the neuroendocrine and neural cells, co-stored with other endocrine substances (like insulin, glucagon, FSH and LH or NPY) in secretory granules. Outside the cell, it has been suggested that this peptide or one of its fragments, obtained by proteolytic cleavage, could act in an autocrine or paracrine way, regulating either the cell function or the contractibility of vascular segments. The purpose of the present study is to determine the distribution of chromogranin-A in the structures of the bovine testis. Immunohistochemical analysis was performed employing the biotin-streptavidin-peroxidase immunostaining technique in tissue specimens obtained at a local abbatoir. A CgA expression was found in the germinal epithelium at several stages of differentiation. Generally the strongest positive reaction was consistently observed in the basal compartment of the seminiferous tubules, with spermatogonia presenting a dense granular immunostaining pattern; a less intense reaction was also consistently recorded in type II spermatocytes and in round spermatids, which showed a more scattered disposition of CgA-positive granules. Clusters of Leydig cells also displayed a faint and homogeneous cytoplasmatic immunoreactivity for chromogranin-A. These results demonstrate a widely distribution of CgA-positive cells in the organism, and its presence in the testis raises the possibility of its participation in the cohort of local factors involved in the regulation of spermatogenesis.

Spermatogenic cells distal to the blood-testis barrier in rats lack C3 convertase regulators and may be at risk of complement-mediated injury.

On most tissues, multiple membrane complement regulators (CReg) protect self-cells from damage by complement. An exception is the brain, where the blood-brain barrier provides a protected environment within which cells survive with little or no protection from complement. The testis has a functionally similar structure, the blood-testis barrier (BTB). Here, we have investigated the expression of C3/C5 convertase CReg and C3 in the normal rat testis at different ages and different spermatogenetic stages, as well as in rats in which spermatogenesis and the BTB were impaired due to a developmental deficit. Immature testis, prior to BTB formation at puberty, displayed broad expression of the ubiquitous rodent CReg Crry on all elements and no expression of CD46 or CD55. Within days of BTB formation, CReg expression was dramatically altered; Crry was expressed only in the spermatogenetic cells external to the BTB in basal layers of adult seminal epithelium. Spermatogenic cells immediately distal to the BTB at first expressed no C3/C5 convertase regulators but later acquired expression of CD46 and CD55. Staining for C3 was widespread pre-puberty, but absent distal to the BTB in mature rats. In rats with defects in spermatogenesis and BTB integrity, expression patterns of CReg and C3 resembled those in pre-pubertal normals. The relative paucity of CReg and absence of C3 synthesis distal to the BTB suggest the presence of a complement-protected environment analogous to that described in the brain, and suggest also that cells enclosed by the BTB may be susceptible to complement damage when the barrier is breached.

Histochemical analysis of glycoconjugates in the domestic cat testis.

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.

Neonatal coadministration of testosterone with diethylstilbestrol prevents diethylstilbestrol induction of most reproductive tract abnormalities in male rats.

The primary purpose of this study was to evaluate whether the coadministration of testosterone (TE; 200 micro g) with 10 micro g of diethylstilbestrol (DES) between days 2 and 12 postnatally could prevent the adverse gross reproductive tract changes and associated loss of androgen receptor (AR) expression induced by DES treatment alone. Various endpoints (rete testis area, efferent duct lumen area, epithelial cell height of efferent ducts, and vas deferens) were quantified to check for the abnormal changes that have been shown to occur after neonatal treatment with a high dose (10 micro g) of DES. Additionally, DES induction of an aberrant pattern of estrogen receptor alpha (ER-alpha) immunoexpression in the vas deferens and seminal vesicles was evaluated. The coadministration of DES with TE prevented the induction of all but one of the abnormalities induced by DES treatment on its own, coincident with the restoration of normal/supranormal TE levels and normal immunoexpression of the AR and ER-alpha in the tissues studied. The exception was DES-induced lumenal distension of the efferent ducts, which was only partially prevented by the coadministration of DES with TE. These evaluations were made on day 18, but the described abnormalities were already somewhat evident by day 8 in DES-treated animals. It was therefore tested whether a delay of TE replacement until days 8-12 was still able to reverse the abnormalities already induced by DES treatment alone. A delayed treatment with TE reversed the adverse changes in epithelial cell height and in ER-alpha and AR immunoexpression in the same tissues by day 18; however, rete testis overgrowth was only partially prevented, and efferent duct distension was not prevented at all. These results provide further evidence that DES-induced disorders of reproductive tract development in the male result from a disturbance of the androgen-estrogen balance rather than from estrogen action alone.

