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[Objectives] This study investigates the capability of exosome secretion by Sertoli cells and its influence on the self-renewal and differentiation status of porcine spermatogonial stem cells(pSSC) using an in vitro model. [Methods] The two-step enzyme digestion method was employed to isolate Sertoli cells and pSSC, and an in vitro co-culture model of Sertoli cells and pSSC was established. Suppression of exosome secretion by Sertoli cells was achieved to examine the effects on pSSC self-renewal and differentiation markers, thereby studying the impact of Sertoli cell-derived exosome on the fate of pSSC. [Results] The isolated Sertoli cells exhibited a Wilm tumor 1(WT1) positive rate of (87.1±0.02)% and an intorgrin β-1(ITGB1) positive rate of (94.2±0.01)%. The isolated pSSC showed a promyelocytic lenkaemia zine finger protein (PLZF) positive rate of (80.6±0.02) %. Presence of exosome secreted by Sertoli cells was confirmed through Western blot analysis and immunofluorescence assays. Treatment with 20 µmol L-1 GW4869 effectively inhibited exosome secretion without compromising Sertoli cells. Co-culture of pSSC with Sertoli cells treated with the inhibitor revealed significant differences in pSSC status compared to the control group. RT-PCR results showed a significant increase in the expression of the PLZF gene, while expression levels of receptor tyrosine kinase gene (KIT), thymus cell antigen 1 gene (THYI), and dead box 4 gene (DDX4) were significantly reduced. Western blot analysis indicated that levels of protein gene product 9.5(PGP9.5) protein expression elevated and DDX4 protein expression level decreased in pSSC co-cultured with inhibitor-treated Sertoli cells. [Conclusions] Exosomes secreted by Sertoli cells had a regulatory role in both self-renewal and differentiation of pSSC in vitro. This finding implied that exosome represented an important means through which Sertoli cells influence the fate of spermatogonial stem cells, offering novel insights for enhancing boar reproductive performance and fertility improvement. [ABSTRACT FROM AUTHOR]

To study the effect of busulfan on the expression and localization of the proliferation and differentiation related proteins PCNA, PLZF, and GATA4 in seminiferous tubules in testis of piglets, in this study, seminiferous tubules of piglets cultured in vitro were selected as the research subjects and divided into control group and experimental group. The experimental group was supplemented with 5 mg/kg of busulfan for 1, 2 and 3 days respectively, and seminiferous tubules were obtained by centrifugation. HE staining was used to detect the morphological changes of chromosomes. Immunohistochemistry, qRT-PCR, and Western blot were used to detect the changes in the expression and localization of PCNA, PLZF, and GATA4. HE staining showed that there were both Sertoli cells and germ cells existing in the seminiferous tubules in all experimental groups. Immunohistochemical studies showed that PCNA protein was localized in the nuclei of germ cells and Sertoli cells in all experimental groups. The staining of PCNA became darker and the expression level increased due to the function of busulfan. In the experimental groups, GATA4 protein was located in the nucleus of Sertoli cell, and there was no significant difference in GATA4 protein staining between the experimental group and the control group. PLZF protein was located in nucleus of germ cells of experimental groups. Compared with the control group, PLZF staining showed no significant difference after 2 days of busulfan treatment, while PLZF staining became darker and expression increased after 1 and 3 days of busulfan treatment. The results of qRT-PCR and Western Blot showed that the mRNA and protein expressions of PCNA and GATA4 in the experimental groups were significantly increased compared with the control group. The expression of PLZF mRNA and protein had no significant differences after 2 days of busulfan treatment. The expression of PLZF mRNA and protein was significantly increased after 1 and 3 days of busulfan treatment. In conclusion, PCNA, PLZF, and GATA4 were expressed in seminiferous tubules of piglets cultured in vitro. The expression levels of PCNA, PLZF, and GATA4 in seminiferous tubules were changed due to the effect of busulfan, suggesting that busulfan damage spermatozoa of porcine testis by affecting the expressions of proteins related to proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Objective To explore the effects of Semen Cuscutae-Fructus Lycii（SC-FL） on testicular structure and sperm quality of oligoasthenozoospermia mouse models induced by Glucosides of Tripterygium wilfordii hook. F（GTW）. Methods A total of 36 male BALB/c mice were randomly divided into the control group（12） and the model group（24）. The mice in the control group were treated with deionized water, and the mice in the model group with GTW for 4 weeks. Two mice from the control group and four mice from the model group were randomly selected for model verification. After the model was successfully validated, the remaining 10 normal mice were used as the control group, and the mice in the model group were divided into the model group and the treatment group according to the random number table method, with 10 mice in each group. The mice in the control group and the model group were given with deionized water, and the mice in the treatment group intragastrically with SC-FL suspension for 4 weeks. At the end of the 8th week of the experiment, the body mass, testicular mass and semen parameters of mice were measured, and the histological morphology of testes was observed by hematoxylin eosin（HE） staining. Results The testicular quality, sperm concentration, and total sperm motility（PR+NP） of mice in the treatment group were higher than those in the spermatogenic failure model group, with significant differences（P＜ 0.01）. Compared with the model group, the Sertoli cells and spermatids were increased in the testicular tissue of the treatment group, and the seminiferous tubules were structurally intact and arranged closely. Conclusion Semen Cuscutae-Fructus Lycii（SC-FL）can effectively alleviate oligoasthenozoospermia, and its mechanism may be related to the repair of testicular tissue structure damage. [ABSTRACT FROM AUTHOR]

