Map-1a regulates Sertoli cell BTB dynamics through the cytoskeletal organization of microtubule and F-actin.

Microtubule-associated protein 1a (Map1a) is a microtubule (MT) regulatory protein that binds to the MT protofilaments in mammalian cells to promote MT stabilization. Maps work with MT cleavage proteins and other MT catastrophe-inducing proteins to confer MT dynamics to support changes in the Sertoli cell shape to sustain spermatogenesis. However, no functional studies are found in the literature to probe its role in spermatogenesis. Using an RNAi approach, coupled with the use of toxicant-induced testis (in vivo)- and Sertoli cell (in vitro)-injury models, RNA-Seq analysis, transcriptome profiling, and relevant bioinformatics analysis, immunofluorescence analysis, and pertinent biochemical assays for cytoskeletal organization, we have delineated the functional role of Map1a in Sertoli cells and testes. Map1a was shown to support MT structural organization, and its knockdown (KD) also perturbed the structural organization of actin, vimentin, and septin cytoskeletons as these cytoskeletons are intimately related, working in concert to support spermatogenesis. More importantly, cadmium-induced Sertoli cell injury that perturbed the MT structural organization across the cell cytoplasm was associated with disruptive changes in the distribution of Map1a and a surge in p-p38-MAPK (phosphorylated p38-mitogen-activated protein kinase) expression but not total p38-MAPK. These findings thus support the notion that p-p38-MAPK activation is involved in cadmium-induced Sertoli cell injury. This conclusion was supported by studies using doramapimod, a specific p38-MAPK phosphorylation (activation) inhibitor, which was capable of restoring the cadmium-induced disruptive structural organization of MTs across the Sertoli cell cytoplasm. In summary: this study provides mechanistic insights regarding restoration of toxicant-induced Sertoli cell and testis injury and male infertility.

The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, ß-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, ß-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

The FAK/occludin/ZO-1 complex is critical for cadmium-induced testicular damage by disruption of the integrity of the blood-testis barrier in chickens.

Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and reproductive function. However, the mechanism of Cd-induced disruption of BTB and testicular damage has not been fully elucidated. Here, our study investigates the effects of Cd on BTB integrity and testicular dysfunction. 80 (aged 1 day) Hy-Line white variety chickens were randomly designed into 4 groups and treated for 90 days, as follows: control group (essential diet), 35 Cd, 70 Cd and 140 Cd groups (35, 70 and 140 mg/kg Cd). The results found that Cd exposure diminished volume of the testes and induced histopathological lesions in the testes. Exposure to Cd induced an inflammatory response, disrupted the structure and function of the FAK/occludin/ZO-1 protein complex and disrupted the tight junction and adherens junction in the BTB. In addition, Cd exposure reduced the expression of steroid-related proteins and inhibited testosterone synthesis. Taken together, these data elucidate that Cd disrupts the integrity of the BTB and further inhibits spermatogenesis by dissociating the FAK/occludin/ZO-1 complex, which provides a basis for further investigation into the mechanisms of Cd-induced impairment of male reproductive function and pharmacological protection.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Cross-talk between Vimentin and autophagy regulates blood-testis barrier disruption induced by cadmium.

The blood-testis barrier (BTB) plays a vital role in mammalian spermatogenesis by separating the seminiferous epithelium into an adluminal and a basal compartment. Cadmium (Cd) is a toxic heavy metal that is widely present in the environment. We observed that Cd can induce BTB disruption, leading to apoptosis of testicular cells. However, the molecular mechanisms contributing to BTB injury induced by Cd have not yet been fully clarified. Vimentin (Vim) is an important desmosome-like junction protein that mediates robust adhesion in the BTB. In this study, we investigated how Vim responds to Cd. We found that Cd treatment led to a significant decrease in Vim expression, accompanied by a marked increase in LC3-II expression and a higer number of autophagosomes. Interestingly, we also observed that Cd-induced autophagy was associated with decreased Vim activity and enhanced apoptosis of testicular cells. To further investigate the role of autophagy in Vim regulation under Cd exposure, we treated cells with an autophagy inhibitor called 3-MA. We found that 3-MA treatment enhanced Vim expression and improved the disruption of the BTB under Cd exposure. Additionally, the inhibition of Vim confirmed the role of autophagy in modulating Vim expression. These results reveal a previously unknown regulatory mechanism of Cd involving the interplay between a heavy metal and a protein.