TGF-beta3 regulates the blood-testis barrier dynamics via the p38 mitogen activated protein (MAP) kinase pathway: an in vivo study.

Recent studies using Sertoli cells cultured in vitro to permit tight junction (TJ) assembly have shown that TJ dynamics are regulated, at least in part, by TGF-beta3 via the p38 mitogen activated protein (MAP) kinase pathway. This in turn regulates the production of occludin, a TJ-integral membrane protein, by Sertoli cells. Yet it is not known if this pathways is used by Sertoli cells to regulate the blood-testis barrier (BTB) function in vivo. Using an in vivo model for studying BTB dynamics, we report herein the CdCl(2)-induced BTB damage in rats was associated with a significant reduction in testicular occludin along with a loss of immunoreactive occludin in the seminiferous epithelium at the site of the BTB. Also, this CdCl(2)-induced occludin loss from the BTB coincided with a surge in testicular TGF-beta3, as well as p-p38 MAP kinase (the phosphorylated/activated form of p38), but not p38 MAP kinase and neither extracellular signal-regulated kinase nor its phosphorylated form (ERK/p-ERK), consistent with results of in vitro studies. More important, intratesticular administration of SB202190, a specific p38 MAP kinase inhibitor, could block the CdCl(2)-induced occludin loss from the BTB. These results illustrate that BTB dynamics in vivo are regulated by the TGF-beta3/p38 MAP kinase pathway, which in turn determines the level of occludin at the site of Sertoli cells TJs.

The Fas system in the seminiferous epithelium and its possible extra-testicular role.

The Fas system is involved in the control of immune system homeostasis and nonfunctional Fas system leads to autoimmune disease in mice and humans. The Fas system is a mechanism through which cells expressing Fas ligand (FasL) induce apoptosis of Fas expressing cells. In mouse and rat, the testis represents the main source of constitutive FasL in the body. The roles so far proposed for this molecule in the testis, such as maintenance of immunoprivilege and regulation of physiological germ cell apoptosis, need to be reconsidered as both hypotheses are based on an erroneous cellular location of FasL in the seminiferous epithelium. Recently, we demonstrated that in rodents FasL mRNA is present in germ cells and not in Sertoli cells, and that FasL protein is displayed on the surface of spermatozoa. Here we propose that, for the mouse spermatozoa, the FasL may represent a self-defence mechanism against lymphocytes present in the female genital tract. To verify this hypothesis, we performed crossings between males gld, with nonfunctional FasL, and syngenic or nonsyngenic females. We observed a significant decrease of litter size in outbred crossings with gld males compared with wild-type males, suggesting a possible role of FasL in immunoprotection of the sperm in the female genital tract. The possibility that in humans, by analogy with mouse, FasL plays a self-protective role for the spermatozoon cannot be excluded, and awaits experimental information on the expression of FasL on human sperm cells.

Activation of mitogen activated protein kinases and apoptosis of germ cells after vasectomy in the rat.

PURPOSE: Vasectomy induces a large amount of germ cell apoptosis. We examined the activation of mitogen activated protein kinases (MAPKs) in association with the apoptosis and proliferation of germ cells after vasectomy in the rat. MATERIALS AND METHODS: Eight-week-old Wistar rats underwent bilateral vasectomy and the testes were harvested 1 to 9 days after vasectomy. Germ cell apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling and electrophoretic assay of DNA fragmentation. Western blotting and immunohistochemistry were used to examine the temporal and spatial activation of signal regulated kinases 1/2, c-Jun-terminal kinases 1/2 and p38. Phospho-specific MAPK antibodies were used to examine their activations. Proliferation of germ cells was evaluated by proliferative nuclear cell antigen expression. RESULTS: Germ cell apoptosis was detected predominantly in primary spermatocytes with a peak 7 days after vasectomy. Signal regulated kinases 1/2, c-Jun-terminal kinases 1/2 and p38 were constitutively expressed in the control testis. Western blotting and immunohistochemistry showed rapid activation of signal regulated kinases 1/2, followed by activation of c-Jun-terminal kinases 1/2 and p38. Immunohistochemical study demonstrated the temporal and spatial relationships of apoptosis and MAPK activation in primary spermatocytes. On the other hand, proliferating cell nuclear antigen expression was enhanced in tetraploid spermatocyte and spermatogonia maximally 5 days after vasectomy. CONCLUSIONS: MAPKs were rapidly activated after vasectomy and germ cell apoptosis was observed after vasectomy. In contrast to the delayed phase up to 24 weeks after vasectomy, we observed hyperdynamic cellular turnover, spermatocyte loss through apoptosis and enhanced germ cell proliferation transiently at the early phase after vasectomy.

