The reproductive system and gonad development and germ cell occurrence of Whitmania pigra have been studied by using tissue section electron microscope techniques. W. pigra has completely independent male and female reproduction system, which lasts 11 months. The development of spermary started before the development of ovary. When egg cell is only a primordial germ cell, sperm has an initially complete form. Meanwhile, sperm cells and egg cells orderly development and synchronously mature. According to the development of sperm cells and egg cells, the development of cycle of the spermary could be divided into 6 stages: proliferating stage (1-3 months of age), growing stage (4-5 months of age), resting stage (6-8 months of age), maturing stage (9 months of age), spawning stage (10 months of age) and degradation stage (11 months of age). The development of cycle of the ovary could be divided into 6 stages: forming stage (1-2 months of age), proliferating stage (3-4 months of age), growing stage (5-8 months of age), maturing stage (9 months of age), spawning stage (10 months of age) and resting stage (11 months of age). W. pigra is a synchronous hermaphrodite animal, the development of cycle of the spermary and ovary each has six stages, sperm cells and egg cells orderly development and synchronously mature.
Animals ; Female ; Gonads ; cytology ; Leeches ; growth & development ; Male ; Ovary ; cytology ; Ovum ; cytology ; Reproduction ; Spermatocytes ; cytology

OBJECTIVETo investigate the effects of single heat stress treatment on spermatogenic cells in mice.

METHODSWe randomly divided 36 C57 male mice into a control and a heat stress treatment group and submerged the lower part of the torso in water at 25 °C and 43 °C, respectively, both for 15 minutes. At 1, 7, and 14 days after treatment, we obtained the testicular organ indexes, observed the changes in testicular morphology by HE staining, and determined the location and expression levels of the promyelocytic leukemia zinc finger (PLZF) and synaptonemal comlex protein-3 (SCP-3) in the testis tissue by immunohistochemistry and Western blot.

RESULTSThe testicular organ index was significantly lower in the heat stress treatment than in the control group (P < 0.05). Compared with the controls, the heat shock-treated mice showed loosely arranged spermatogenic cells scattered in the seminiferous tubules at 1 day after heat stress treatment, atrophied, loosely arranged and obviously reduced number of spermatogenic cells at 7 days, and relatively closely arranged seminiferous tubules and increased number and layers of spermatogenic cells at 14 days. The number of SCP-3 labelled spermatocytes obviously decreased in the heat stress-treated animals at 1 and 7 days and began to increase at 14 days. The PLZF protein expression was significantly reduced in the heat stress treatment group at 1 day as compared with that in the control (0.19 ± 0.12 vs 0.64 ± 0.03, P < 0.01), but elevated to 0.77 ± 0.02 at 7 and 14 days, even remarkably higher than in the control animals (P < 0.01).

CONCLUSIONHeat stress treatment can induce short-term dyszoospermia in mice, which can be recovered with the prolonged time after treatment.

Animals ; Blotting, Western ; Hot Temperature ; Immunohistochemistry ; Male ; Mice ; Nuclear Proteins ; metabolism ; Promyelocytic Leukemia Protein ; Seminiferous Tubules ; cytology ; Spermatocytes ; cytology ; pathology ; Testis ; metabolism ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism

Uchl1 was found to be involved in spermatocyte apoptosis. The aim of the present study was to test whether Uchl1 and its associated proteins Jab1 and p27(kip1) were involved in spermatogenic damages in response to heat-stress in cryptorchidism. Hematoxylin and eosin (HE) staining and DNA end labeling (TUNEL) were used to observe morphological and apoptotic characteristics of spermatogenic cells; Immunohistochemical analysis was used to detect changes of Uchl1 and its associated proteins Jab1 and p27(kip1) in response to heat-stress from cryptorchidism leading to spermatocyte losses; And protein affinity analysis (pull-down) and immunofluorescence co-localization were used to verify the relevance among the three proteins in spermatocytes. The results showed that, Jab1 and p27(kip1), in parallel to Uchl1, increased in spermatocytes of apoptotic appearances in response to heat-stress, but not in multinucleated giant cells; Jab1 bound to Uchl1 in testis protein extracts, and co-localized with Uchl1 and p27(kip1) specifically in spermatocytes with apoptotic appearances. These results suggest that the accumulation of Uchl1 protein is involved in the heat-stress-induced spermatocyte apoptosis through a new pathway related with Jab1 and p27(kip1), but not the formation of multinucleated giant cells.
Animals ; Apoptosis ; COP9 Signalosome Complex ; Cryptorchidism ; pathology ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Hot Temperature ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Mice ; Peptide Hydrolases ; metabolism ; Spermatocytes ; cytology ; metabolism ; Stress, Physiological ; Ubiquitin Thiolesterase ; metabolism

OBJECTIVETo study the promoting effects of the epidermal growth factor (EGF) and stem cell factor (SCF) on the proliferation and differentiation of spermatogenic cells in mice.