Inhibition of miR-143-3p Restores Blood-Testis Barrier Function and Ameliorates Sertoli Cell Senescence.

Due to the increasing trend of delayed childbirth, the age-related decline in male reproductive function has become a widely recognized issue. Sertoli cells (SCs) play a vital role in creating the necessary microenvironment for spermatogenesis in the testis. However, the mechanism underlying Sertoli cell aging is still unclear. In this study, senescent Sertoli cells showed a substantial upregulation of miR-143-3p expression. miR-143-3p was found to limit Sertoli cell proliferation, promote cellular senescence, and cause blood-testis barrier (BTB) dysfunction by targeting ubiquitin-conjugating enzyme E2 E3 (UBE2E3). Additionally, the TGF-ß receptor inhibitor SB431542 showed potential in alleviating age-related BTB dysfunction, rescuing testicular atrophy, and reversing the reduction in germ cell numbers by negatively regulating miR-143-3p. These findings clarified the regulatory pathways underlying Sertoli cell senescence and suggested a promising therapeutic approach to restore BTB function, alleviate Sertoli cell senescence, and improve reproductive outcomes for individuals facing fertility challenges.

Eugenol alleviates acrylamide-induced rat testicular toxicity by modulating AMPK/p-AKT/mTOR signaling pathway and blood-testis barrier remodeling.

This study aimed to investigate the effects of eugenol treatment on reproductive parameters in acrylamide (ACR)-intoxicated rats. The study evaluated alterations in relative testes and epididymides weights, sperm quality, serum hormonal status, seminal plasma amino acids, testicular cell energy and phospholipids content, oxidative and nitrosative stress parameters, adenosine monophosphate-activated protein kinase/ phosphoinositide 3-kinase/phosphor-protein kinase B/mammalian target of rapamycin (AMPK/PI3K/p-AKT/mTOR) signaling pathway, blood-testis barrier (BTB) remodeling markers, testicular autophagy and apoptotic markers, as well as histopathological alterations in testicular tissues. The results revealed that eugenol treatment demonstrated a significant improvement in sperm quality parameters, with increased sperm cell concentration, progressive motility live sperm, and a reduction in abnormal sperm, compared to the ACR-intoxicated group. Furthermore, eugenol administration increased the levels of seminal plasma amino acids in a dose-dependent manner. In addition, eugenol treatment dose-dependently improved testicular oxidative/nitrosative stress biomarkers by increasing oxidized and reduced glutathione levels and reducing malondialdehyde and nitric oxide contents as compared to ACRgroup. However, eugenol treatment at a high dose restored the expression of AMPK, PI3K, and mTOR genes, to levels comparable to the control group, while significantly increasing p-AKT content compared to the ACRgroup. In conclusion, the obtained findings suggest the potential of eugenol as a therapeutic agent in mitigating ACR-induced detrimental effects on the male reproductive system via amelioration of ROS-mediated autophagy, apoptosis, AMPK/p-AKT/mTOR signaling pathways and BTB remodeling.

PFOS exposure destroys the integrity of the blood-testis barrier (BTB) through PI3K/AKT/mTOR-mediated autophagy.

Perfluorooctanesulfonate or perfluorooctane sulfonic acid (PFOS), a type of perfluorinated compound, is mainly found in consumer products. Exposure to PFOS could cause male reproductive toxicity by causing injury to the blood-testis barrier (BTB). However, the specific mechanisms through which PFOS affects male reproduction remain unclear. The mammalian target of rapamycin (mTOR) is a vital protein kinase that is believed to be a central regulator of autophagy. In this study, we established in vivo and in vitro models to explore the effects of PFOS on the BTB, autophagy, and the regulatory role of the mTOR signaling pathway. Adult mice were developmentally exposed to 0, 0.5, 5, and 10 mg/kg/day PFOS for five weeks. Thereafter, their testicular morphology, sperm counts, serum testosterone, expression of BTB-related proteins, and autophagy-related proteins were evaluated. Additionally, TM4 cells (a mouse Sertoli cell line) were used to delineate the molecular mechanisms that mediate the effects of PFOS on BTB. Our results demonstrated that exposure to PFOS induced BTB injury and autophagy, as evidenced by increased expression of autophagy-related proteins, accumulation of autophagosomes, observed through representative electron micrographs, and decreased activity of the PI3K/AKT/mTOR pathway. Moreover, treatment with chloroquine, an autophagy inhibitor, alleviated the effects of PFOS on the integrity of TM4 cells in the BTB and the PI3K/AKT/mTOR pathway. Overall, this study highlights that exposure to PFOS destroys the integrity of the BTB through PI3K/AKT/mTOR-mediated autophagy.