New method of quantitative analysis of seminiferous epithelium in male guinea pigs.

The aim of the study was the comparison of a newly developed method of quantitative analysis of the seminiferous epithelium (SE) cells with the traditional method based on stage classification according to Clermont. In 4000 sections of seminiferous tubules the following parameters have been evaluated: in procedure I--the frequency of cells at the particular stages of the SE cycle, in procedure II--the frequency of particular cell types forming SE. In the two procedures, 17,726 and 14,670 cells were assessed, respectively. In both procedures, the mean values of the studied cells per animal (I--886.3 +/- 15.4; II--733.5 +/- 96.9) and the distribution of the particular types of cells were similar. The new method of quantitative analysis is less laborious and cheaper. It may be applied instead of the method used so far.

Expression of neurotrophin receptors in rat testis. Upregulation of TrkA mRNA with hCG treatment.

We report the expression of TrkA, TrkB and TrkC mRNAs in adult rat testis. With in situ hybridisation a low signal for TrkB and TrkC could be seen in postmeiotic cells of the seminiferous epithelium, whereas no signal for TrkA could be observed in untreated animals. Animals treated with hCG showed an induction of TrkA mRNA in premeiotic cells 12 h after the treatment, whereas an injection with EDS had no effect on the expression of Trk mRNAs. With the RNAse protection assay a low signal for TrkA was seen in whole testis of hCG treated animals. In staged tubules low expression was seen at stages VII-XI of untreated animals. Animals injected with hCG revealed that TrkA induction was highest during stages VIIcd and VIII of the cycle. The distinct expression pattern of these high-affinity neurotrophin receptors suggests different roles for neurotrophins during spermatogenesis. Induction of TrkA mRNA by hCG suggests that high-affinity binding of NGF during stages VIIcd-VIII in premeiotic cells is under control of the hypothalamic-pituitary-testicular axis.

Identification of N- and O-linked oligosaccharides in human seminal vesicles.

The main oligosaccharide residues and the saccharide linkage in infantile and adult human seminal vesicles were studied by means of lectin histochemistry at light and electron microscopy levels. In adult glands, the epithelial cell cytoplasm and luminal content reacted positively to the following residues: (GlcNAc)n (WGA), Galbeta1,3GalNAc (PNA), GalNAcalpha1,3Gal (SBA), GalNAcalpha1,3GalNAc (HPA), Fucalpha1,2Galbeta1,4GlcNAc (UEA-I), and alphaL-Fuc1,6DGlcNAc-O-Melibiosc (AAA). The presence of intense staining in the luminal content suggest that glycoproteins containing these oligosaccharide moieties are secreted by epithelial cells. Adult epithelial cells also reacted to Neu5Acalpha2,6Gal (SNA), Neu5Acalphaa2,3Galbeta1,4GlcNAc (MAA), Galbeta1,4GlcNAc (DSA), branched mannose chains (ConA), Man1,3Man (GNA), and Fucalpha1,2Galbeta1,4GlcNAcFucalpha1,3GlcNAc (LTA) but reaction to these residues was weak (MAA, DSA, ConA, and LTA) or absent (SNA and GNA) in the gland lumen, which suggests that they belong to intracytoplasmic proteins. The chemical and enzymatic treatments used suggest that the residues recognized by SNA, MAA, PNA, DSA, HPA, and SBA belong to O-linked oligosaccharides; those residues localized by ConA and GNA have an N-glycosidic linkage, and those bound by WGA, LTA, UEA-I, and AAA are linked to both N- and O-oligosaccharides. In prepubertal seminal vesicles, reaction in the epithelial cell cytoplasm was similar to that observed in adults, except for GNA and HPA, which showed a weaker reaction. However, the lumen of prepubertal seminal vesicles showed intense reaction to WGA and SBA only. The chemical and enzymatic treatments suggest that the scanty glycoproteins secreted by the prepubertal glands belong to the mucin-type.