Spermatogenesis is a complex regulatory process depending on a variety of hormones (such as FSH, LH, T, and 17beta estradiol), cytokines, and genes. Research on gene regulation in spermatogenesis has become a hot spot and revealed some spermato-genesis-related genes, such as AYZ, DAZ, YRRM, NOSTRIN, and so on. Reports are rarely seen on the role of CR16 in male reproduction, and its action mechanism in spermatogenesis is not yet clear. This article updates the role of CR16 in spermatogenesis in the male reproductive system from the perspective of Sertoli cells forming a blood-testis barrier.
Animals ; Blood-Testis Barrier ; Humans ; Male ; Microfilament Proteins ; Sertoli Cells ; Spermatogenesis ; Testis ; cytology

OBJECTIVESTo develop a simple and effective method by which spermatids can be isolated from mouse testis.

METHODSCombination of enzymatic digestion was used to prepare suspension of spermatogenic cells from adult mouse testis, and then a modified discontinuous Percoll gradient (15%, 22%, 30%, 40%, 50%, 60%) centrifugation method was introduced to isolate spermatids from the cellular suspension. The content of spermatids in each isolated fraction by Percoll method was determined by morphology (Wright-Giemsa staining) and flow cytometry analysis, and the viability of spermatogenic cells was assessed using Eosin Y exclusion test.

RESULTSMore than 97% of the testicular cells remained their viability after enzymatic digestion. After Percoll centrifuged, six fractions were formed. In each isolated fraction, the 22% fraction contained mostly spermatids(mean 86.7%) and cell viability was more than 85.5%. While in the 30% fraction, immature spermatogenic cells were present, and more than 92% of the cells remained their viability.

CONCLUSIONSA large of relatively purified spermatids can be isolated from mouse testis by enzymatic digestion combined discontinuous Percoll gradient centrifugation method.

Animals ; Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Male ; Mice ; Spermatids ; cytology ; Testis ; cytology

OBJECTIVETo study the feasibility of spermatogonial stem cell allotransplantation.

METHODSThe spermatogonial stem cell allotransplantation was performed, without the use of immune inhibitor, in KM mice of similar gene types, and the spermatogenesis in recipients' testes was evaluated. The right testes were pierced for transplantation while the left ones were taken as control.

RESULTSAllotransplant germ cells in KM mice can recover normal function of spermatogenesis in the transplanted testis without any immune suppression.

CONCLUSIONAllospermatogonial stem cells can be transplanted successfully among KM mice.

Animals ; Male ; Mice ; Models, Animal ; Spermatogonia ; cytology ; transplantation ; Stem Cell Transplantation ; Testis ; cytology ; Transplantation, Homologous

Lectins are glycoproteins of plant and animal origin that have the ability to bind specific carbohydrate residues of cell glycoconjugates, particularly in terminal positions. In this study, the binding of lectins, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Bandeiraea simplicifolia BS-1 (isolectin B4), Triticum vulgaris (WGA), Arachis hypogaea (PNA), and Ulex europaeus (UEA-I), was studied in the reproductive systems of male thoroughbred horses.DBA was detected in the stereocilia of the caput and corpus epididymis, and in the vas deferens. It was weakly detected in connective tissue of the corpus epididymis. Strong SBA staining was seen in epithelial cells in the testis, stereocilia of the corpus and cauda epididymis, and in the vas deferens. There were intense positive reactions for isolectin B4 in interstitial cells in all tissue and serosa of the vas deferens. PNA staining was seen only in stereocilia in the caput and corpus epididymis, and in the vas deferens. Strong WGA staining was seen throughout the testis, except in Sertoli cells, stereocilia, and connective tissue. UEA-I was detected in secondary spermatids, stereocilia, and epithelial cells of the cauda epididymis.These results show that degenerating cells in the testis, epididymal tubules, and vas deferens have differential affinities for lectins, and suggest that lectins play a role in the reproductive system of the horse. The heterogeneity of the lectin staining pattern in the reproductive tubules of adult horses suggests that the carbohydrate composition of each cell type is region specific.
Animals ; Epididymis/cytology/*metabolism ; Horses/*metabolism ; Immunohistochemistry/veterinary ; Lectins/*metabolism ; Male ; Testis/cytology/*metabolism ; Vas Deferens/cytology/*metabolism

OBJECTIVETo investigate the differences in the development of primordial germ cells (PGCs) between male and female mouse embryos.