A short-term high-fat diet alters rat testicular activity and blood-testis barrier integrity through the SIRT1/NRF2/MAPKs signaling pathways.

Background: Overweight/obesity are metabolic disorder resulting from behavioral, environmental, and heritable causes. WHO estimates that 50% of adults and 30% of children and adolescents are overweight or obese, and, in parallel, an ongoing decline in sperm quality and male fertility has been described. Numerous studies demonstrated the intimate association between overweight/obesity and reproductive dysfunction due to a highly intricate network of causes not yet completely understood. This study expands the knowledge on the impact of a short-term high-fat diet (st-HFD) on rat testicular activity, specifically on steroidogenesis and spermatogenesis, focusing on the involved molecular mechanisms related to mitochondrial dynamics, blood-testis barrier (BTB) integrity, and SIRT1/NRF2/MAPKs pathways. Methods: Ten adult Male Wistar rats were divided into two groups of five and treated with a standard diet or an HFD for five weeks. At the end of the treatment, rats were anesthetized and sacrificed by decapitation. Blood was collected for serum sex hormone assay; one testis was stored at -80ÅãC for western blot analysis, and the other, was fixed for histological and immunofluorescence analysis. Results: Five weeks of HFD results in reduced steroidogenesis, increased apoptosis of spermatogenic cells, and altered spermatogenesis, as highlighted by reduced protein levels ofmeiotic and post-meiotic markers. Further, we evidenced the compromission of the BTB integrity, as revealed by the downregulation of structural proteins (N-Cadherin, ZO-1, occludin, connexin 43, and VANGL2) other than the phosphorylation of regulative kinases (Src and FAK). At the molecular level, the impairment of mitochondrial dynamics (fission, fusion, andbiogenesis), and the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways, were evidenced. Interestingly, no change was observed in the levels of pro-inflammatory markers (TNFα, NF-kB, and IL-6). Conclusions: The combined data led us to confirm that overweight is a less severe state than obesity. Furthermore, understanding the molecular mechanisms behind the association between metabolic disorders and male fertility could improve the possibility of identifying novel targets to prevent and treat fertility disorders related to overweight/obesity.

[Role of autophagy in cadmium-induced damage to the blood-testis barrier in mice].

OBJECTIVE: To investigate the role of autophagy in cadmium chloride (CdCl2)-induced damage to the blood-testis barrier (BTB) in mice. METHODS: Twenty four-week-old male C57BL/6 mice were randomly divided into four groups and intraperitoneally injected with CdCl2 at 0 mg/kg/d (the control), 0.5 mg/kg/d (low-dose), 1.0 mg/kg/d (medium-dose) and 2.0 mg/kg/d (high-dose) respectively for 28 consecutive days. Then the morphological changes of the testis tissue was observed by HE staining, the integrity of BTB measured with the biotracer, and the expressions of the BTB components ZO-1 and N-Cadherin proteins detected by Western blot. The TM4 Sertoli cells were treated with CdCl2at 0, 2.5, 5 and 10 µmol/L respectively for 24 hours, followed by determination of the expression levels of ZO-1 and N-Cadherin as well as the autophagy-related proteins LC3II and p62. Then the cells were again treated with CdCl2 in the presence of the autophagy inhibitor chloroquine (CQ) at 5 µmol/L or the autophagy inducer rapamycin (Rap) at 50 nmol/L for 24 hours, followed by measurement of the expressions of LC3II, p62, ZO-1 and N-Cadherin by Western blot. RESULTS: Compared with the control group, the cadmium-exposed mice showed increased interstitial space in the seminiferous tubules, formation of intracellular cavitation in the germ cells with decreased layers and disordered arrangement, and damaged integrity of the BTB. The expressions of the ZO-1 and N-Cadherin proteins were significantly down-regulated in the testis tissue of the mice in the medium- and high-dose CdCl2 groups (P < 0.05), and even more significantly in the CdCl2-exposed cells in comparison with those in the control mice (P < 0.01), while the expressions of the LC3II and p62 proteins were remarkably up-regulated (P < 0.05). The expressions of ZO-1, N-Cadherin, LC3II and p62 were also up-regulated in the cells co-treated with CQ and CdCl2 (P < 0.01), those of ZO-1, N-Cadherin and p62 down-regulated (P< 0.05) and that of LC3II up-regulated (P < 0.05) in the cells co-treated with Rap and CdCl2. CONCLUSION: CdCl2 can damage the integrity of the mouse BTB, which may be attributed to its ability to enhance the autophagy in Sertoli cells and regulate the expressions of BTB proteins.

