Bisphenols as promoters of the dysregulation of cellular junction proteins of the blood-testis barrier in experimental animals: A systematic review of the literature.

Daily, people are exposed to chemicals and environmental compounds such as bisphenols (BPs). These substances are present in more than 80% of human fluids. Human exposure to BPs is associated with male reproductive health disorders. Some of the main targets of BPs are intercellular junction proteins of the blood-testis barrier (BTB) in Sertoli cells because BPs alter the expression or induce aberrant localization of these proteins. In this systematic review, we explore the effects of BP exposure on the expression of BTB junction proteins and the characteristics of in vivo studies to identify potential gaps and priorities for future research. To this end, we conducted a systematic review of articles. Thirteen studies met our inclusion criteria. In most studies, animals treated with bisphenol-A (BPA) showed decreased occludin expression at all tested doses. However, bisphenol-AF treatment did not alter occludin expression. Cx43, ZO-1, ß-catenin, nectin-3, cortactin, paladin, and claudin-11 expression also decreased in some tested doses of BP, while N-cadherin and FAK expression increased. BP treatment did not alter the expression of α and γ catenin, E-cadherin, JAM-A, and Arp 3. However, the expression of all these proteins was altered when BPA was administered to neonatal rodents in microgram doses. The results show significant heterogeneity between studies. Thus, it is necessary to perform more research to characterize the changes in BTB protein expression induced by BPs in animals to highlight future research directions that can inform the evaluation of risk of toxicity in humans.

Effects of bisphenols on Blood-Testis Barrier protein expression in vitro: A systematic review and meta-analysis.

Bisphenols are a group of environmental endocrine-disrupting chemicals that produce alterations in the expression of intercellular junction proteins of the Blood-Testis Barrier (BTB) involved in spermatogenesis. The association between bisphenol exposure and BTB protein expression is controversial. Therefore, we performed this systematic review and meta-analysis to clarify bisphenol effects on Sertoli cell BTB protein expression in vitro. The Standardized Mean Difference (SMD) with a 95 % confidence interval (95 % CI) was used to evaluate the association between alterations in the BTB protein expression and bisphenol exposure in vitro. Six articles were included in the meta-analysis. Bisphenol-A (BPA) exposure at 200 µM was associated with significant decrease in BTB protein expression (SMD = -2.70, 95 %CI: -3.59, -1.80, p het = 0.46, p = <0.00001). In the moderate (40-50 µM) and low dose (<25 µM), no significant associations were obtained. We also found a non-monotonic dose-response curve of bisphenol effect in ZO-1 protein expression; low and high doses presented a significant decrease compared to control, while moderate dose presented no change. The current temporary Tolerable Daily Intake (tTDI) of BPA is 4 µg/kg bw/day. The 5-25 µM doses of BPA are equivalent to ∼1-5 mg/kg bw, respectively. Although the low dose group (<25 µM) assessed doses below the previous NOAEL value, these doses are above the current tTDI. Thus, it is necessary to conduct more studies with lower bisphenol concentrations to avoid underestimating the potential adverse effects of bisphenols at doses below tTDI.

Low Doses of Glyphosate/Roundup Alter Blood-Testis Barrier Integrity in Juvenile Rats.