METHODSThe morphological changes of genital ridge development were detected in C57BL/6J mouse embryos of 11-13.5 days, and the changes of PGCs quantity and proliferation were compared between the male and female embryos using immunofluorescence histochemistry.

RESULTSThe PGCs was the most numerous at 13.5 days in male and female embryos, and the quantity of proliferating PGCs reached the maximum at 13 days. The quantity of PGCs and proliferating PGCs in male embryos at 13 days was significantly larger than that in female embryos.

CONCLUSIONThe development of PGCs is characterized by a gender differences in early development of mouse embryos (11-13.5 days).

Animals ; Cell Proliferation ; Embryo, Mammalian ; cytology ; Female ; Gene Expression Regulation, Developmental ; Germ Cells ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Ovary ; cytology ; Sex Factors ; Testis ; cytology

Male germ-line stem cells (mGSCs) have the capability of self-renewal and latent capability of differentiation. mGSCs is the unique diploid immortal cell which can transfer genetic information to filial generation. The combination of transgenic technology and mGSCs heterotransplanting will supply new opportunities and paths to cloning animal, transgenic animal and gene therapy of some human hereditary disease. We studied the isolation and cultivation of mGSCs that were isolated and purified from 5-6 month old bovine fetal testis, new born bovine testis by adopting mixed enzymes digestion and different attaching velocities methods. The results showed that Sertoli cells were indispensable to mGSC's proliferation and differentiation in vitro. The Sertoli cells in logarithmic phase have a significant effect on mGSC's attaching, proliferation and differentiation. Co-culture with Sertoli cells, mGSCs differentiated to long sperm after 16 days. A preliminary system for mGSC's inducing differentiation was established.
Animals ; Cattle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Male ; Sertoli Cells ; cytology ; Spermatozoa ; cytology ; Stem Cells ; cytology ; Testis ; cytology

OBJECTIVETo investigate the ectopic grafts of mouse testicular cells by observing the reconstruction of seminiferous tubules, colonization of spermatogenic cells and spermatogenesis using immunodeficient mice as recipients.

METHODSThe testes of newborn male ICR mice were digested to obtain single cell suspension. The cells were then mixed with matrigel and subcutaneously grafted into the dorsal region of the male nude mice. The mice were castrated after the operation and the grafts were dissected from 5 of the nude mice at 4, 6, 8 and 10 weeks, respectively. The success rates of transplantation and the graft diameters were calculated, and the structure of the reconstituted seminiferous tubules, colonization of the germ cells and spermatogenesis were observed by HE staining and immunohistochemistry.

RESULTSAll the mice recipients survived after the testicular cell transplantation. Within 10 weeks after the operation, tissue masses could be observed, with the diameter increased from (3.91 +/- 0.71) mm at 4 weeks to (6.69 +/- 0.50) mm. Neovascularization was detected at the surface of the masses and seminiferous tubule structures found in the grafts. The germ cells that developed from spermatogonia to round spermatids were observed, but with no sperm in the tubules. Germ cells, Sertoli cells and Leydig cells were identified by immunochemical detection of Mvh, Gata4 and P450Scc in the grafts at 8 weeks.

CONCLUSIONSeminiferous tubules could be ectopically reconstructed from suspension of neonatal mouse testicular cells. Ectopic grafting provided a preferable model for the studies on testis tissue engineering and interactions between testicular cells during testicular development and spermatogenesis.