WIN18,446 enhances spermatogonial stem cell homing and fertility after germ cell transplantation by increasing blood-testis barrier permeability.

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation.

Nickel induces blood-testis barrier damage through ROS-mediated p38 MAPK pathways in mice.

Nickel (Ni) is an essential common environmental contaminant, it is hazardous to male reproduction, but the precise mechanisms are still unknown. Blood-testis barrier (BTB), an important testicular structure consisting of connections between sertoli cells, is the target of reproductive toxicity caused by many environmental toxins. In this study, ultrastructure observation and BTB integrity assay results indicated that NiCl2 induced BTB damage. Meanwhile, BTB-related proteins including the tight junction (TJ), adhesion junction (AJ) and the gap junction (GJ) protein expression in mouse testes as well as in sertoli cells (TM4) were significantly decreased after NiCl2 treatment. Next, the antioxidant N-acetylcysteine (NAC) was co-treated with NiCl2 to study the function of oxidative stress in NiCl2-mediated BTB deterioration. The results showed that NAC attenuated testicular histopathological damage, and the expression of BTB-related proteins were markedly reversed by NAC co-treatment in vitro and vivo. Otherwise, NiCl2 activated the p38 MAPK signaling pathway. And, NAC co-treatment could significantly inhibit p38 activation induced by NiCl2 in TM4 cells. Furthermore, in order to confirm the role of the p38 MAPK signaling pathway in NiCl2-induced BTB impairment, a p38 inhibitor (SB203580) was co-treated with NiCl2 in TM4 cells, and p38 MAPK signaling inhibition significantly restored BTB damage induced by NiCl2 in TM4 cells. These results suggest that NiCl2 treatment destroys the BTB, in which the oxidative stress-mediated p38 MAPK signaling pathway plays a vital role.

Polystyrene nanoplastics aggravated dibutyl phthalate-induced blood-testis barrier dysfunction via suppressing autophagy in male mice.

Nanoplastics (NPs) frequently cause adverse health effects by transporting organic pollutants such as dibutyl phthalate (DBP) into organisms by utilizing their large specific surface area, large surface charge, and increased hydrophobicity. However, the effects of NPs combined with DBP on the reproductive systems of mammals are still unclear. The present investigation involved the administration of polystyrene NPs (PS-NPs) to BALB/c mice via gavage, with a size of 100 nm and at doses of 5 mg/kg/day or 50 mg/kg/day, along with DBP at a dose of 0.5 mg/kg/day, or a combination of PS-NPs and DBP, for 30 days, to assess their potential for reproductive toxicity. The co-exposure of mice to PS-NPs and DBP resulted in a significant increase in reproductive toxicities compared to exposure to PS-NPs or DBP alone. This was demonstrated by a marked decrease in sperm quality, significant impairment of spermatogenesis, and increased disruption of the blood-testis barrier (BTB). Furthermore, a combination of in vivo and in vitro investigations were conducted to determine that the co-exposure of DBP and PS-NPs resulted in a noteworthy reduction in the expressions of tight junction proteins (ZO-1 and occludin). Moreover, the in vitro findings revealed that monobutyl phthalate (MBP, the active metabolite of DBP, 0.5 µg/mL) and PS-NPs (30 µg/mL or 300 µg/mL) inhibited autophagy in Sertoli cells, thereby increasing the expression of matrix metalloproteinases (MMPs). The study found that PS-NPs and DBP co-exposure caused harmful effects in male reproductive organs by disrupting BTB, which may be alleviated by reactivating autophagy. The paper's conclusions provided innovative perspectives on the collective toxicities of PS-NPs and other emerging pollutants.