It has been postulated that glyphosate (G) or its commercial formulation Roundup (R) might lead to male fertility impairment. In this study, we investigated the possible effects of G or R treatment of juvenile male rats on blood-testis barrier function and on adult male sperm production. Pups were randomly assigned to the following groups: control group (C), receiving water; G2 and G50 groups, receiving 2 and 50 mg/kg/day G respectively; and R2 and R50 groups receiving 2 and 50 mg/kg/day R respectively. Treatments were performed orally from postnatal day (PND) 14 to 30, period of life that is essential to complete a functional blood-testis barrier. Evaluation was done on PND 31. No differences in body and testis weight were observed between groups. Testis histological analysis showed disorganized seminiferous epithelium, with apparent low cellular adhesion in treated animals. Blood-testis barrier permeability to a biotin tracer was examined. A significant increase in permeable tubules was observed in treated groups. To evaluate possible mechanisms that could explain the effects on blood-testis barrier permeability, intratesticular testosterone levels, androgen receptor expression, thiobarbituric acid reactive substances (TBARS) and the expression of intercellular junction proteins (claudin11, occludin, ZO-1, connexin43, 46, and 50 which are components of the blood-testis barrier) were examined. No modifications in the above-mentioned parameters were detected. To evaluate whether juvenile exposure to G and R could have consequences during adulthood, a set of animals of the R50 group was allowed to grow up until PND 90. Histological analysis showed that control and R50 groups had normal cellular associations and complete spermatogenesis. Also, blood-testis barrier function was recovered and testicular weight, daily sperm production, and epididymal sperm motility and morphology did not seem to be modified by juvenile treatment. In conclusion, the results presented herein show that continuous exposure to low doses of G or R alters blood-testis barrier permeability in juvenile rats. However, considering that adult animals treated during the juvenile stage showed no differences in daily sperm production compared with control animals, it is feasible to think that blood-testis barrier impairment is a reversible phenomenon. More studies are needed to determine possible damage in the reproductive function of human juvenile populations exposed to low doses of G or R.

Distribution pattern of ZO-1 and claudins in the epididymis of vampire bats.

Epithelial cells connect with each other by tight junctions (TJs) in several tissues. In epididymides, TJs proteins form the blood-epididymis barrier (BEB), which is crucial for male fertility. However, little is known about BEB morphological and physiological aspects in wild animals. This study examines the region-specific distribution pattern of TJs proteins in D. rotundus' epididymis, assessing their regulation in rainy and dry season. The expression of zonula occludens-1 (ZO-1), and claudins (Cldn)-1, -3, and -4 were evaluated by confocal immunofluorescence and ELISA analysis. Herein, ZO-1 was strictly expressed in TJs, whereas Cldns were expressed in TJs and basolateral membranes of epithelial cells. Their co-localization and intensity of expression varied in the epididymal regions examined. The effect of season on protein expression was detected mainly in TJ proteins located in the proximal regions. As such, in the initial segment (IS), Cldn-3 and -4 were detected at low levels in basolateral membranes in the rainy season compared to the dry season. Furthermore, in the distal IS, Cldn-1 expression was lower in TJs of epithelial cells during the rainy season than the dry season. ZO-1 expression was higher in the cauda region than the corpus region by ELISA analysis. Additionally, in the corpus region, ZO-1 expression was higher in TJs during dry season compared to the rainy season. Our study sheds light on the understanding of BEB in D. rotundus, improving the knowledge of their reproductive biology.

Enzyme-Linked Immunosorbent Assay and Quantitative Reverse Transcription PCR as a Technique to Analyze Inflammation.

Inflammation is part of a defense reaction of live tissues that is triggered by pathogens, chemical reagents, trauma, and radiation. Understanding the inflammatory process triggered by Zika virus (ZIKV) is important to better understand the pathogen-host interaction. The evaluation of this process can be done using tools such as enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription PCR (RT-qPCR). Both techniques have been an indispensable tool not just for immunologists but for all interested in understanding the inflammatory process.

Methyl parathion causes genetic damage in sperm and disrupts the permeability of the blood-testis barrier by an oxidant mechanism in mice.

Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells.

In vitro effects of glyphosate and Roundup on Sertoli cell physiology.