Animals ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred ICR ; Mice, Nude ; Seminiferous Tubules ; cytology ; Sertoli Cells ; cytology ; transplantation ; Spermatids ; cytology ; Spermatogenesis ; Testis ; cytology ; transplantation ; Transplantation, Heterologous

Men with non-obstructive azoospermia (NOA) can achieve fertility by testicular sperm extraction (TESE) coupled with intracytoplasmic sperm injection (ICSI), the key to which is the successful retrieval of sperm from the testis. Although improved testicular sperm extraction techniques have increased the chances of sperm retrieval, to predict preoperatively the success of sperm retrieval from NOA patients remains challenging. A non-invasive diagnostic technique predicting the presence of sperm in the testis would be useful for avoiding possible surgical intervention. At present, some preoperative variables, such as serum FSH, inhibin B level, testis volume, genetic analysis, histopathology on diagnostic biopsy, Raman Spectroscopy, and molecular and protein markers, have provided new insights into the chances of successful sperm retrieval in NOA males. This review aims to evaluate the preoperative factors currently available for predicting the outcomes of sperm retrieval from NOA patients.
Azoospermia ; therapy ; Biomarkers ; Biopsy ; Genetic Testing ; Humans ; Inhibins ; blood ; Male ; Sperm Injections, Intracytoplasmic ; Sperm Retrieval ; Spermatozoa ; cytology ; Testis ; cytology

OBJECTIVETo explore the action mechanisms of temperature in male infertility or subfertility by observing the effects of different temperatures on the proliferation of and occludin (OCLN) expression in rat Sertoli cells in vitro.

METHODSWe isolated Sertoli cells from the testis of male Wistar rats, and performed oil red O staining and immunohistochemistry to identify their FasL. We cultured the Sertoli cells at 34 degrees C (control group) and at 35, 36, 37, 38 and 39 degrees C (experimental groups) for 4 days. Then we measured their proliferation by CCK-8 assay, observed their morphology and structure by hematoxylin-eosin staining, and determined their OCLN expression level by Western blotting and immunofluorescence.

RESULTSThe purity of the isolated Sertoli cells was (96.20 +/- 1.95)%. CCK-8 assay indicated that the proliferation of the Sertoli cells was increased between 34 and 36 degrees C, and decreased at 36-39 degrees C. The pyknotic nuclei and fragmentation of the Sertoli cells were more obvious at > 36 degrees C. Western blot and immunofluorescence showed the highest level of OCLN expression at 36 degrees C, which, however, decreased while the temperature rose above 36 degrees C (P < 0. 01).

CONCLUSIONHigh temperature (> 36 degrees C) inhibited the proliferation of rat Sertoli cells in vitro, and decreased the expression of OCLN, which suggests that a higher temperature above 36 degrees C may reduce male fertility by affecting the proliferation of Sertoli cells and integrity of the tight junction among Sertoli cells or Sertoli cells and other cells.

Animals ; Cell Proliferation ; Male ; Occludin ; metabolism ; Rats ; Rats, Wistar ; Sertoli Cells ; cytology ; metabolism ; Temperature ; Testis ; cytology ; metabolism

This study was aimed to explore a simple and applicable method of separating mature sperms from human testicular tissue for intracytoplasmic sperm injection and embryo transfer. The suspension of human testicular tissue was cultured in 10% human serum albumin and human tubule fluid with different concentrations (0 u/ml; 50 u/ml; 100 u/ml; 150 u/ml; 200 u/ml) of hyaluronidase for 24 h, and then the Percoll gradient centrifugation was processed to separate the sperms; meanwhile the sperms were counted and graded according to their motility. The difference in quality and quantity among the groups and the difference between the groups and the zero-hour culturing group were detected. It was shown that the four hyaluronidase-treated groups contained large quantity and high quality of sperms as compared with the two contrast groups (P<0.01). The groups in the solution of 50 u/ml, 100 u/ml and 150 u/ml concentrations of hyaluronidase had almost the same amount of sperms that displayed higher motility as compared against the sperms in the group treated with 200 u/ml concentration of hyaluronidase (P<0.01). There was no difference between the two contrast groups (P>0.05), or among the groups treated with 50 u/ml, 100 u/ml, and 150 u/ml of hyaluronidase concentration (P>0.05). This method of adopting hyaluronidase with Percoll gradient centrifugation in the process for separating mature sperms from human testicular tissue is applicable. It can increase the quantity and quality of sperms separated from testicular tissue suspensions when adequate concentrations of hyaluronidase is used.
Adult ; Cell Separation ; methods ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Hyaluronoglucosaminidase ; pharmacology ; Male ; Spermatozoa ; cytology ; Testis ; cytology