Sertoli cell-derived extracellular vesicles traverse the blood-testis barrier and deliver miR-24-3p inhibitor into germ cells improving sperm mobility.

Asthenozoospermia, characterized by poor sperm motility, is a common cause of male infertility. Improving energy metabolism and alleviating oxidative stress through drug regimens are potential therapeutic strategies. In this study, we observed upregulated miR-24-3p levels in asthenozoospermia spermatozoa, contributing to energy metabolism disorder and oxidative stress by reducing GSK3ß expression. Thus, reducing miR-24-3p levels using drugs is expected to improve sperm motility. The blood-testis barrier (BTB) protects the testis from xenobiotics and drugs. In this study, we found that Sertoli cell-derived small extracellular vesicles (SC-sEV) can traverse the BTB and enter germ cells. We successfully loaded miR-24-3p inhibitor into SC-sEV, creating the nano-drug SC-sEV@miR-24-3p inhibitor, which effectively delivers miR-24-3p inhibitor into germ cells. In a gossypol-induced mouse asthenozoospermia model, administration of SC-sEV@miR-24-3p inhibitor significantly improved sperm motility, in vitro fertilization success, and blastocyst formation rates. As anticipated, it also improved the litter size of asthenozoospermia mice. These results suggest that SC-sEV@miR-24-3p inhibitor holds promise as a potential clinical treatment for asthenospermia.

Deoxynivalenol Induces Blood-Testis Barrier Dysfunction through Disrupting p38 Signaling Pathway-Mediated Tight Junction Protein Expression and Distribution in Mice.

Deoxynivalenol (DON) is widely present in cereals and processed grains. It can disrupt the blood-testicular barrier (BTB), leading to sterility in males; however, the mechanism is unknown. In this study, 30 Kunming mice and TM4 cells were exposed to 0 or 4.8 mg/kg (28 d) and 0-2.4 µM (24 h) of DON, respectively. Histopathological findings showed that DON increased BTB permeability in mice, leading to tight junction (TJ) structural damage. Immunofluorescence results indicated that DON disrupted the localization of zonula occludens (ZO)-1. The results of protein and mRNA expression showed that the expression of ZO-1, occludin, and claudin-11 was reduced, and that the p38/GSK-3ß/snail and p38/ATF-2/MLCK signaling pathways were activated in mouse testes and TM4 cells. Pretreatment with the p38 inhibitor SB203580 maintained TJ integrity in TM4 cells after exposure to DON. Thus, DON induced BTB dysfunction in mice by disrupting p38 pathway-mediated TJ expression and distribution.

[Effects of cadmium chloride on testicular autophagy and blood-testis barrier integrity in prepubertal male rats].

Objective: To study the effects of cadmium chloride (CdCl(2)) exposure on testicular autophagy levels and blood-testis barrier integrity in prepubertal male SD rats and testicular sertoli (TM4) cells. Methods: In July 2021, 9 4-week-old male SD rats were randomly divided into 3 groups: control group (normal saline), low dose group (1 mg/kg·bw CdCl(2)) and high dose group (2 mg/kg·bw CdCl(2)), and were exposed with CdCl(2) by intrabitoneal injection. 24 h later, HE staining was used to observe the morphological changes of testis of rats, biological tracer was used to observe the integrity of blood-testis barrier, and the expression levels of microtubule-associated protein light chain 3 (LC3) -Ⅰ and LC3-Ⅱ in testicular tissue were detected. TM4 cells were treated with 0, 2.5, 5.0 and 10.0 µmol/L CdCl(2) for 24 h to detect the toxic effect of cadmium. The cells were divided into blank group (no exposure), exposure group (10.0 µmol/L CdCl(2)), experimental group[10.0 µmol/L CdCl(2)+60.0 µmol/L 3-methyladenine (3-MA) ] and inhibitor group (60.0 µmol/L 3-MA). After 24 h of treatment, Western blot analysis was used to detect the expression levels of LC3-Ⅱ, ubiquitin binding protein p62, tight junction protein ZO-1 and adhesion junction protein N-cadherin. Results: The morphology and structure of testicular tissue in the high dose group were obvious changed, including uneven distribution of seminiferous tubules, irregular shape, thinning of seminiferous epithelium, loose structure, disordered arrangement of cells, abnormal deep staining of nuclei and vacuoles of Sertoli cells. The results of biological tracer method showed that the integrity of blood-testis barrier was damaged in the low and high dose group. Western blot results showed that compared with control group, the expression levels of LC3-Ⅱ in testicular tissue of rats in low and high dose groups were increased, the differences were statistically significant (P<0.05). Compared with the 0 µmol/L, after exposure to 5.0, 10.0 µmol/L CdCl(2), the expression levels of ZO-1 and N-cadherin in TM4 cells were significantly decreased, and the expression level of p62 and LC3-Ⅱ/LC3-Ⅰ were significantly increased, the differences were statistically significant (P<0.05). Compared with the exposure group, the relative expression level of p62 and LC3-Ⅱ/LC3-Ⅰ in TM4 cells of the experimental group were significantly decreased, while the relative expression levels of ZO-1 and N-cadherin were significantly increased, the differences were statistically significant (P<0.05) . Conclusion: The mechanism of the toxic effect of cadmium on the reproductive system of male SD rats may be related to the effect of the autophagy level of testicular tissue and the destruction of the blood-testis barrier integrity.