Roundup (R), a formulation that contains glyphosate (G) as the active ingredient, is a commonly used nonselective herbicide that has been proposed to affect male fertility. It is well known that an adequate Sertoli cell function is essential to maintain germ cell development. The aim of the present study was to analyze whether G and R are able to affect Sertoli cell functions, such as energy metabolism and blood-testis barrier (BTB) integrity. Sertoli cell cultures from 20-day-old rats were exposed to 10 and 100 ppm of G or R, doses which do not decrease cell viability. Neither G nor R caused impairment in lactate production or fatty acid oxidation. G and R decreased Transepithelial Electrical Resistance, which indicates the establishment of a Sertoli cell junction barrier. However, neither G nor R modified the expression of claudin11, ZO1 and occludin, proteins that constitute the BTB. Analysis of cellular distribution of claudin11 by immunofluorescence showed that G and R induced a delocalization of the signal from membrane to the cytoplasm. The results suggest that G and R could alter an important function of Sertoli cell such as BTB integrity and thus they could compromise the normal development of spermatogenesis.

Torsión testicular, anticuerpos antiesperma y repercusión clínica / Testicular Torsion, Antisperm Antibodies, and Clinical Repercussions

La torsión testicular es una patología que afecta directamente la calidad del esperma de los pacientes debido a la ruptura de la barrera hematotesticular, lo cual hace que se formen anticuerpos antiesperma, que inclusive afectan al testículo contralateral. Múltiples estudios tanto en modelos animales como humanos, permiten hacer evidente la relación entre la torsión testicular y la formación de anticuerpos antiesperma y su repercusión clínica, principalmente, en la fertilidad.

Testicular torsion is a condition that directly affects the quality of the sperm of patients due to rupture of the blood-testis barrier; it leads to the formation of antisperm antibodies and can even affect the contralateral testis. This condition has been demonstrated previously, and multiple studies have been conducted in both animal and human models. In this review, the evidence from studies of testicular torsion, the formation of antisperm antibodies, and the clinical repercussions, mainly with respect to fertility, is presented.

The organophosphate pesticide methamidophos opens the blood-testis barrier and covalently binds to ZO-2 in mice.

Methamidophos (MET) is an organophosphate (OP) pesticide widely used in agriculture in developing countries. MET causes adverse effects in male reproductive function in humans and experimental animals, but the underlying mechanisms remain largely unknown. We explored the effect of MET on mice testes (5 mg/kg/day/4 days), finding that this pesticide opens the blood-testis barrier and perturbs spermatogenesis, generating the appearance of immature germ cells in the epididymis. In the seminiferous tubules, MET treatment changed the level of expression or modified the stage-specific localization of tight junction (TJ) proteins ZO-1, ZO-2, occludin, and claudin-3. In contrast, claudin-11 was barely altered. MET also modified the shape of claudin-11, and ZO-2 at the cell border, from a zigzag to a more linear pattern. In addition, MET diminished the expression of ZO-2 in spermatids present in seminiferous tubules, induced the phosphorylation of ZO-2 and occludin in testes and reduced the interaction between these proteins assessed by co-immunoprecipitation. MET formed covalent bonds with ZO-2 in serine, tyrosine and lysine residues. The covalent modifications formed on ZO-2 at putative phosphorylation sites might interfere with ZO-2 interaction with regulatory molecules and other TJ proteins. MET bonds formed at ZO-2 ubiquitination sites likely interfere with ZO-2 degradation and TJ sealing, based on results obtained in cultured epithelial cells transfected with ZO-2 mutated at a MET target lysine residue. Our results shed light on MET male reproductive toxicity and are important to improve regulations regarding the use of OP pesticides and to protect the health of agricultural workers.

In Vitro Evaluation of Damage by Heavy Metals in Tight and Gap Junctions of Sertoli Cells.

