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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.
Subject(s)
Rats , Male , Animals , Testis , Cadmium Chloride/metabolism , Cadmium , Blood-Testis Barrier/metabolism , Rats, Sprague-Dawley , Cadherins/metabolism , AutophagyABSTRACT
Cadmium (Cd) is a well - known environmental toxin that is naturally present in air, water and soil. The reproductive system is most vulnerable to oxidative damage and therefore most affected by Cd. Zinc (Zn) is an essential antioxidant and a chelating agent that is capable of protecting the testis from Cd induced toxicity. Apium graveolens commonly known as Celery is a herbal plant rich in antioxidants and it improves various sperm parameters. MethodsMale Wistar albino rats were randomly divided into 7 groups. Control received 0.5 % Carboxy-Methyl Cellulose (CMC) in distilled water; the experimental groups namely Cd received 10 mg / Kg body weight of CdCl2; Cd + Zn received 10 mg / Kg bodyweight of CdCl2 + 40 mg / Kg body weight of ZnCl2; Cd + AG 200 received 10 mg/Kg bodyweight of CdCl2 + 200 mg / Kg body weight of Apium graveolens; Cd + AG400 received 10 mg/Kg body weight of CdCl2 + 400 mg / Kg body weight of Apium graveolens; Cd + AG 200 + Zn received 10 mg / Kg bodyweight of CdCl2 + 200 mg / Kg body weight of Apium graveolens + 40 mg / Kg body weight of ZnCl2; Cd + AG 400 + Zn received 10 mg / Kg bodyweight of CdCl2 + 400 mg/Kg body weight of Apium graveolens + 40 mg / Kg body weight of ZnCl2 all in 0.5% CMC. Hydroalcoholic extract of Apium graveolens was used in this experiment. The experiment was conducted for a duration of 56 days. Histopathology, sperm analysis, lipid peroxidation and hormone assays were performed. The therapeutic potential of Apium graveolens at two doses (200 and 400 mg / Kg body weight) with and without Zn supplementation was evaluated in this experiment. ResultsRats treated with Cd showed severe testicular damages. Zn offered protection from the damages done by cadmium. The hydroalcoholic extract of Apium graveolens at doses of 200 mg / Kg body weight showed better protective effect than 400 mg / Kg body weight and the protecting nature was enhanced by zinc supplementation.
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Aim To investigate the changes of autoph- A gy in rat testis and its effect on blood-testis barrier during aging. Methods HE staining was used to observe the morphological changes of testis in SD male rats at 6, 12, 18 and 24 months old. Western blot was used to detect the relative expression of autophagy-re-lated proteins Beclinl, ATG5, ATG7 and 1X13II, and the blood-testis barrier related connexins Occludin and (3-catenin. Immunofluorescence was used to detect the expression and localization of the autophagy-associated proteins Beclinl and LC3, as well as the protein of the cell connexin p-catenin. Results HE staining showed that the morphology and structure of rat testis changed significantly during the aging process, the seminiferous tubules atrophied, the number of spermatogenic cells decreased, the gap between cells increased, and partial shedding occurred. Western blot results showed that the relative expression of autophagy-related proteins Beclinl, ATG5, ATG7 and LC3II, and the blood-testis barrier-related proteins Occludin and p-catenin gradually decreased during aging. The results of immunofluorescence showed that the autophagy marker proteins Beclinl and LC3 were down-regulated in rat seminiferous epithelial cells during the aging process, and the expression of the blood-testis barrier marker protein p-catenin also gradually decreased. Conclusions During the aging process, the spermato-genic function of the testis is reduced in rats, and the mechanism is related to the decrease of the autophagy level of testicular spermatogenic epithelial cells, which in turn destroys the integrity of the blood testis barrier.
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During spermatogenesis, developing germ cells that lack the cellular ultrastructures of filopodia and lamellipodia generally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These include the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell-cell and Sertoli-germ cell interface also undergo rapid remodeling, involving disassembly and reassembly of cell junctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the involving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protein S6 (rpS6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mTORC1/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubule-based cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.
