OBJECTIVETo investigate the differentiation of human testicular spermatogenic cells during in vitro culture.

METHODSTesticular cells of obstructive azoospermic patients' testis biopsies were dispersed employing mechanic methods. Then, (1) mixed testicular cells were applied to in vitro culture, and changes of the ratio of elongating spermatids and all round cells were analyzed during mixed cell culture; (2) round spermatids were picked up from the mixed cells employing micromanipulator, followed by differentiation of the isolated round spermatids during microdrop culture.

RESULTSThe ratio of the elongating spermatids increased significantly (P < 0.05) after 24 hours of mixed cell culture in HTF medium supplemented with FSH and testosterone. During single round spermatid culture, transformation of the round spermatid to elongating spermatid with newly formed flagellum was observed, and the transformation ratio within 48 hours of microdrop culture was 3.54%. The differentiation of human testicular spermatogenic cells cultured in Vero cell conditioned medium was similar to that cultured in HTF medium.

CONCLUSIONHuman testicular round spermatids can differentiate to elongating spermatids during in vitro culture. Vero cell conditioned medium does not promote the differentiation of human testicular round spermatids to elongating spermatids.

Animals ; Cell Differentiation ; Cells, Cultured ; Cercopithecus aethiops ; Humans ; Infertility, Male ; pathology ; Male ; Spermatids ; cytology ; Testis ; cytology ; Vero Cells

OBJECTIVESTo develop a method by which large and purified populations of spermatids can be isolated in semen of male infertile patients.

METHODSA total of fifteen ejaculates containing cellular elements from infertile patients with various andrological pathologies were obtained after a 24-hour abstinence. A modified discontinuous Percoll gradient (15%, 22%, 30%, 40%, 50%, 60%) centrifugation method was used to isolate the spermatids. After centrifugation at 2,000 r/min for 30 min at 18 degrees C, the single Percoll fractions were separated and analyzed in order to select the one with the greatest purity of spermatid. The germinal cells in each isolated fraction were counted using a Macro sperm counting chamber, then the contents of spermatids were determined by morphology (Wright-Giemsa staining method) and flow cytometry (FCM) analysis, while the contaminated leukocytes were assessed by anti-CD45 immunocytochemistry.

RESULTSAfter Percoll centrifuged, six single fractions were obtained. Morphology and FCM analysis showed that the 22% fraction contained mostly spermatids [(91.85 +/- 5.18)%, P < 0.005] and the mean density in this fraction was (1.010 +/- 0.786) x 10(5)/ml. While in the 30% fraction, various immature spermatogenic cells including spermatids were present and leukocytes mostly presented in the 60% fraction.

CONCLUSIONSA large population of relatively purified spermatids can be isolated from the ejaculates of infertile patients by using this modified discontinuous Percoll gradient centrifugation method.

Cell Separation ; methods ; Centrifugation, Density Gradient ; methods ; Flow Cytometry ; Humans ; Immunohistochemistry ; Infertility, Male ; pathology ; Leukocyte Common Antigens ; analysis ; Leukocytes ; chemistry ; cytology ; Male ; Semen ; cytology ; Spermatids ; chemistry ; cytology

OBJECTIVETo observe the apoptosis of spermatogenic cells and the expressions of Bcl-2 and Bax proteins after burying the testis in the inguinal pocket, and to investigate their relationship.

METHODSWe randomly divided 36 healthy male New Zealand white rabbits into an experimental group (n = 18) and a control group (n = 18). Models were established by burying testes in the inguinal pocket in the experimental group, while the controls were left untreated. At the end of the 8th week after surgery, 6 animals were randomly taken from each group for measurement of the testis surface temperature and testicular biopsy. The apoptosis of spermatogenic cells in the testis tissues was detected by TUNEL assay, and the expressions of Bcl-2 and Bax proteins determined by immunohistochemistry and imaging analysis.

RESULTSAt 8 weeks after burying the testis in the inguinal pocket, the testicular surface temperature was significantly higher in the experimental group than in the control ([ 38.02 +/- 0.36] degrees C vs [36.15 +/- 0.64 ] degrees C, P < 0.05), and so was the apoptosis index (AI) of spermatogenic cells ([89.69 +/- 3.76] % vs [7.73 +/- 4.95 ] %, P < 0.05). The expression of the Bax protein in the testis was significantly increased, while that of the Bcl-2 protein remarkably decreased in the experimental group as compared with the control group (P < 0.05). The apoptotic cells were mostly primary spermatocytes and round spermatids.

