Nanos2, a member of the Nanos2 gene family, is a specific gene in male germ cells and encodes an evolutionarily conserved RNA binding protein expressed in male primordial germ cells (PGCs) during the embryonic period as well as in the spermatogonial stem cells (SSCs) of the testis. In the embryonic period, Nanos2 promotes the development of male PGCs and inhibits them from meiosis. In the process of spermatogenesis, Nanos2 suppresses the differentiation of SSCs in the testis and maintains the stability of the SSC pool. The knockout of Nanos2 may cause the disappearance of germ cells and sterility in male mice while its overexpression in the testis may lead to accumulation of SSCs in seminiferous tubules. Besides, Nanos2 is involved in the degradation of specific RNAs and possibly associated with some diseases of the male reproductive system. This review focuses on the recent progress in the studies of Nanos2 in the male reproductive system.
Animals ; Cell Differentiation ; Gene Knockout Techniques ; Male ; Meiosis ; Mice ; RNA ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Spermatogenesis ; physiology ; Spermatogonia ; Spermatozoa ; Testis ; cytology

BACKGROUNDIt is still controversial whether the serum inhibin B level is a superior predictor of the presence of sperm in testicular sperm extraction (TESE) in azoospermic men compared with serum follicle-stimulating hormone (FSH). In this study, we evaluated the diagnostic accuracy of serum inhibin B levels as a predictor of the outcome of TESE in Chinese non-obstructive azoospermic men and compared it with the traditional marker serum FSH and testicular volumes.

METHODSBasal values of serum hormone levels, testicular volumes and histological evaluation of 305 Chinese non-obstructive azoospermic men were analyzed. The level of inhibin B was measured using a three-step enzyme-linked immunoassay before sperm extraction, and the diagnostic accuracy of prediction of the outcome of TESE was compared for different markers by the receiver operating characteristics (ROC) curve analysis.

RESULTSTesticular sperm was successfully retrieved in 137 of 305 patients (44.9%). The serum level of inhibin B, the FSH and the testicular volume were significantly different between the successful TESE group and the unsuccessful group. According to the ROC curve analysis, for inhibin B, the cut-off value for discriminating between successful and failed TESE was 28.39 pg/ml (sensitivity 83.5%, specificity 79.1%). For FSH, the best cut-off value for discriminating was 11.05 pg/ml (sensitivity 83.5%, specificity 74.5%). The area under the ROC curve of serum inhibin B was similar to that of FSH. Combining the serum inhibin B with FSH levels did not improve the predictive value for successful TESE.

CONCLUSIONSSerum inhibin B and FSH levels are correlated with spermatogenesis. However, inhibin B is not superior to FSH in predicting the presence of sperm in TESE. And the combination of them does not improve the diagnostic accuracy on TESE outcome.

Adult ; Azoospermia ; blood ; Biomarkers ; blood ; Follicle Stimulating Hormone ; blood ; Humans ; Inhibins ; blood ; Male ; Sperm Retrieval ; Spermatogenesis ; physiology ; Testis ; cytology

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and alpha-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both alpha-SMA and SM22-alpha proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.
Animals ; Animals, Newborn ; Biological Markers/metabolism ; Cell Differentiation/physiology ; Embryonic Stem Cells/cytology/physiology ; Endothelial Cells/*cytology/physiology ; Gene Expression ; Gene Expression Profiling ; Humans ; Immunohistochemistry ; Male ; Mice ; Muscle, Smooth, Vascular/*cytology/physiology ; Myocytes, Smooth Muscle/*cytology/physiology ; Pluripotent Stem Cells/*cytology/physiology ; Real-Time Polymerase Chain Reaction ; Spermatogonia/*cytology/physiology ; Testis/*cytology/physiology