The Sertoli cell plays a vital role during the spermatogenesis process and has been identified as one of the main targets of the toxic action of heavy metals on the seminiferous epithelium. In the present work, the effect of lead (Pb), Arsenic (As), and Cadmium (Cd) in primary cultures of Sertoli cells was analyzed by measuring the expression of the genes Cldn11, Ocln, and Gja1 that participate in the tight and gap junctions, which are responsible for maintaining the blood-testis barrier. Sertoli cells were isolated from the testes of Wistar rats. Sertoli cell cultures were exposed separately and at the same concentrations of three heavy metals for 48 h. Subsequently, gene expression was measured by real-time polymerase chain reaction. In the morphological analysis of the cultures, after 24 h, the cultures exposed to Cd showed greatest detachment of the monolayer, followed by those exposed to As and Pb. As for gene expression patterns, As induced a decrease in the expression of the Cldn11 gene at 24 and 48 h (p < 0.01) and in that of Ocln at 24 (p < 0.001) and 48 h (p < 0.01), whereas Cd induced overexpression of the Gja1 gene from day 1 of exposure (p < 0.001) and subexpression of the Ocln gene (p < 0.05) at 24 h. Because each of these three metals generated different expression patterns in the three genes, we can postulate that the mechanisms of damage that they induce are different; therefore, the effect that they exert on the Sertoli cell occurs through different pathways, generating changes in structural proteins, altering Sertoli cell morphology, and compromising its function in the regulation of the spermatogenesis process.

Regulatory and junctional proteins of the blood-testis barrier in human Sertoli cells are modified by monobutyl phthalate (MBP) and bisphenol A (BPA) exposure.

The blood-testis barrier (BTB) is responsible for providing a protected environment and coordinating the spermatogenesis. Endocrine disruptors (EDs) might lead to infertility, interfering in the BTB structure and modulation. This study aimed to correlate the actions of two EDs, monobutyl phthalate (MBP) and bisphenol A (BPA) in different periods of exposure, in a low toxicity dose to the human Sertoli cells (HSeC) and its effects on the proteins of the BTB and regulatory proteins involved in its modulation. HSeC cells were exposed to MBP (10µM) and BPA (20µM) for 6 and 48h. Western Blot assay indicated that MBP was able to reduce the expression of occludin, ZO-1, N-cadherin and Androgen Receptor (AR), while BPA leads to a reduction of occludin, ZO-1, ß-catenin and AR. TGF-ß2 and F-actin were not modified. Phalloidin and Hematoxylin and Eosin assay revealed phenotically disruption in Sertoli cells adhesion, without changes in F-actin expression or localization. Our data suggested both EDs present potential for disrupting the structure and maintenance of the human BTB by AR dependent pathway.

Polyglucosan Molecules Induce Mitochondrial Impairment and Apoptosis in Germ Cells Without Affecting the Integrity and Functionality of Sertoli Cells.

Glycogen is the main storage form of glucose; however, the accumulation of glycogen-like glucose polymers can lead to degeneration and cellular death. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase enhances the apoptosis of pre-meiotic male germ cells and a complete disorganization of the seminiferous tubules. Here we sought to further identify the effects of glycogen storage in cells from the seminiferous tubules and the mechanism behind the pro-apoptotic activity induced by its accumulation. Using an in vitro culture of Sertoli cells (line 42GPA9) and spermatocyte-like cells (line GC-1) expressing a superactive form of glycogen synthase or the Protein Targeting to Glycogen (PTG), we found that glycogen synthesized in both cell lines is poorly branched. In addition, the immunodetection of key molecules of apoptotic events suggests that cellular death induced by polyglucosan molecules affects GC-1 cells, but not 42GPA9 cells by mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood-testis barrier. The results using a non-permeable fluorescent molecule showed that, in conditions of over-synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier and the levels of connexin43, occludin, and ZO1 proteins were not affected. These results suggest that the accumulation of polyglucosan molecules has a selective effect-triggered by the intrinsic activation of the apoptotic pathway-in germ cells without directly affecting Sertoli cells. J. Cell. Physiol. 231: 2142-2152, 2016. © 2016 Wiley Periodicals, Inc.

Behavior of calf Sertoli cells and fibroblast cells transfected with the human HNP-1 gene.