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Objective To observe the effects of high fat dietinduced elevation of blood glucose on the microvascular function of testis and male reproduction in C57BL/6 mice. Methods A total of 40 male C57BL/6 mice were randomly divided into control group and high fat diet (HFD) group (n =20). The mice in HFD group were fed with high fat diet for 20 weeks. Blood glucose and body weight were measured weekly. The permeability of bloodtestis barrier was evaluated by intraperitoneal injection of Evans blue. The blood flow of testicular microcircu-lation and the frequency and amplitude of microvascular vasomotion were detected by laser Doppler blood flow ima-ging system. The morphology of testicular tissue was observed by HE staining. The expressions of platelet- endothelial cell adhesion molecule-1 (PECAM-1/CD31) in testicular microvascular endothelial cells and prolifera-ting cell nuclear antigen (PCNA) in spermatogenic cells were detected by immunohistochemistry. The apoptosis of spermatogenic cells was observed by TUNEL staining. Results The body weight and blood glucose of HFD group were significantly higher than those in control group (P＜0.01). Evans blue staining showed that the integrity of blood-testis barrier of HFD group was damaged, and increased permeability was observed in seminiferous tubules. In HFD group, the mean blood flow of testis and the frequency and amplitude of microvascular vasomotion were sig-nificantly lower than those in control group (P＜0.01). The number of spermatogenic epithelial cells and the thickness of seminiferous epithelium decreased. The expressions of CD31 in microvascular endothelial cells and PCNA in spermatogenic cells were significantly lower in HFD group than those in control group (P＜0.01). The apoptosis level of spermatogenic cells was higher than that in the control group (P <0.01). Conclusions Increased blood glucose level induced by high fat diet in mice can impair the testicular microvasculature and damage the integrity of blood-testis barrier and injure the structure of seminiferous epithelium in mice.
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The Sertoli cell tight junction (TJ) is the key component of the blood-testis barrier, where it sequesters developing germ cells undergoing spermatogenesis within the seminiferous tubules. Hormonally regulated claudin-11 is a critical transmembrane protein involved in barrier function and its murine knockout results in infertility. We aimed to assess quantitatively the significance of the contribution of claudin-11 to TJ function, in vitro, using siRNA-mediated gene silencing. We also conducted an analysis of the contribution of occludin, another intrinsic transmembrane protein of the TJ. Silencing of claudin-11 and/or occludin was conducted using siRNA in an immature rat Sertoli cell culture model. Transepithelial electrical resistance was used to assess quantitatively TJ function throughout the culture. Two days after siRNA treatment, cells were fixed for immunocytochemical localization of junction proteins or lyzed for RT-PCR assessment of mRNA expression. Silencing of claudin-11, occludin, or both resulted in significant decreases in TJ function of 55% (P < 0.01), 51% (P < 0.01), and 62% (P < 0.01), respectively. Data were concomitant with significant decreases in mRNA expression and marked reductions in the localization of targeted proteins to the Sertoli cell TJ. We provide quantitative evidence that claudin-11 contributes significantly (P < 0.01) to Sertoli cell TJ function in vitro. Interestingly, occludin, which is hormonally regulated but not implicated in infertility until late adulthood, is also a significant (P < 0.01) contributor to barrier function. Our data are consistent with in vivo studies that clearly demonstrate a role for these proteins in maintaining normal TJ barrier structure and function.