METHODSWe cocultured in vitro spermatogenic cells of male mice aged 7 - 8 days in the medium with EGF and/or SCF at the concentrations of 5, 10, 20, 40 and 100 ng/ml, respectively. Then we observed the survival rate and morphological changes of the spermatogenic cells, detected the expressions of the pachytene-specific phosphoprotein gene (P19) and haploid sperm cell-specific transition protein gene (TP1), and analyzed the ploidy of the cells.

RESULTSAfter cocultured with EGF or SCF for 2 - 4 days, the spermatogenic cells began to proliferate in masses or chains in all concentration groups, most obviously in the 20 ng/ml EGF and 40 ng/ml SCF groups. At 7 days, both the number and survival rate of spermatogenic cells were significantly higher in the 20 ng/ml EGF and 40 ng/ml SCF groups than in the others (P < 0.05), and meanwhile, the P19/TP1 ratio was obviously decreased and the rate of haploid sperm markedly increased in the 40 ng/ml SCF group (P < 0.05). Combination of EGF and SCF remarkably promoted the proliferation of the spermatogenic cells (P < 0.05).

CONCLUSIONBoth EGF and SCF could increase the number and survival rate of spermatogenic cells. SCF could promote the formation of haploid sperm, and the combination of the two cytokines could enhance the effect on the proliferation of spermatogenic cells.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chromosomal Proteins, Non-Histone ; metabolism ; Epidermal Growth Factor ; pharmacology ; Male ; Mice ; Spermatocytes ; cytology ; drug effects ; Stem Cell Factor ; pharmacology

OBJECTIVETo construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.

METHODSPIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot.

RESULTSWe successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.

CONCLUSIONThe construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.

Animals ; Cell Line ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Mice ; Plasmids ; Protein Inhibitors of Activated STAT ; genetics ; Spermatocytes ; cytology ; Transfection

OBJECTIVETo primarily study the influence of recombination abnormality in human spermatocyte meiosis on the pathology of the patients with non-obstructive azoospermia (NOA).

METHODSWe obtained testis tissues from 6 NOA patients by testicular biopsy and divided the tissue of each patient into 2 portions, one for pathological examination and the other for immunofluorescent staining. We observed the synaptonemal complex and the numbers of the recombination sites on homologous chromosomes, and analyzed the relationship between abnormal recombination and pathological findings.

RESULTSPathological examination showed that the basement membrane of the seminiferous tubules was thickened in 3 of the cases and atrophied in the other 3, the number of autosomal MLH1 foci in a spermatocyte ranging from 10 to 50 in the former 3, and from 30 to 50 in the latter 3.

CONCLUSIONThe increased range of the homologous chromosomal recombination frequency may be one of the possible factors for the thickening of seminiferous tubule basement membrane and even lumen occlusion in NOA patients.

Adult ; Azoospermia ; genetics ; pathology ; Chromosome Aberrations ; Humans ; Male ; Meiosis ; Recombination, Genetic ; Spermatocytes ; cytology ; Young Adult

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEA-treated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.
Animals ; Antigens, CD95/*immunology ; Apoptosis/*drug effects/immunology ; Estrogens, Non-Steroidal/*toxicity ; Fas Ligand Protein/*immunology ; Histocytochemistry ; Immunoblotting ; In Situ Nick-End Labeling ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatocytes/cytology/*drug effects/immunology ; Spermatogenesis/drug effects/immunology ; Spermatogonia/drug effects/immunology ; Testis/cytology/*drug effects/immunology ; Zearalenone/*toxicity

OBJECTIVETo investigate the expression of transient receptor potential melastatin (TRPM) and transient receptor potential vanilloid (TRPV) channel family genes in rat spermatogenic cells.

METHODSRat spermatogenic cells were isolated by a mechanical procedure and the total RNA was extracted using TRIzol reagent. TRPM and TRPV channel family genes were amplified by RT-PCR and the presence of the target genes was detected by agarose gel electrophoresis. The relative gene expression levels were measured by real-time quantitative RT-PCR.

