During spermatogenesis, developing germ cells that lack the cellular ultrastructures of filopodia and lamellipodia generally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These include the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell-cell and Sertoli-germ cell interface also undergo rapid remodeling, involving disassembly and reassembly of cell junctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the involving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protein S6 (rpS6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mTORC1/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubule-based cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.
Actins/metabolism* ; Animals ; Blood-Testis Barrier/metabolism* ; Cells, Cultured ; Male ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Permeability ; Rats ; Ribosomal Protein S6/metabolism* ; Seminiferous Epithelium/metabolism* ; Sertoli Cells/metabolism* ; Signal Transduction/physiology*

Cimetidine is an H2 receptor antagonist that has an antiandrogenic effect. It intervenes with the conversion of testosterone into estrogen in the Sertoli cells with accompanying testicular structural changes. In the present study, the microscopic and the ultrastructural changes induced by cimetidine and the effect of vitamin B12 as a protective agent on rat testes were studied. Immunoexpression of estrogen receptor β (ERβ) in testes was evaluated. Twenty-four adult male rats were divided into four groups: control, cimetidine-treated, vitamin B12 treated, and combined cimetidine and vitamin B12 treated. The experimental rats were administered with cimetidine and/or vitamin B12 for 52 days. Group II rats showed marked atrophy of the seminiferous tubules with a significant increase in tubular diameter and decrease in the tubular luminal and epithelial areas. Ultrastructure of this group showed irregular Sertoli cells with basal cytoplasmic vacuolation and significantly thickened basement membrane. ERβ immunoexpression was similar to controls. Group III rats showed near normal seminiferous tubular structures with minimal cellular alterations and the immunoreactivity of the testicular sections was very close to normal. However, group IV rats showed markedly immunopositive detached cells, spermatids, and primary spermatocytes. Cimetidine interferes with the control of spermatogenesis as evidenced by microscopic and ultrastructural studies and affection of ERβ receptors and vitamin B12 has a protective action against this harmful effect.
Adult ; Animals ; Basement Membrane ; Cimetidine ; Cytoplasm ; Estrogens ; Humans ; Male ; Phenobarbital ; Rats* ; Seminiferous Epithelium* ; Seminiferous Tubules ; Sertoli Cells ; Spermatids ; Spermatocytes ; Spermatogenesis ; Testis ; Testosterone ; Vitamin B 12* ; Vitamins*

BACKGROUND: Mesenchymal stem cells (MSCs), have been suggested as a potential choice for treatment of male infertility. Yet, the effects of MSCs on regeneration of germinal epithelium of seminiferous tubules and recovery of spermatogenesis have remained controversial. In this research, we have evaluated and compared the fate of autologous bone marrow (BM)-MSCs during three different periods of time- 4, 6 and 8 weeks after transplantation into the testes of busulfan-induced infertile male rats. METHODS: Rats BM samples were collected from tibia bone under anesthesia. The samples were directly cultured in culture medium. Isolated, characterized and purified BM-MSCs were labeled with PKH26, and transplanted into the testes of infertile rats. After 4, 6 and 8 weeks, the testes were removed and underwent histological evaluations. RESULTS: Immunohistochemical analysis showed that transplanted BM-MSCs survived in all three groups. Some of the cells homed at the germinal epithelium and expressed spermatogonia markers (Dazl and Stella). The number of homed spermatogonia-like cells in 4-week testes, was more than the 6-week testes. The 8-week testes had the least numbers of homed cells (p<0.05). Immunostaining for vimentin showed that BM-MSCs did not differentiate into the sertoli cells in the testes. CONCLUSIONS: From our results, it could be concluded that, autologous BM-MSCs could survive in the testis, migrate onto the seminiferous tubules basement membrane and differentiate into spermatogonia. Although, no more differentiation was observed in the produced spermatogonia, generation of such endogenous GCs would be a really promising achievement for treatment of male infertility using autologous stem cells.
Anesthesia ; Animals ; Basement Membrane ; Bone Marrow* ; Epithelium ; Germ Cells* ; Humans ; Infertility, Male ; Male* ; Mesenchymal Stromal Cells* ; Rats* ; Regeneration ; Seminiferous Tubules ; Sertoli Cells ; Spermatogenesis ; Spermatogonia ; Stem Cells ; Testis* ; Tibia ; Transplantation ; Vimentin