Objective To establish a simple and efficient method for isolation and culture of mouse spermatogonial stem cell（mSSC） in vitro. Method（s） The cell suspension from testis of male fetal Kunming mice and C57BL/6mice was obtained by mechanical separation and enzyme digestion. The cell suspension containing mSSC cells and Sertoli cells and other cells was co-cultured until the clone of mSSC formed. When Sertoli cells were not in period of proliferation, the clones of mSSC were removed to the feeding layer cells. The isolated mSSC cells were further identified by molecular markers and induced differentiation. Result（s） The mSSC cells isolated by this co-culturing method from different genetic backgrounds of male fetal Kunming and C57BL/6 mice could be cultured for several generations in vitro. The result of RT-PCR showed that specific molecular markers, such as Plzf, Gfrα-1 and Ngn3 were expressed in mSSC cells. mSSC cells treated with retinoic acid presented the phenotype of sperm-like cells. The amount of haploid in induced mSSC cells accounted for 9.52%, while no haploid in the control mSSC cells. The induced mSSC expressed differentiation-associated genes such as Acrosin, Th2b and Sycp3. Conclusion（s） In this study, we developed the co-culturing method which could isolate and culture mSSC cells from different genetic background mice. This method not only simplifies the separation steps, but also saves the cost of culture, which will provide basis for the study of mSSC cells. [ABSTRACT FROM AUTHOR]

Objective　To investigate blood-testis barrier injury in C57BL/6J mice induced by one-time total-body irradiation (TBI) with 6 Gy 60Co γ ray, and the ability of Yiqi Jiedu decoction to protect against this injury. Methods 　Eighty male C57BL/6J mice were randomly assigned to the following groups according to body mass: blank group, model group, anduolin group (0. 54 g/ kg), and Yiqi Jiedu decoction group (16. 58 g/ kg), with 20 mice per group. After a 10-day pretreatment, the mice (except the blank group) were given one-time TBI with 6 Gy 60Co γ ray, following which, treatment was administered from the 4th day to 11th day after radiation. The mice were sacrificed separately on the 2nd day and 12th day after radiation. The testis and epididymis were weighed, and their pathological changes were examined using HE staining. Additionally, the diameter of the seminiferous tubules and the thickness of the seminiferous epithelium were measured. In addition, the serum inhibin B (INH B) was detected using ELISA, and the expressions of claudin-11 and occludin in the testis were determined using Western blotting. Results　Compared with the blank group, the weight of the testis and epididymis in the model group, anduolin group, and Yiqi Jiedu decoction group did not change significantly on the 2nd day after the TBI, yet all decreased on the 12th day after TBI (P < 0. 01). The testis and epididymis in the model group showed pathological damages on the 2nd day that were aggravated on the 12th day, while the diameter of the seminiferous tubules and thickness of the seminiferous epithelium were decreased compared with those of the blank group at both time points (P<0. 01). The pathological damages to the testis and epididymis in the Yiqi Jiedu decoction group were both improved when compared with those of the model group, and the diameter of the seminiferous tubules and thickness of the seminiferous epithelium increased compared with the model group at both time points(P<0. 01) . On the 12th day after TBI, the INH B level in the model group was lower than that in the blank group (P < 0. 05), while that in the Yiqi Jiedu decoction group was increased compared with the model group (P < 0. 01). At both time points, the expressions of claudin-11 and occludin in the model group was decreased compared with that in the blank group (P<0. 05, P<0. 01), while the expressions of claudin-11 and occludin in the Yiqi Jiedu decoction group increased significantly compared with the model group (P<0. 01). Conclusion　One-time TBI with 6 Gy 60Co γ ray induced significant damage to the blood-testis barrier of male C57BL/6J mice, while the Yiqi Jiedu decoction effectively alleviated this damage and promoted the recovery of spermatogenic function with a certain protective effect. [ABSTRACT FROM AUTHOR]

【Objective】 This study aimed to establish an effective method for isolation and culture of testicular Sertoli cells （SCs） from the Congjiang Xiang pig in vitro, provide cell material for the following reproduction of male Xiang pig, and provide reference for the isolation and cultivation of SCs from same type of Xiang pig. 【Method】 Using the combined enzyme digestion method of 0.1% collagenase Ⅳ and 0.25% trypsin-EDTA, testicular tissues were digested to isolate SCs. SCs were purified by differential adhesion method and cultured in the complete medium containing 10% fetal bovine serum. The morphological characteristics and growth curve of SCs from the Congjiang Xiang pig were observed, and SCs was identified by hematoxylin-eosin staining, RT-PCR and immunofluorescence staining. Identifying SCs by hematoxylin-eosin staining, RT-PCR and immunofluorescence staining.【Result】 A large number of spermatogenic epithelial cell suspensions could be obtained after combined enzyme digestion. The isolated SCs were round or oval, and the adherent cells after culturing 5-6 hours were triangular or irregular. SCs showed tight junction after 3-4 days, and subsequently, the cell proliferation rate decreased after 7-8 days and the growth curve of primary Sertoli cells was S-shaped. HE staining showed that the nucleus of SCs was dark purple and the cytoplasm was light red; RT-PCR showed that Sertoli cell specific genes WT1 and SOX9 were expressed, and the positive rate of GATA-4（a SC-specific maker） was more than 90% in immunofluorescence staining.【Conclusion】 Aculture method of testicular SCs of the Congjiang Xiang pig in vitro is successfully established. [ABSTRACT FROM AUTHOR]

Objective: To explore the protective effect and its mechanism of the protein and peptide extracts from the muscle of Chinese giant salamanders (SP) on the protection of bisphenol A (BPA)-induced sperm disorder in mice. Methods: Forty male C57BL/6 mice were divided into four groups, Control group, BPA group, BPA+CS group and BPA+SP group. BPA group was injected with 50 mg/kg/d of BPA, which dissolved in corn oil. Control group was injected with corn oil only. The BPA+CS group was given compound substances, which have been proved to be beneficial for spermatogenesis, and were used as controls. The BPA+SP group was given the above mentioned compounds plus SP dissolved in physiological saline. Mice received the compounds plus SP by gavage. The doses were 4 g/kg/d for both groups. The model was established continuously for 28 days, during which the body weight of mice was measured weekly. Testicular volume and testosterone-body ratio were measured after establishment of animal model. Epididymal sperms were collected to measure sperm number and sperm motility by using the computer aided analysis system (CASA). The amount of testosterone in the serum was determined by ELISA. HE staining and TUNEL staining were used to analyze testicular histopathology. Finally, immunofluorescence staining and Western blotting were used to analyze the expression of metastasis associated protein 2 (MTA2), and Oil Red O staining was used to observe the phagocytosis of Sertoli cells in vitro and in vivo. Results: Compared with the control group, the testicular volume (P<0.05), testicular weight (P<0.05), testosterone-body ratio (P<0.05), sperm number (P<0.05), sperm motility (P<0.05), and the amount of testosterone in the serum of mice (P<0.01) in BPA group were significantly decreased. Testicular morphology was damaged and the number of apoptotic spermatogenic cells in BPA group was increased compared with control group(P<0.01). At the same time, the expression of MTA2 and the phagocytosis of Sertoli cells in BPA group were decreased compared with control group (P<0.01). There was no difference between BPA + CS group and BPA group. Surprisingly, the above changes in BPA group were significant difference by using SP(P<0.05). Conclusion: SP has obvious protective effect on BPA-induced sperm disorder in mice, and the mechanism of action may be related to enhanced phagocytosis of Sertoli cells by improving the expression of MTA2. [ABSTRACT FROM AUTHOR]

[Objective] To analyze the protective effect of tacrine on lipopolysaccharide (LPS)-induced inflammatory damage in mouse testicular sertoli cells (TM4), and to further explore the function of tacrine from an inflammatory perspective, in order to provide more drug options for the use of anti-inflammatory drugs related to reproductive diseases and sperm quality enhancement in male animals during animal production. [Method] The CCK8 method was used to detect the appropriate damage conditions for TM4 induced by 0. 01, 0. 10, l. 00, 10. 00 and 100. 00 µ.g/mL LPS for 3. 0, 6. 0, 12. 0 and 24. 0 hours, respectively; And to analyze the appropriate protection conditions for TM4 pretreated by 0. 01,0. 10, 1. 00, 10. 00, 100. 00, 1000. 00,10 000. 00 and 100 000. 00 µ.g/mL tacrine for 0. 5, 1. 0 and 2. 0 hours prior to the addition of LPS or treated for 0. 5, 1. 5, 3. 0, 6. 0, 12. 0 and 24. 0 hours after the addition of LPS. The mRNA expression levels of TNF-α and IL-6 genes were measured by fluorescent quantitative PCR(qRT-PCR). The expression levels of TNF-α and IL-6 proteins in cell supernatants were further evaluated by ELISA, and the expression of NF - KB protein were analyzed by immunoprotein blotting (WB). [Result] There were differences in the effects of LPS on the proliferation of TM4 under different concentrations and different treatment times, and the inflammatory damage induced by 1. 00 µ.g/mL LPS treatment of TM4 for 12 hours was significantly lower than that in the blank group (P < 0. 01, the same as below). The protective effect of tacrine on TM4 was time and dose-dependent, with the TM4 proliferation rate of 10 000. 00 µ.g/mL tacrine pretreatment for 2. 0 hours being significantly (P < 0. 05, the same as below) lower than that of the blank group, and 100 000. 00 µ.g/ mL tacrine pretreatment for 0. 5 and 1. 0 hours or post-treatment for 0. 5, 1. 5,3. 0,6. 0, 12. 0 and the TM4 proliferation rate at 24. 0 hours was highly significantly lower than that of the blank group, while the best protection was achieved by 0. 01 µ.g/mL tacrine post-treatment for 6. 0 hours, which had a significantly higher TM4 proliferation rate than that of the blank group. Compared with the blank group, LPS significantly up-regulated the mRNA and protein expression of TM4 IL-6 and TNF-α genes, while 0. 01 µ.g/mL tacrine significantly down-regulated their expression compared with the LPS group. LPS significantly up-regulated NF-KB protein expression in TM4 compared to the blank group; 1. 00, 100. 00 and 10 000. 00 µ.g/mL tacrine significantly up-regulated NF-KB protein expression, while 0. 01 µ.g/mL tacrine decreased NF-KB protein expression compared to the LPS group but the difference was not significant (P >0. 05). [Conclusion] 0. 01 µ.g/mL tacrine post-treatment for 6. 0 hours showed the best protective effect on TM4, and the mechanism may be related to the down-regulation of mRNA and protein expression of IL-6 and TNF-α genes. [ABSTRACT FROM AUTHOR]

Objective To determine the value of pelvic MRI for assessing the gonads in complete androgen insensitivity syndrome (CAIS). Methods The clinical data and pelvic MRI of 10 men with CAIS diagnosed by clinical, imaging and surgical pathology were retrospectively analyzed. Results 9 patients underwent gonadectomy, and 1 patient underwent vulvoplasty with gonads temporarily retained. Of the 18 pathologically confirmed testes, 8 were located above the internal inguinal ring, 5 in the subcutaneous space of the inguinal canal, 3 in the iliac fossa, and 2 medial to the external iliac vessels. Eight testes contained tumors including highly differentiated sertoli-leydig cell tumors (4), sertoli cell tumors (2), highly differentiated sertoli-leydig cell tumor with seminoma and fibroma (1), highly differentiated sertoli-leydig cell tumor and fibroma (1). Three testes showed mesonephric duct cysts. Bilateral ectopic testicles were observed on MRI in all 10 patients. The 10 testes without tumors were well-defined with uniform Tt isointensity, uniform slight hyperintensity on T2- and diffusion-weighted images, and uniform contrast enhancement. Of 8 testes with sex cord stromal tumor and/or seminoma, 4 were homogeneous slightly T2 hyperintense and 4 were heterogeneous with internal nodular low signal. All 8 lesions manifested heterogeneous nodular or septal enhancement. No uterus and bilateral appendages were noted in all 10 patients. Inferior vagina was found in 9 patients. Conclusion MRI can delineate the location of ectopic testis and any associated tumor in CAIS. [ABSTRACT FROM AUTHOR]

Objective To explore the pharmacological mechanism of Wuzi Yanzong Pill in the treatment of male spermatogenic dysfunction by network pharmacology in rat sertoli cells. Methods Targets of anti-spermatogenic dysfunction in Wuzi Yanzong Pill were screened and the biological function and signal pathway were analyzed by online database. The heat stress model was constructed with rat sertoli cells to detect changes in the production of reactive oxygen species (ROS) and the expression of Hsp70. Results The results of network pharmacology showed that Wuzi Yanzong Pill mainly improved male spermatogenesis dysfunction by regulating ROS level and extracellular stimulus response etc. The ROS fluorescence probe results showed that，compared with the blank group，the ROS production was significantly increased in the model group，and significantly decreased in the Wuzi Yanzong Pill group. Western blot results showed that the expression of Hsp70 in Wuzi Yanzong Pill group was higher than that in the blank group and lower than that in the model group. Conclusion Wuzi Yanzong Pill seems to protect rat sertoli cells under heat stress by reducing ROS production and Hsp70 expression. [ABSTRACT FROM AUTHOR]

[Objectives]The purpose : of this study is to investigate the effect of zearalcnone( ZKAJon lactic acid production in Sertoli cells (SC). [Methods]The SC of Vi istar rats were cultured in vitro as test materials. 0(control), 0.1,1,10,20,30 μ.mol·L-1 ZEA were used to detect the cytotoxicity of ZKA against SC by I.DH release assay kit. The lactic acid test (lactic acid kit) was used to observe the effect of ZKA on lactic acid production in SC. The pyruvate test was performed to observe the effect of ZKA on pyruvate production in SC. The Western blot was used to detect the expression of lactic acid prodi jcing relative proteins. The immunofluorescence assay was performed aiming at detecting the key prot ein I.DH of lactic acid produc tion in SC. [Results] With the increase of ZKA concentration, the cytotoxicity of ZKA on SC gradually increased( P<0.01), and the content of lactic acid inside and outside the cell, and tl: ic concentration of pyruvate in the cell showed a downward trend (P< 0.05 or P<0.01 ). The expressions of lactic acid production-related proteins GLUT1 and I.DH exposed to ZKA at 1,10,20,30 μ.mol·L group and above significantly decreased (P<0.01), and the expression of MCT4 at the 30μmol·L-1 ZKA group significantly decreased ( P<0.05). The immunofluorescence assay showed that the fluorescence intensitv of I.DH decreased with the increase of the concentration. Compared with the control group, the protein expressions in the eells exposed to ZEA at 10,20,30 (μmol·L-1 groups, and above significantly decreased (f<0.01). [ Conclusions ] The above results indicate ZEA can inhibit the producti on of lactic acid in SC, and interfere with the energy supply of SC to germ eells, whieh damage* > the mal e reproductive function. [ABSTRACT FROM AUTHOR]

Sertoli cells, the only somatic cell in seminiferous tubule, play an important role in the process of spermatogenesis. The functions of Sertoli cells are regulated by several hormones. For example, follicle - stimulating hormone (FSH) can regulate the cell proliferation and facilitate the cellular communication, and contribute to form the blood-testis barrier (BTB) as well. Androgen can down-regulate the level of anti-Mullerian hormone (AMH) to promote the development of Sertoli cells and maintain the integrity of BTB. Estrogen can induce apoptosis and survival of Sertoli cells at different doses, and regulate the expression of aquaporin and further to affect the BTB construction. In this paper, we reviewed the influences of hormones and their receptors on the structure and function of Sertoli cells and the specific molecular mechanisms. We think that it is available for both understanding the normal processes of spermatogenesis and developing possible treatment of male infertility caused by spermatogenic disorders. [ABSTRACT FROM AUTHOR]

Objective To discuss the effect and mechanism of Wuzi Yanzong Wan (Five Seeds for Multiplying Pills) in the proliferation of Sertoli cells in infant rats. Methods The Sertoli cells were separated from male SD rats (20 d old) and cultured, and then randomly divided into normal group, low-dose Wuzi Yanzong Wan group (5 g/L, low-dose group), mid-dose Wuzi Yanzong Wan group (10 g/L, mid-dose group) and high-dose Wuzi Yanzong Wan group (15 g/L, high-dose group). The changes of optical density (OD) of Sertoli cells were detected by using methyl thiazolyl tetrazolium test (MTT) in all groups. The expression of Ki67 positive granules in Sertoli cells were detected by using immunohistochemistry technique in all groups. The expressions of Akt and p-Akt in Sertoli cells were detected by using Western blot method. Results Compared with normal group, the proliferation of Sertoli cells treated with Wuzi Yanzong Wan was promoted significantly (P<0.05 or P<0.01). The expression of Ki67 positive granules increased significantly (P<0.05 or P<0.01), and relative expression of p-Akt increased significantly (P<0.05 or P<0.01). Conclusion Wuzi Yanzong Wan can possibly promote the proliferation of Sertoli cells in infant rats through activating Akt signal transduction pathway and up-regulating p-Akt expression. [ABSTRACT FROM AUTHOR]

Objective To study the mechanism of Wuzi Yanzong Wan (Five-Seed Progeny Pill) on im­proving heat stress resistance of Sertoli cells in rats based on data from the whole genome microarray. Methods Sertoli cells in primary culture were randomly divided into Wuzi Yanzong Wan (WZYZW) group, normal group and model group. The cells in three groups were given WZYZW solution and equal volume of PBS respectively. After 24 hours, rats in WZYZW group and model group were placed in water bath at 43 °C for 15 minutes, while rats in normal group were put in water bath at 37 °C for 15 minutes, then total RNA of three groups were extracted respectively and data was analyzed with gene chip technolo­gy. Results Compared with the model group, there were totally 1,203 differently genes in WZYZW group based on Sertoli cell gene expression profiles, in which 697 were down regulated and 506 were up-regulated. There were totally 473 differently expressed genes in the normal group, including 168 up-regu-later! genes and 305 down regulated genes. Among these down-regulated gene expressions. RGD1305298. Trptl, D113, Abi3 and Ppplrl2b were significant in the WZYZW group and norma! group, while Dusp7 was significant among up-regulated gene expressions. The genes mentioned above were related to cell cycle, apoptosis, cell metabolism, cell adhesion and Notch signal pathway, regula­tion of act in cytoskeleton, MAPK signaling pathway and tight junction pathway, etc. Conclusion The mechanism of Wuzi Yanzong pill in improving heat stress resistance of Sertoli cells may involve mainte­nance of tight junctions between Sertoli cells, inhibition of Sertoli cells apoptosis and promotion of cell dif­ferentiation and maturation. [ABSTRACT FROM AUTHOR]

Mammalian target of rapamycin （mTOR）, one member of the family of phosphatidylinositol kinase, is a kind of highly conservative Ser/Thr protein kinase. It regulates many physiological functions, such as protein synthesis, metabolism and oxidative stress,which plays an important role in cell proliferation, growth and differentiation. It was confirmed that mTOR signaling pathway can regulate the proliferation and differentiation of Sertoli cell. The mTOR signaling pathway regulates the formation of blood-testis barrier ,but also mTORC1 and mTORC2 respectively regulate the lock and open of blood-testis barrier. The mTOR pathway regulates proliferation and self-renewal of spermatogenia stem cell,and the meiosis of spermatocyte in the process of spermatogenesis. The review discussed the effects of mTOR signaling pathway in regulating the proliferation of sertoli cell,the formation of blood-testis barrier and spermatogenesis. [ABSTRACT FROM AUTHOR]