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Objective To investigate the regulation of blood-testis barrier by Rab13-PKA pathway in rats.Meth-od First, shRNA vector targeting at Rab13 was constructed and then the Rab13 shRNA was transfected into the rat testis by injection.Western blot was used to detect the knock-down effect of Rab13 and the expression of blood-testis barrier ( BTB) constituent proteins.PKA activity was detected by autoradiography and scintillation counting.Further, immunoflu-orescence analysis and phalloidin staining were applied to observe the distribution of occludin and F-actin, respectively. Results The expression level of Rab13 in the testis was reduced by approximately 70%after transfection of Rab13 shRNA as compared with the non-targeted control group ( P<0.01 ) , while the expression of BTB constituent proteins remained unchanged.PKA activity was significantly increased after Rab13 RNAi transfection (P<0.01).The distribution of occlu-din at BTB was remarkably increased after Rab13 RNAi silencing around stage VIII but not at other stages of the seminifer-ous epithelial cycle.The assembly of F-actin at BTB was also intensified in Rab13-silenced testis.Both the changes of dis-tribution of occludin and F-actin induced by Rab13 shRNA were found to be antagonized by the PKA specific inhibitor H89.Conclusions Rab13 can modulate the distribution of occludin and F-actin at the blood-testis barrier in rats by regu-lating PKA activity, which may participate in the regulation of BTB function.
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Objective To investigate the changes of Claudin-3 and Claudin-11,two key components of blood-testis barrier (BTB) on male infertility induced by γ-ray irradiation.Methods Fortytwo KunMing male mice (20-25 g) were divided into one control group,three γ-ray irradiation groups and three estrodiol (E2) intervention groups randomly:Group A,sham controlled; the lower abnominal and scrotal area of the mice in Group B,C,D were irradiated with single dose of 2,6 or 10 Gy 60Co γ-ray after anaesthetizd; 17β-estradiol intervention were initiated in Group E,F,G after 6 Gy γ-ray irradiation via hypodermic injection for 4w at the dose of 1,2,4 μg/d,respectively.Mice were sacrificed 2 w after the last E2 administration.The tubule differentiation index (TDI) was counted in testis sections.InhibinβB,Claudin-3 and Claudin-11 transcription levels were assayed with semiquantitative real time PCR.Claudin-11 protein levels in testis were generated by western blot.Results Compared with sham control group,TDI in three γ-ray irradiation groups were markedly reduced (68.5 ± 6.4,35.0± 6.1,16.3 ± 5.7 vs 100.0,all P＜0.05).InhibinββB mRNA expression level in testis of gourp D was markedly decreased (0.5±0.2 vs 1.0±0.1,P＜0.05).Claudin-3 mRNA levels of group C and D were up-regulated to 2.17 and 3.49 times,respectively.Claudin-11 protein levels were significantly increased to 2.18 and 2.23 times.Compared with group C,TDI in three E2 intervention groups were improved,which were obvious in group F and G (61.7±7.2,55.8±11.9 vs 35.0±6.1,P＜0.05).The InhibinβB mRNA levels were increased,though there were no significant differences (all P ＞0.05).Claudin-3 mRNA levels in group F and G were down-regulated (1.3± 0.2,1.6±0.3 vs.2.2 ± 0.2,all P＜0.05).In group F significantly reduced mRNA level and protein level of Claudin-11 were observed (mRNA:1.2±0.2 vs.1.8±0.2,P＜0.05; Protein:1.5±0.5 vs.2.2±0.3,P＜0.05).It was negatively correlated TDI with mRNA expression levels of Claudin-3 and Claudin-11 in the irradiated testis (rs =-0.884,P＜0.05; rs=-0.758,P ＜0.05,respectively).Conelusions Irradiation could elevated the expression of claudin-3 and claudin-11.
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Objective To explore the effects on dogs' testis by microbubbles excitated by ultrasound.Methods Sixteen male dogs were divided into 3 groups randomly.Pulsed ultrasound irradiation and intravenous microbubbles injection were both applied in the microbubble enhanced ultrasound group (MEUS),pulsed ultrasound irradiation and intravenous microbubbles injection were individually applied in the ultrasound group (TUS) and the simple microbubbles group (MB).Results In MEUS group,the layers of seminiferous tubules decreased and the germ cells were arranged disorderly and bubbles appeared in the cells.The appearances of focal ultra-structural damage such as intercellular space widening,baseline broken and tight-connection of sustentacular cells disappearing were observed by transmission electron microscope,which were slightly or hardly demonstrated in other groups.Conclusions Microbubbles excitated by ultrasound can affect testicular germ cells and the integrity of bloold-testis harrier,which providing new train of thought of target treatments of testis diseases.