The present study examined the efficacy of Ocimum basilicum (basil) extract, a natural herb, with antioxidant properties, against testicular toxicity induced by cadmium (Cd), which is one of the most important toxic heavy metals. The intoxicated rats showed significant alterations in the testicular tissue including decreased seminiferous epithelium height and changes in the arrangement of spermatogenic layers. Hypospermatogensis with cytoplasmic vacuolization and pyknotic nuclei were observed. Intertubular hemorrahage and absence of spermatozoa were noted. Decreased cell proliferation was reflected by a decrease in Ki-67 expression, whereas the increase in apoptotic rate was associated with a decrease in the Bcl/Bax ratio. Concomitant treatment with aqueous basil extract led to an improvement in histological, morphometrical and immunohistochemical changes induced by Cd. The beneficial effects of basil extract could be attributed to its antioxidant properties.
Animals ; Apoptosis ; Cadmium ; Cell Proliferation ; Cytoplasm ; Metals, Heavy ; Ocimum ; Ocimum basilicum ; Rats ; Seminiferous Epithelium ; Spermatozoa ; Testis

OBJECTIVE: Concerns are growing about the decrease in male reproductive health. Caffeine is one of the popular nutrients that has been implicated as a risk factor for infertility. In the present study, we examined whether in utero and lactational exposure to caffeine affects the reproductive function of the offspring of rats. METHODS: Pregnant rats received caffeine via drinking water during gestation (26 and 45 mg/kg) and lactation (25 and 35 mg/kg). Body and reproductive organ weight, seminiferous tubule diameter, germinal epithelium height, sperm parameters, fertility rate, number of implantations, and testosterone level of the offspring were assessed from birth to adulthood. RESULTS: Significant dose-related decreases were observed in the body and reproductive organ weight, seminiferous tubule diameter, and germinal epithelium height of the offspring. Sperm density had declined significantly in offspring of the low-dose and high-dose groups, by 8.81% and 19.97%, respectively, by postnatal day 150. The number of viable fetuses had decreased significantly in females mated with male offspring of the high-dose group at postnatal days 60, 90, 120, and 150. There were also significant reductions in testosterone levels of high-dose group offspring from birth to postnatal day 150. CONCLUSION: It is concluded that maternal caffeine consumption impairs gonadal development and has long-term adverse effects on the reproductive efficiency of male offspring rats.
Animals ; Birth Rate ; Caffeine ; Drinking Water ; Epithelium ; Female ; Fertility ; Fetus ; Gonads ; Growth and Development ; Humans ; Infertility ; Lactation ; Male ; Organ Size ; Parturition ; Pregnancy ; Rats ; Reproduction ; Reproductive Health ; Risk Factors ; Seminiferous Tubules ; Spermatozoa ; Testis ; Testosterone

OBJECTIVETo explore the effects of different methods of scrotal reconstruction on the apoptosis of spermatogenic cells and expression of the bcl-2 protein in patients with third-degree scrotal burns.

METHODSForty male and 24 female 2-month-old Guizhou mini-pigs were used in this study, the former equally randomized to groups I (normal control), II (natural healing), III (skin grafting) and IV (skin flap grafting). Ten months after the establishment of the model of third-degree burns, 6 male pigs from each group were paired with the female pigs and fed for 3 weeks. Then the female pigs were fed for another 4 months, followed by observation of their reproductivity. At 12 months, the bilateral testes were taken from the male pigs for detection of the apoptosis index of spermatogenic cells by TUNEL and determination of the expression of the bcl-2 protein by immunohistochemistry. The data obtained were subjected to single factor analysis of variance.