Germ cells make two major decisions when they move from an indeterminate state to their final stage of gamete production. One decision is sexual commitment for sperm or egg production, and the other is to maintain mitotic division or entry into meiosis. It is unclear whether the two decisions are made as a single event or separate events, because there has been no evidence for the presence of germ cell sex prior to meiosis. Here we report direct evidence in the fish rainbow trout that gonia have distinct sexuality. We show that dazl expression occurs in both male and female gonia but exhibits differential intracellular distribution. More strikingly, we show that boule is highly expressed in male gonia but absent in female gonia. Therefore, mitotic gonia possess sex, sperm/egg decision and mitosis/meiosis decision are two independent events, and sperm/egg decision precedes mitosis/meiosis decision in rainbow trout, making this organism a unique vertebrate model for mechanistic understanding of germ cell sex differentiation and relationship between the two decisions.
Animals ; Female ; Fish Proteins ; genetics ; Gene Expression Regulation ; Male ; Meiosis ; Oncorhynchus mykiss ; genetics ; physiology ; Ovary ; cytology ; metabolism ; Ovum ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA-Binding Proteins ; genetics ; Sex Determination Processes ; Spermatozoa ; cytology ; metabolism ; Testis ; cytology ; metabolism

The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein (GFP)-expressing recombinant baculovirus (GFP-baculovirus), in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus (CMV)-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineal injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction (RT-PCR), and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. Intra-tunica albuguineal injection of GFP-baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein (IGFBP)-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intra-tunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.
Animals ; Baculoviridae ; genetics ; COS Cells ; Cercopithecus aethiops ; Cytomegalovirus ; genetics ; Gene Expression ; physiology ; Gene Transfer Techniques ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Injections ; Insulin-Like Growth Factor Binding Protein 5 ; genetics ; metabolism ; Male ; Mice ; Microscopy, Fluorescence ; Promoter Regions, Genetic ; genetics ; Rats ; Testis ; cytology ; physiology

AIMTo characterize mouse capping protein alpha3 (CPalpha3) during spermatogenesis and sperm maturation.

METHODSWe produced rat anti-CPalpha3 antiserum and examined the expression of CPalpha3 in various mouse tissues using Western blot analysis and the localization of CPalpha3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPalpha3 and beta-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPalpha3 antiserum and anti-actin antibody.

RESULTSWestern blot analysis using specific antiserum revealed that CPalpha3 was expressed specifically in testes. Interestingly, the molecular weight of CPalpha3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPalpha3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPalpha3 and ACTB localization was virtually identical to the localization before the acrosomal reaction.

CONCLUSIONCPalpha3 might play an important role in sperm morphogenesis and/or sperm function.

Acrosome Reaction ; physiology ; Actins ; metabolism ; Animals ; Blotting, Western ; CapZ Actin Capping Protein ; metabolism ; Cells, Cultured ; Epididymis ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sperm Head ; metabolism ; Sperm Tail ; metabolism ; Spermatogenesis ; physiology ; Spermatozoa ; cytology ; metabolism ; Testis ; cytology ; metabolism

AIMTo show whether molecular motor dynein on a microtubule track, molecular motor myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interacting protein (MyRIP), and vesicle receptor Rab27b on an F-actin track were present during human and monkey spermiogenesis involving intramanchette transport (IMT).

METHODSSpermiogenic cells were obtained from three men with obstructive azoospermia and normal adult cynomolgus monkey (Macaca fascicularis). Immunocytochemical detection and reverse transcription-polymerase chain reaction (RT-PCR) analysis of the proteins were carried out. Samples were analyzed by light microscope.

RESULTSUsing RT-PCR, we found that dynein, myosin Va, MyRIP and Rab27b were expressed in monkey testis. These proteins were localized to the manchette, as shown by immunofluorescence, particularly during human and monkey spermiogenesis.

CONCLUSIONWe speculate that during primate spermiogenesis, those proteins that compose microtubule-based and actin-based vesicle transport systems are actually present in the manchette and might possibly be involved in intramanchette transport.

Actins ; metabolism ; Adult ; Animals ; Biological Transport ; physiology ; Dyneins ; metabolism ; Humans ; Macaca fascicularis ; Male ; Microtubules ; metabolism ; Myosin Heavy Chains ; metabolism ; Myosin Type V ; metabolism ; Myosins ; metabolism ; Spermatids ; cytology ; metabolism ; Spermatogenesis ; physiology ; Testis ; cytology ; metabolism ; Transport Vesicles ; physiology ; Vesicular Transport Proteins ; metabolism ; rab GTP-Binding Proteins ; metabolism

AIMTo assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone (FSH) or both agents given together.