CONCLUSIONElevated local temperature of the testis buried in the inguinal pocket increases the apoptosis of spermatogenic cells, and the spermatogenic cell apoptosis is highly correlated with the decreased expression of Bcl-2 and increased expression of Bax. The changes in the expressions of Bcl-2 and Bax proteins were a main mechanism behind the temperature elevation-induced apoptosis of spermatogenic cells.

Animals ; Apoptosis ; Groin ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rabbits ; Spermatids ; metabolism ; Temperature ; Testis ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the reproductive system impairment induced by cocaine in adult male rats and the possible underlying mechanism. METHODS: Thirty adult male rats were randomly divided into experimental and control groups, with 15 rats in each group. Rats of the experimental group were injected cocaine hydrochloride (15 mg/kg body weight) subcutaneously daily for four weeks. The weight of body and testis, as well as the level of serum hormone of the rats were examined. In addition, the apoptosis rate of testicular tissue by TUNEL and the expression of Fas gene in testicular tissue were examined by immunohistochemistry. RESULTS: Compared with the control, the weight of testis in the cocaine exposed group decreased significantly (P<0.05), and the serum testosterone level decreased significantly (P<0.05). Moreover, both the apoptosis rate and the expression of Fas gene increased in the testicular tissue of rats in the cocaine exposed group in comparison to the control group (P<0.05). The apoptosis rate was significantly correlated with the expression of Fas gene (r=0.9012, P<0.05). CONCLUSION Cocaine may cause reproductive system injury in adult male rats, and Fas-mediated apoptosis may be one of the functional mechanisms involved in the reproductive system injuried by cocaine.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis/drug effects* ; Cocaine/adverse effects* ; Forensic Toxicology ; Male ; Molecular Chaperones ; Nuclear Proteins/metabolism* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatids/pathology* ; Substance-Related Disorders ; Testis/pathology* ; Testosterone/blood*

AIMTo evaluate the treatment outcome of antegrade internal spermatic vein sclerotherapy in men with non-obstructive azoospermia or severe oligoteratoasthenospermia (OTA) as a result of varicocele.

METHODSBetween September 1995 and January 2004, 47 patients (mean age 33.8 +/- 6.3 years) underwent antegrade internal spermatic vein sclerotherapy for the treatment of varicocele with azoospermia (14 patients) or severe OTA (33 patients). Testicular core biopsy was also performed in complete azoospermic patients who provided informed consent. The outcome was assessed in terms of improvement in semen parameters and conception rate.

RESULTSForty-two (89.4%) of 47 patients had bilateral varicocele. Serum follicle stimulating hormone (FSH) did not differ between patients with azoospermia and severe OTA. After the follow-up of 24.8 +/- 9.2 months, significant improvement was noted in mean sperm concentration, motility and morphology in 35 patients (74.5%). Comparison between groups during the follow-up revealed significantly higher values of sperm concentration, motility and normal morphology in the severe OTA group. Pregnancy was achieved in 14 cases (29.8%). Testicular histopathology of the azoospermic patients with postoperative induction of spermatogenesis revealed maturation arrest at spermatid stage, Sertoli-cell-only (SCO) with focal spermatogenesis or hypospermatogenesis. None of the patients with pure SCO pattern or maturation arrest at spermatocyte stage achieved spermatogenesis after the treatment. Preoperative serum FSH levels didn't relate to treatment outcome.

CONCLUSIONAntegrade internal spermatic vein sclerotherapy is an easy and effective treatment for symptomatic varicocele. It can significantly reverse testicular dysfunction and improve spermatogenesis in men with severe OTA, as well as induce sperm production in men with azoospermia, improving pregnancy rates in subfertile couples.

Adult ; Costs and Cost Analysis ; Female ; Functional Laterality ; Germany ; Humans ; Infertility, Male ; etiology ; Male ; Oligospermia ; etiology ; Pregnancy ; Retrospective Studies ; Sclerotherapy ; economics ; Sperm Count ; Spermatids ; pathology ; Spermatogenesis ; Testis ; blood supply ; Treatment Outcome ; Varicocele ; therapy

AIMTo investigate the effects of rat Erythropoietin (Epo) on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation.

METHODSSprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups: the first group was given intratesticular injections of pCAGGS-Epo (pCAGGS-Epo group), the second group was given intratesticular injections of pCAGGS (pCAGGS group), and the third group were given intratesticular injections of phosphate-buffered saline (PBS group). At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells (G/S ratio) was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point.

RESULTSThe testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was (0.85+/-0.08) g and (0.83+/-0.03) g, respectively, at week 1 (P = 0.788) and (0.62+/-0.06) g and (0.52+/-0.02) g, respectively, at week 2 (P = 0.047). At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 +/-6.80 vs. 18.63+/-5.30 at week 1 (P = 0.0078) and 7.16+/-3.06 vs. 6.05+/-1.58 at week 2 (P = 0.1471), respectively.

CONCLUSIONThe transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

Animals ; Cryptorchidism ; pathology ; therapy ; Electroporation ; methods ; Erythropoietin ; genetics ; Genetic Therapy ; methods ; Lac Operon ; Male ; Organ Size ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Risk Factors ; Sertoli Cells ; cytology ; Spermatids ; pathology ; Spermatogenesis ; Spermatozoa ; pathology ; Testis ; pathology ; physiology

RNA binding proteins (RBPs) regulate the function of cells by interacting with nascent transcripts and therefore are receiving increasing attention from researchers for their roles in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. Further investigations on the post-transcriptional regulation mechanisms and isoforms of PTB proteins in the spermatogenesis show that PTB protein 1 (Ptbp1) is a predominant isoform in mitotic cells (spermatogonia), while Ptbp2 predominates in meiotic spermatocytes and postmeiotic spermatids and binds to the specific 3' untranslated region (3' UTR) of the phosphoglycerate kinase 2 (Pgk-2) mRNA, which helps to stabilize Pgk-2 mRNA in male mouse germ cells. In case of Ptbp2 inactivation in the testis, the differentiation of germ cells arrests in the stage of round spermatids, with proliferation of multinucleated cells in the seminiferous tubule, increased apoptosis of spermatocytes, atrophy of seminiferous tubules, and lack of elongating spermatids, which consequently affects male fertility. This article presents an overview on the structure of the PTB protein and its role in regulating mammalian spermatogenesis.
Animals ; Atrophy ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoproteins ; metabolism ; physiology ; Homeostasis ; Isoenzymes ; metabolism ; Male ; Mice ; Nerve Tissue Proteins ; metabolism ; physiology ; Phosphoglycerate Kinase ; metabolism ; Polypyrimidine Tract-Binding Protein ; metabolism ; physiology ; RNA, Messenger ; metabolism ; RNA-Binding Proteins ; Seminiferous Tubules ; pathology ; Spermatids ; metabolism ; Spermatocytes ; metabolism ; Spermatogenesis ; physiology ; Spermatogonia ; metabolism ; Testis ; metabolism

AIMTo investigate the stage-specific localization of transforming growth factor (TGF) beta1 and beta3 during spermatogenesis in adult human testis.

METHODSThe localization of TGFbeta1 and beta3 was investigated by immunohistochemical staining method employing specific polyclonal antibodies.

RESULTSBoth TGFbeta1 and beta3 and their receptors were preponderant in the Leydig cells. TGFbeta1 could not be detected in the seminiferous tubules. TGFbeta3 and TGFbeta-Receptor (R) I were mainly seen in the elongated spermatids, while TGFbeta-RII in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli cells. Only TGFbeta-RII was detected in the Sertoli cells. TGFbeta3, TGFbeta-RI and TGFbeta-RII showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle.

CONCLUSIONTGFbeta isoforms and their receptors are present in the somatic and germ cells of the adult human testis, suggesting their involvement in the regulation of spermatogenesis.

Adult ; Humans ; Immunohistochemistry ; Leydig Cells ; metabolism ; Ligands ; Male ; Middle Aged ; Orchiectomy ; Prostatic Neoplasms ; pathology ; Receptors, Transforming Growth Factor beta ; metabolism ; Seminiferous Epithelium ; cytology ; metabolism ; Spermatids ; metabolism ; Spermatogenesis ; physiology ; Testis ; metabolism ; physiology ; Transforming Growth Factor beta ; metabolism ; Transforming Growth Factor beta1 ; Transforming Growth Factor beta3