RESULTSThe apoptosis indexes of spermatogenic cells were (7.07 +/- 3.5), (40.34 +/- 4.85), (15.14 +/- 1.36) and (39.29 +/- 5.73)% in groups I , II, III and IV, respectively, significantly higher in groups II , III and IV than in I (P<0.05), with statistically significant differences between group III and groups II and IV (P<0.05) but not between II and IV (P>0.05). The expression rates of the bcl-2 protein were (75.07 +/- 3.74), (54.93 +/- 4.03), (66.85 +/- 3.06) and (53.33 +/- 5.22)% in groups I, II, III, and IV, respectively, remarkably higher in I than in the other three (P<0.05), with significant differences between group III and groups II and IV (P<0.05) but not between II and IV (P>0.05). Pregnancies were found in all the female pigs of group I with 10.0 +/- 1.18 newborns and in 4 of group III with 9.92 +/- 1.31 newborns, but in none of groups II and IV, with significant differences between group I and the other three (P<0.05) as well as between group III and groups II and IV (P<0.05), but not between II and IV (P>0.05).

CONCLUSIONAll the three methods of reconstruction for the scrotum with third-degree burns can suppress spermatogenic function, increase the apoptosis of spermatogenic cells and decrease the expression of the bcl-2 protein, among which, skin grafting least affects spermatogenic function.

Animals ; Apoptosis ; Burns ; surgery ; Disease Models, Animal ; Female ; Male ; Reconstructive Surgical Procedures ; methods ; Scrotum ; injuries ; metabolism ; surgery ; Seminiferous Epithelium ; cytology ; metabolism ; Skin Transplantation ; methods ; Spermatogenesis ; Swine ; Swine, Miniature

OBJECTIVETo explore a simple and feasible technique to locate proteins during spermatogenesis.

METHODSVarious germ cells and somatic cells were separated by collagenase I and DNase I after the albuginea was removed. The cells were then smeared, dyed, and observed directly under fluorescence and confocal microscopy.

RESULTSGerm cells at different steps were successfully identified by specific dyestuffs for acrosome and nucleolus.

CONCLUSIONA simple method for locating proteins during spermatogenesis was successfully developed.

Animals ; Cell Separation ; methods ; Cells, Cultured ; Male ; Mice ; Seminiferous Epithelium ; cytology ; Spermatozoa ; cytology

Spermatogenesis is a particularly difficult process to study the unique multiple cellular associations within the seminiferous epithelium. Laser capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. The superoxide dismutase (SOD) is the first and most important enzyme of antioxidant defense systems against superoxide anion. The aim of this study was to investigate the quantitative changes of SOD gene expression according to the spermatogenic cycle in mouse testes using LCM and real-time polymerase chain reaction (PCR) techniques. Frozen sections (10 micrometer) were obtained from the testes of 8-weeks-old ICR mice. LCM was used to capture all cells in cross-sectioned seminiferous tubules which were grouped into stages I-V, VII-VIII, and IX-XI. The expression level of cytoplasmic Cu, Zn-SOD (SOD1) mRNA was remarkably higher than those of mitochondrial Mn-SOD (SOD2) and extracellular Cu, Zn-SOD (SOD3) mRNAs in mouse testes. During spermatogenesis, the expressions of SOD1 and SOD2 mRNAs were highest on stages I-V, began to decrease after stage VII, and showed a lowest level on stage IX-XI. However, the expression of SOD3 mRNA was highest on stages VII-VIII. These findings suggest that the subtypes of SOD are expressed differentially in mouse testes during spermatogenesis.
Animals ; Cytoplasm ; Frozen Sections ; Gene Expression ; Laser Capture Microdissection ; Mice ; Mice, Inbred ICR ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Seminiferous Epithelium ; Seminiferous Tubules ; Spermatogenesis ; Superoxide Dismutase ; Superoxides ; Testis

OBJECTIVETo investigate the impact of experimental varicocele (EV) on the ipsilateral testis in rats.

METHODSEV was induced by partial ligation of the left renal vein in male SD rats, the control rats subjected to sham operation, and the testes of the EV models and controls were extirpated 6, 12, and 18 weeks later. Johnson's score, ultrastructure of seminiferous tubules, intratesticular testosterone concentration (ITC) and germ cell apoptotic index (AI) of each left testis were evaluated.