SARS-CoV-2 Structural Proteins Modulated Blood-Testis Barrier-Related Proteins through Autophagy in the Primary Sertoli Cells.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disrupts the blood-testis barrier (BTB), resulting in alterations in spermatogenesis. However, whether BTB-related proteins (such as ZO-1, claudin11, N-cadherin, and CX43) are targeted by SARS-CoV-2 remains to be clarified. BTB is a physical barrier between the blood vessels and the seminiferous tubules of the animal testis, and it is one of the tightest blood-tissue barriers in the mammalian body. In this study, we investigated the effects of viral proteins, via ectopic expression of individual viral proteins, on BTB-related proteins, the secretion of immune factors, and the formation and degradation of autophagosomes in human primary Sertoli cells. Our study demonstrated that ectopic expression of viral E (envelope protein) and M (membrane protein) induced the expressions of ZO-1 and claudin11, promoted the formation of autophagosomes, and inhibited autophagy flux. S (spike protein) reduced the expression of ZO-1, N-cadherin, and CX43, induced the expression of claudin11, and inhibited the formation and degradation of autophagosomes. N (nucleocapsid protein) reduced the expression of ZO-1, claudin11, and N-cadherin. All the structural proteins (SPs) E, M, N, and S increased the expression of the FasL gene, and the E protein promoted the expression and secretion of FasL and TGF-ß proteins and the expression of IL-1. Blockage of autophagy by specific inhibitors resulted in the suppression of BTB-related proteins by the SPs. Our results indicated that SARS-CoV-2 SPs (E, M, and S) regulate BTB-related proteins through autophagy.

The effect of semen cuscutae flavonoid on Sertoli cells and blood-testis barrier in male infertility: integrating network pharmacology and experimental verification.

CONTEXT: Semen cuscutae is commonly used to treat male infertility (MI), and semen cuscutae flavonoid (SCF) is the main active component of semen cuscutae. The therapeutic mechanism of SCF on MI is still unclear. OBJECTIVE: To clarify the mechanisms of SCF against MI. MATERIALS AND METHODS: Network pharmacology and molecular docking were used to predict the potential pathways of SCF against MI. Primary Sertoli cells (SCs) were extracted from testis of 60-day-old rats and divided into Control, Model, and 3 treatment groups. The Control and Model groups were given normal medium, the treatment groups were treated with various concentrations of SCF-containing medium (200, 400, and 800 µg/mL). After 24 h, the Model and treatment groups were exposed to heat stress at 43 °C for 15 min. Western blotting and immunohistochemistry were used to detect the expression of targets. RESULT: Network pharmacology indicated that the treatment of SCF on MI was closely related to PI3K-AKT signaling pathway. The in vitro experiments showed that SCF could up-regulated the expression of AKT, AR, occludin, and Ki67, and down-regulated the expression of CK-18 in SCs after heat stress. The AKT inhibitor could block this process. CONCLUSIONS: SCF can treat MI by regulating the proliferation and differentiation of SCs and the integrity of the blood-testis barrier. The study could provide experimental basis for clinical research.

mTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.

Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.

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.