METHODSFrom postnatal day (PND) 5 to 15 male rats were daily injected with 12.5 microg of 17beta-estradiol benzoate (EB) or 7.5 IU of human purified FSH (hFSH) or EB + hFSH or solvents (control). On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the seminiferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUNEL method.

RESULTSAlthough EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB + hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB + hFSH inhibited Sertoli cell proliferation. All treatments significantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB + hFSH.

CONCLUSIONAt puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Estradiol ; pharmacology ; Follicle Stimulating Hormone ; pharmacology ; Male ; Rats ; Rats, Wistar ; Seminiferous Tubules ; cytology ; drug effects ; Sexual Maturation ; drug effects ; physiology ; Spermatogenesis ; drug effects ; physiology ; Spermatozoa ; cytology ; drug effects ; Testis ; cytology ; drug effects

AIMto investigate the effects of crude garlic on adult male rat reproductive functions.

METHODSThirty male rats were divided into five groups: group 1 (untreated) and groups 2, 3, 4 and 5 were fed for 30 days with 5%, 10%, 15% and 30% crude garlic, respectively. Testes and accessory organs were weighed and some markers were assessed. Light and electron microscopy observations were also performed.

RESULTSA significant decrease was observed in the body weight of groups 4 (14%; P < 0.01) and 5 (20%; P < 0.01); of the prostate weight in group 5 (29.1%; P < 0.05) and of seminal vesicle weight in groups 3 (14.4%; P < 0.01), 4 (18.3%; P < 0.01) and 5 (27.3%; P < 0.01). In contrast, testis and epididymis weights were unchanged. In epididymis tissue, the alpha glucosidase activity and the spermatozoa density were unchanged. The treatment resulted in a significant decrease in testosterone serum levels in groups 3 (77.3%; P < 0.01), 4 (77.3%; P < 0.01) and 5 (90.9%; P < 0.01), associated with a significant increase in LH serum levels (P < 0.01). Testicular histology showed a dose-dependent increase in the percentage of empty seminiferous tubules. Moreover, testicular function was affected; a significant decrease in phosphatase acid activity (P < 0.01) and testosterone (P < 0.05) contents were observed.

CONCLUSIONCrude garlic consumption during 1 month reduced testosterone secretion and altered spermatogenesis at 10%, 15% and 30% doses.

Animals ; Dose-Response Relationship, Drug ; Epididymis ; drug effects ; physiology ; Garlic ; adverse effects ; Leydig Cells ; drug effects ; physiology ; Luteinizing Hormone ; blood ; Male ; Plant Preparations ; pharmacology ; Prostate ; drug effects ; physiology ; Rats ; Rats, Wistar ; Reproduction ; drug effects ; physiology ; Seminal Vesicles ; drug effects ; physiology ; Sertoli Cells ; drug effects ; physiology ; Sperm Count ; Spermatogenesis ; drug effects ; physiology ; Testis ; cytology ; drug effects ; metabolism ; Testosterone ; blood

This article introduces the structure and function of the Sertoli cell cytoskeleton of the testis and the research progress in this aspect, focusing on the description of the function of vimentin, with some illustrations on the impact of physical and chemical factors on cytoskeleton, especially the structural changes of vimentin cell microfilament under simulated microgravity and space true microgravity. It for the first time proposes that the Sertoli cell cytoskeleton can be detected in semen, with a view to involving more researchers in further studies in this field.
Actin Cytoskeleton ; metabolism ; physiology ; Animals ; Apoptosis ; physiology ; Cytoskeleton ; metabolism ; physiology ; Humans ; Male ; Mice ; Rats ; Semen ; cytology ; metabolism ; Sertoli Cells ; cytology ; metabolism ; Testis ; cytology ; metabolism ; Vimentin ; metabolism