RESULTSTRPV5, TRPM3, TRPM4 and TRPM7 mRNAs were expressed in rat spermatogenic cells, but TRPV1, TRPV2, TRPV3, TRPV4, TRPV6, TRPM1, TRPM2, TRPM5, TRPM6, TRPM7 and TRPM8 mRNAs were not detected. The relative expressions of TRPM and TRPV mRNA were determined by quantitative real-time RT-PCR. TRPM7 expression was the highest among all the TRPM subtypes in rat spermatogenic cells, at a level equivalent to (0.0430-/+0.0034)% of beta-actin expression. TRPM3 and TRPM4 were also highly expressed, but their expression levels were only approximately 56% and 63% of that of TRPM7, respectively. For the TRPV subfamily, only TRPV5 mRNA was abundantly expressed at the level of (0.0157-/+0.0029)% relative to that of beta-actin.

CONCLUSIONTRPV5, TRPM3, TRPM4 and TRPM7 mRNAs were coexpressed in spermatogenic cells in rats, among which TRPM4 and TRPM7 mRNA were expressed at high levels. TRPM4 and TRPM7 channels may be involved in the regulation of growth, differentiation and maturation of rat spermatogenic cells and are associated with the generation of the sperms.

Animals ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spermatocytes ; cytology ; metabolism ; Spermatogonia ; cytology ; metabolism ; TRPM Cation Channels ; genetics ; metabolism ; TRPV Cation Channels ; genetics ; metabolism

There has been no study aimed at directly determiningof the periods of Sertoli cell proliferation in birds evendomestic fowl. The aims of this study were to observe thecessation of post-hatching mitotic proliferation of Sertolicells in domestic fowl, and to determine the volumedensity of Sertoli and germ cells during this period. Atotal of 50 Leghorn chicks were used in this study. Thetestes sections of the animals were immunostained withBrdU to observe the proliferation of cells from one to 10weeks of age. The volume density of the Sertoli and germcells were determined using the standard point countingmethod. The volume density of the germ cell nuclei wasinitially less than that of the Sertoli cells but the volumedensity converged by week 6, and remained relativelyconstant until the commencement of meiosis. Clearlabeling of Sertoli and germ cells was observed from week1 to week 7. The only those cells still labeled after 8 weekswere germ cells, indicating that Sertoli cell proliferationhad ceased. Therefore, it is recommended that anyresearch into the testes of domestic fowl should considerthe cessation of Sertoli cell proliferation by approximately8 weeks.
Animals ; Bromodeoxyuridine/metabolism ; Cell Differentiation/physiology ; Cell Growth Processes/physiology ; Chickens/*physiology ; Histocytochemistry/veterinary ; Male ; Mitosis/physiology ; Sertoli Cells/*cytology/metabolism ; Spermatocytes/cytology ; Testis/*cytology/metabolism

OBJECTIVETo investigate the effects of Jujingwan on the spermatozoal ultrastructure and apoptosis of germ cells in oligospermia patients.

METHODSWe treated 50 oligospermia patients with Jujingwan and observed the spermatozoal ultrastructure, the apoptosis of germ cells and the changes in the DNA ploidy proportion of spermatogenic cells by electron microscopy and FCM before the treatment and 3, 6, 9 and 12 months after it.

RESULTSJujingwan increased sperm acrosome base density 6 months after the treatment and remarkably improved the integrity of acrosome membrane 12 months after it, with no obvious pathological changes in the nuclei and tails. Three months after the treatment, cell debris and apoptotic cells decreased significantly as compared with pre-treatment (P < 0. 05) , and very significantly 12 months after the treatment (P <0. 01). The proportion of haploid spermatozoa increased very significantly (P <0.01) , and the lost primary spermatocytes decreased significantly (P <0. 05) compared with pre-treatment.

CONCLUSIONJujingwan can increase the density of sperm acrosome base and improve the pathological changes of acrosome membrane in oligospermia patients; it can improve the activity of acrosome enzyme and the integrity of acrosome membrane, decrease the apoptosis rate of germ cells and sperm and increase the percentage of haploid spermatozoa; it can also reduce the percentage of apoptotic bodies and diploid sperm cells. It is indicated that Jujingwan can inhibit the apoptosis of germ cells and sperm and improve spermatogenesis in oligospermia patients.

Acrosome ; pathology ; Adult ; Apoptosis ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Infertility, Male ; drug therapy ; pathology ; Male ; Oligospermia ; drug therapy ; pathology ; Phytotherapy ; Sperm Count ; Spermatocytes ; cytology ; Spermatozoa ; ultrastructure