RESULTSJohnson's scores were (6.92 +/- 0.52), (4.83 +/- 0.41) and (2.95 +/- 0.26), ITCswere (6.32 +/- 0.85), (5.17 +/- 0.76) and (4.11 +/- 0.69) and AIs were (5.32 +/- 1.23), (15.21 +/- 0.97) and (21.13 +/- 1.12) respectively in the 6 w , 12 w and 18 w EV groups, significantly lower than in the corresponding control groups, (9.56 +/- 0.35, 9.63 +/- 0.31, 9.39 +/- 0.46), (9.64 +/- 1.23, 9.38 +/- 0.69, 9.73 +/- 0.49) and (3.21 +/- 1.15, 3.43 +/- 1.21, 3.61 +/- 1. 15) (P < 0.05), the former two showing a gradual decline while the latter a significant elevation with the increasing duration of varicocele. The damage to the ultrastructure of seminiferous tubules was aggravated with the prolonging of varicocele.

CONCLUSIONEV can cause a progressive decline of ITC, dyszoospermia and increased AI of germ cells.

Animals ; Apoptosis ; Disease Models, Animal ; Infertility, Male ; Male ; Rats ; Rats, Sprague-Dawley ; Seminiferous Epithelium ; cytology ; ultrastructure ; Testis ; chemistry ; metabolism ; Testosterone ; metabolism ; Varicocele ; metabolism ; physiopathology

OBJECTIVETo explore the apoptosis and the express of proliferating cell nuclear antigen (PCNA) in spermatogenic cells, and study generation function of the rabbit after scrotal reconstruction with flaps.

METHODSThe 48 New Zealand white rabbits were randomly divided into three groups as experimental group (the scrotum reconstructed with flaps, n = 48), the control group (the sham operated group, n = 24) and the blank group (n = 18). The apoptosis and the expression of PCNA in the spermatogenic cells were detected with TUNEL and the immunohistochemistry from the 3rd to the 8th week after operation. 8 weeks later, 12 animals in each group were fed respectively with one female rabbit to observe the procreation.

RESULTSThe apoptotic index of the spermatogenic cells in blank group was 7.73 +/- 4.95. 3 weeks after operation, the apoptotic index of spermatogenic cells was 22.59 +/- 3.04 in the experimental group, and 21.13 +/- 1.68 in control group, showing no significant difference between the two groups (P > 0.05). 8 weeks after operation, the apoptotic index of spermatogenic cells was 71.85 +/- 2.69 in the experimental group, and 13.64 +/- 2.09 in control group, show a significant difference between them (P < 0.05). The apoptotic index in experimental group increased gradually from the 3rd to 8th week after scrotal reconstruction , which was markedly higher than that in the blank group (P < 0.05). The apoptotic index in control group was higher than that in the blank group at the 3rd week (P < 0.05), but not at the 8th week (P > 0.05). The proliferation index of spermatogenic cells was 9.32 +/- 9.30 and 12.52 +/- 3.87 in experimental group at the 3rd and 4th week, respectively, which was significantly lower than that in blank group (43.07 +/- 2.25) and control group (45.69 +/- 4.98) at the 3rd week (P < 0.05). The proliferation index of spermatogenic cells was 46.98 +/- 18.92 and 49.53 +/- 9.79 in experimental group at the 7th and 8th week, respectively, 39.90 +/- 5.10 in control group at the 8th week, showing no difference between the two groups (P > 0.05). The proliferation index of spermatogenic cells in control group at the 3rd and 8th week was not different from that in the blank group (P > 0.05). The female pairing rabbits in the blank and control group were all pregnant, and the average childbirths were 6.0 +/- 1.28 and 5.92 +/- 1.31 respectively, with no difference between the two groups (P > 0.05). All the female pairing rabbits in the experimental group were not pregnant, showing a significant difference from those in the blank and control group (P < 0.05).

CONCLUSIONSThe rabbit generation functional disturbance after scrotal reconstruction with flaps is due to the excessive apoptosis of spermatogenic cell. The spermatogenic cell proliferation is affected only in the early postoperative period, but can recover later.

Animals ; Apoptosis ; Cell Proliferation ; Male ; Proliferating Cell Nuclear Antigen ; metabolism ; Rabbits ; Scrotum ; surgery ; Seminiferous Epithelium ; cytology ; Skin Transplantation ; Surgical Flaps