Human neutrophil peptide-1 (HNP-1) is an important defense molecule in neutrophils and Sertoli cells and plays an important role in the blood-testis barrier. In this study, we investigated the behavior of Sertoli cells transfected with the HNP-1 gene and compared the ability of Sertoli cells and fibroblast cells to resist transfection. Total RNA was isolated from human blood. The DNA coding sequence of HNP-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the eukaryotic expression vector pEGFP-N3-HNP-1 was identified by PCR, endonuclease digestion, and sequencing. Bovine Sertoli cells and fibroblast cells were transfected with pEGFP-N3-HNP-1 using Liposome reagent. The transfection efficiency and the behavior of the transfected cells were evaluated at 24, 48 and 72 h as well as at other times after transfection. The plasmid pEGFP-N3-HNP-1 was successfully constructed. The cells achieved maximum transfection efficiency at 48 h. Two weeks after transfection, the cells began to stop dividing. The ability of Sertoli cells to resist transfection was higher compared to fibroblast cells. The ability of the 2 cell types to resist transfection was higher with plasmid pEGFP-N3-HNP-1 than with the plasmid pEGFP-N3. The injury to Sertoli cells caused by transfection with the HNP-1 gene was less pronounced than in fibroblast cells, which may be closely correlated with the physiological function of Sertoli cells.

Loss of occludin expression and impairment of blood-testis barrier permeability in rats with autoimmune orchitis: effect of interleukin 6 on Sertoli cell tight junctions.

Inflammation of the male reproductive tract is accepted as being an important etiological factor of infertility. Experimental autoimmune orchitis (EAO) is characterized by interstitial lymphomononuclear cell infiltration and severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. Because the blood-testis barrier (BTB) is relevant for the protection of haploid germ cells against immune attack, the aim of this study was to analyze BTB permeability and the expression of tight junction proteins (occludin, claudin 11, and tight junction protein 1 [TJP1]) in rats during development of autoimmune orchitis. The role of IL6 as modulator of tight junction dynamics was also evaluated because intratesticular content of this cytokine is increased in EAO rats. Orchitis was induced in Sprague-Dawley adult rats by active immunization with testicular homogenate and adjuvants. Control rats (C) were injected with saline solution and adjuvants. Untreated (N) rats were also studied. Concomitant with early signs of germ cell sloughing, a reduced expression of occludin and delocalization of claudin 11 and TJP1 were detected in the testes of rats with EAO compared to C and N groups. The use of tracers showed increased BTB permeability in EAO rats. Intratesticular injection of IL6 induced focal testicular inflammation, which is associated with damaged seminiferous tubules. Rat Sertoli cells cultured in the presence of IL6 exhibited a redistribution of tight junction proteins and reduced transepithelial electrical resistance. These data indicate the possibility that IL6 might be involved in the downregulation of occludin expression and in the modulation of BTB permeability that occur in rats undergoing autoimmune orchitis.

Involvement of soluble Fas Ligand in germ cell apoptosis in testis of rats undergoing autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas-Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.

Blood-tissue barriers: morphofunctional and immunological aspects of the blood-testis and blood-epididymal barriers.

The blood-testis barrier (BTB) is known for its ability to create an immune privilege site in the seminiferous epithelium, but less is known of the blood-epididymal barrier (BEB). It is already established that the fully functional BTB and BEB are much more complex and consist of anatomical/physical (tight junctions, basolateral and apical membranes), physiological and immunological components, which are all necessary to make a functioning barrier in the testis and epididymis. However, comparative data for metazoans suggest that an effective Sertoli cell barrier is not entirely necessary for the development of germ cells during spermatogenesis or that our knowledge about the barrier structure/function in metazoans is still immature. This chapter compares the unique barrier formed by the Sertoli cells of the testis to that formed by the apical junctional complexes of the epididymal epithelium.

The actin filament network associated to Sertoli cell ectoplasmic specializations

Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.

The actin filament network associated to Sertoli cell ectoplasmic specializations

Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle

The actin filament network associated to Sertoli cell ectoplasmic specializations

Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle