10-7 M genistein partially alleviates 10-7 M MEHP unfavorable effects in a new modified fetal rat testis culture system.

Background: Recent studies revealed that some common endocrine-disrupting chemicals (EDCs) including phthalates and phytoestrogens may exhibit low-dose effects properties. However, how low dose of these EDCs and their mixture would affect fetal rat testis development still needs further investigation. Moreover, testis organ culture system also needs further modification to provide an effective tool for ex vivo EDCs study. Methods: We firstly modified the agarose organ culture system, in which fetal rat testes were cultured for 4 days (d1 to d4) on agarose gels held by Millicell inserts. Then we used the modified agarose culture system to study the combined effects of multiple EDCs exposure. 15.5 dpc fetal rat testes were isolated and treated with vehicle, MEHP (0.1 µmol/L), GEN (0.1 µmol/L) or MEHP (0.1 µmol/L) + GEN (0.1 µmol/L). Parameters concerning testicular cell development and function were evaluated, trying to gain insight into the early molecular events after multiple EDCs exposure. Results: The development of somatic, germ cells and seminiferous tubule in 15.5 dpc fetal rat testis was better sustained in the modified agarose culture system. Based on the modified system, we found that MEHP at 0.1 µmol/L induced alterations in gonocyte markers, antioxidative enzyme activity as well as transient reduction of testosterone production, accompanied by mitochondria swelling in gonocytes and Sertoli cells. No obvious morphological and histological alterations were observed in all treated groups. However, coadministration of genistein at 0.1 µmol/L partially alleviated MEHP-induced fetal testis damage ex vivo through enhancement of antioxidative action. MEHP at low dose still showed weak endocrine disrupting properties but did not exhibit typical low-dose effects. Conclusion: Our findings indicated that the modified agarose culture system could better mimic testicular microenvironment without obvious hypoxic cell damage. Furthermore, low dose of MEHP induced mild disruption to fetal testis development, cotreatment of genistein at low dose attenuated MEHP induced fetal testis injuries in part by balancing redox state, indicating that low dose of genistein may partially protect fetal testis from phthalates induced injury.

Oxidative Stress Disrupted Prepubertal Rat Testicular Development after Xenotransplantation.

In the past two decades, testicular tissue grafting and xenografting have been well established, with the production of fertilization-competent sperm in some studies. However, few studies have been carried out to observe the development of grafted prepubertal testicular tissue of rats and compare the biological differences between in situ testis and grafted testis. In this study, we established the prepubertal testicular tissue xenografting model using a 22-day-old rat and evaluated certain parameters, including testicular histology, testosterone production, and ultrastructure of the grafted testes. We also assessed gene expression of cell proliferation markers, testicular cell markers, and antioxidative defense system. Our results showed that 47 days after transplantation, intratesticular testosterone concentration was not significantly altered; however, cell proliferation, spermatogenesis, and Sertoli cell markers in the transplanted testes were significantly disrupted compared with the control group, accompanied by aggravated apoptosis and oxidative damage. Moreover, the transplanted testes showed smaller tubular diameter and disrupted spermatogenic epithelium with apparent vacuoles, distorted and degenerated germ cells with obscure nuclear margin, and no spermatids in the center of the tubules. Although testis xenografting has been extensively tested and attained great achievement in other species, the prepubertal rat testicular tissue xenografting to immunodeficient mice exhibited obvious spermatogenesis arrest and oxidative damage. The protocol still needs further optimization, and there are still some unknown factors in prepubertal rat testes transplantation.

Low Dose of Genistein Alleviates Mono-(2-Ethylhexyl) Phthalate-Induced Fetal Testis Disorder Based on Organ Culture Model.

Mono-(2-ethylhexyl) phthalate (MEHP) and genistein have been classified as endocrine-disrupting chemicals (EDCs) which interfere with the differentiation and development of the male reproductive system. However, how these two EDCs would affect fetal rat testis development at a low dose was rarely studied. In this study, we established the organ culture system and applied it to evaluate testicular effects following multiple EDC exposure at a low dose. 15.5 days postcoitum fetal rat testes were dissected, cultured, and exposed to vehicle (control), GEN (1 µmol/L, G), MEHP (1 µmol/L, M), or GEN (1 µmol/L)+MEHP (1 µmol/L, G+M). Testicular cell markers, testosterone concentration, redox state, testicular histology, and testicular ultrastructure were evaluated. Our results showed that a low dose of MEHP suppressed the development of Sertoli cells, Leydig cells, and gonocytes by triggering oxidative injuries, which was consistent with the ultrastructural findings. However, coadministration of genistein at a low dose could partially attenuate MEHP-induced fetal testis damage through antioxidative action. Cotreatment of genistein at a low dose may have a promising future on its protecting role for attenuating other EDC-induced reproductive disorders during early life. Based on the results, it can be speculated that dietary intake of isoflavones may make the fetal testis less susceptible to phthalate-induced injury.

Anatomic characteristics of epididymis based on histology, proteomic, and 3D reconstruction.

BACKGROUND: The epididymis is a popular research topic in urology and reproduction. OBJECTIVES: To explore and identify the anatomical characteristics of the epididymis based on histology, proteomics, and 3D reconstruction of epididymal tubules. MATERIALS AND METHODS: A 3D reconstruction of epididymal tubules was generated based on 7-µm-thick transverse serial sections of an epididymis. The proteins in the subcompartments of the epididymis were obtained and analyzed by non-labeled sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Protein function, signaling pathways, protein expression, and the histology in different subcompartments were analyzed. RESULTS: The caput (Cap), corpus (Cor), and cauda (Cau) of the epididymis were divided into 6, 10, and 4 subcompartments, respectively, and the subcompartment between the Cap and Cor is mixed together. A total of 3411 proteins were identified, and 854 proteins were accurately quantified after screening. When the subcompartment Cap 5 transitioned to Cap 6 and Cap 6 to Cap 7, 87 and 52 proteins were upregulated and 14 and 7 proteins were downregulated, respectively. The Cor 9 transition to Cau 1 was marked by 230 proteins that were downregulated, while 74 proteins were upregulated. At the junction of the cauda and the vas deferens, 57 proteins were downregulated, and 410 proteins were upregulated. Cap 6 histology was consistent with that of Cor 1. DISCUSSION AND CONCLUSION: The epididymis contains distinct connective tissue septa that can be identified under a surgical tabletop microscope, enabling it to be divided into 20 subcompartments.

Surgical management of retroperitoneal schwannoma complicated with severe hydronephrosis: A case report.

RATIONALE: Schwannomas are usually benign tumors arising from well-differentiated schwann cells, which rarely occur in the retroperitoneal space. The lack of specific signs and radiologic imaging characteristics makes preoperative diagnosis rather difficult. Most retroperitoneal schwannomas are benign and the primary treatment choice for retroperitoneal schwannomas is surgical excision, however, the involvement of the urinary system is scarcely reported. PATIENT CONCERNS: A 34-year-old woman presented with progressive left abdominal pain and rebound abdominal mass at the left lower quadrant for 1 month. Radiological imaging suggested capsulated solid mass with cystic and necrotic areas in the retroperitoneum accompanied by severe left kidney hydronephrosis and preoperative biopsy result was inconclusive. DIAGNOSES: We believe this is a rare case of retroperitoneal schwannoma complicated with severe hydronephrosis. INTERVENTIONS: After preparation, the patient underwent laparoscopy exploration and converted to open surgical exploration. The patient accepted complete surgical excision of the retroperitoneal tumor and left kidney. Postoperative pathology diagnosis of the mass was proven to be benign retroperitoneal schwannoma. OUTCOMES: Postoperative course of the patient was uneventful and the left abdominal pain was greatly improved. After 12-month follow up, no evidence of recurrence or any other complication including renal failure was observed. LESSONS: Preoperative imaging and preoperative ultrasound-guided biopsy are helpful to make accurate diagnosis. The final diagnosis is based on postoperative histological and immunohistochemical findings. The primary treatment option is complete surgical resection of the retroperitoneal schwannoma and the involved upper urinary system when severe hydronephrosis occured. Local recurrence and overall survival are closely correlated with negative resection margins and pathology types.

Genistein attenuates di­(2­ethylhexyl) phthalate-induced testicular injuries via activation of Nrf2/HO­1 following prepubertal exposure.

Di­(2­ethylhexyl) phthalate (DEHP) and genistein (GEN) are of the most common endocrine disrupting chemicals (EDCs) present in the environment or the diet. However, investigation of the effects of acute exposure to these two EDCs during prepuberty has been lacking. In this study, DEHP and GEN were administrated to prepubertal male Sprague­Dawley rats by gavage from PND22 to PND35 with vehicle control, GEN 50 mg/kg body weight (bw)/day, DEHP50, 150 and 450 mg/kg bw/day, and combined treatment. Reproductive parameters including testis weight, anogenital distance and organ coefficient were evaluated on PND36. Enzyme activity involved in the regulation of testicular redox state as well as expression of genes and proteins related to anti-oxidative ability and apoptosis were also investigated. The results revealed that by PND36, DEHP treatment had significantly decreased the testis weight, organ coefficient, testicular anti-oxidative enzyme activities and caused tubular vacuolation; however, co­administration of GEN partially alleviated DEHP­induced testicular injuries and enhanced testicular anti­oxidative enzyme activities and upregulated the expression of NF­E2 related factor 2 and heme oxygenase­1, which indicated that GEN partially attenuated DEHP­induced male reproductive system damage through anti­oxidative action following acute prepubertal exposure to DEHP. Thus, GEN may have use in attenuating the damaging effects of other EDCs that lead to reproductive disorders.

[Establishment of a method for in vitro culture of fetal rat testis tissue].

OBJECTIVE: To establish a method for in vitro culture of the fetal rat testis tissue. METHODS: Nine sexually mature specific-pathogen-free rats, 3 males and 6 females, weighing 200－250 g, were used for this study. The estrus of the female rats was determined according to the results of the vaginal smear test. The female rats were mated with the male ones in proestrus and estrus at night in the ratio of 2∶1 and observed the following day for conception (0.5 day post-conception ［dpc］) based on the presence of sperm in the vaginal smear. At 15.5 dpc, the fetal testes were isolated under the anatomical microscope, some for HE staining and the rest divided into a control and an hCG group to be cultured in a soft agar culture system at 37 ℃ in a humidified atmosphere containing 5% CO2. From the first day of culture (d 0), the development of the testes was observed under the inverted microscope, the culture medium collected and replaced on d 1, d 2, d 3 and d 4, and the testis tissue obtained on d 4. The concentration of testosterone in the culture medium was determined and the testis tissues were fixed, dehydrated and embedded for histological examination. RESULTS: Fetal rats were successfully obtained with the vaginal smear at 15.5 dpc, and the fetal testes effectively isolated, which were well developed, with gradual increase of their volume and enlargement of convoluted seminiferous tubules under the inverted microscope. Testosterone was observed in the culture medium, its concentration gradually increasing and reaching the peak on d 3, and its secretion stimulated by hCG. At 15.5 dpc. The fetal testes showed a histomorphological integrity, with typical seminiferous tubules, gonocytes, Sertoli cells and Leydig cells, but no central necrosis. Transmission electron microscopy revealed gonocytes and Sertoli cells within and Leydig cells between the seminiferous tubules, without obvious swelling of the mitochondria and endoplasmic reticula in the cells. CONCLUSIONS: The fetal rat testis tissue cultured in the soft agar culture system can develop well, retain its normal activity, and excrete testosterone into the culture medium.

Protective Effects of Genistein against Mono-(2-ethylhexyl) Phthalate-Induced Oxidative Damage in Prepubertal Sertoli Cells.

Mono-(2-ethylhexyl) phthalate (MEHP) and genistein are two of the most prevalent endocrine-disrupting chemicals (EDCs) that present in the environment and food. However, how these two EDCs would affect prepubertal Sertoli cells development was rarely studied. In this study, primary prepubertal Sertoli cells were isolated from 22-day-old Sprague Dawley rats and exposed to MEHP at 1 µmol/L, 10 µmol/L, and 100 µmol/L (M1, M10, and M100), genistein at 10 µmol/L (G), and their combination (G + M1, G + M10, and G + M100). Cell proliferation inhibition rate, apoptosis and necrosis rate, and cellular redox state were evaluated. Our results revealed that MEHP could significantly increase cell proliferation inhibition rate, apoptosis rate, necrosis rate, and intracellular reactive oxidative species level. However, coadministration of genistein could partially alleviate MEHP-induced prepubertal Sertoli cells oxidative injuries via enhancement of testicular antioxidative enzymes activities and upregulation of Nrf2 and HO-1, indicating that genistein could partially attenuate MEHP-induced prepubertal Sertoli cells damage through antioxidative action and may have promising future on its curative role for attenuating other EDCs-induced reproductive disorders.

[Novel methods for studies of testicular development and spermatogenesis: From 2D to 3D culture].

The two-dimensional model of cell culture is an important method in the study of testicular development and spermatogenesis but can not effectively mimic and regulate the testicular microenvironment and the whole process of spermatogenesis due to the lack of relevant cell factors and the disruption of a three-dimensional spatial structure. In the past 20 years, the development and optimization of the in vitro model such as testis organotypic culture and in vivo model such as testis transplantation achieved a transformation from two- to three-dimension. The maintenance and optimization of the testicular niche structure could mimic the testicular microenvironment and cell types including Leydig, Sertoli and germ cells, which showed similar biological behaviors to those in vivo. Besides, the cell suspension or tissue fragment floats in the gas-liquid interface so that the development of somatic and germ cells is well maintained in vitro whilst the feedback linkage between grafted testis tissue and hypothalamus-pituitary of the host rebuilt in the in vitro model provides an endocrinological basis for spermatogenesis, which serves as an effective methodology to better understand the organogenesis and development of the testis as well as testicular function regulation, advancing the concept of treatment of male infertility. Al- though each of the methods may have its limitations, the progress in the processing, freezing, thawing, and transplantation of cells and tissues will surely promote their clinical application and present their value in translational medicine.