HUMAN SPERM MEMBRANE PROTEIN (hSMP-1): A DEVELOPMENTAL TESTIS-SPECIFIC COMPONENT DURING GERM CELL DIFFERENTIATION

Serum was obtained from an infertile woman having antibodies with sperm agglutinating activity. The antibodies interacted with a human sperm membrane protein (hSMP-1) with an estimated M_r of 55 kD. The gene (HSD-1) coding hSMP-1 was isolated from a human testis cDNA expression library and assigned the accession number U12978. The cDNA was conjugated to a prokaryotic expression vector to construct the recombinant vector, pRSET-HSD-I, which was expressed in Escherichia coli. The recombinant hSMP-1 was isolated and used to immunize rabbits to raise polyclonal antibodies. Using an immunocytochemical technique, hSMP-1 protein was immunolocalized in germ cells of human testis at all stages of spermatogenesis. mRNAs were prepared from 16 different human tissues and analyzed by Northern blot using HSD-1 as probe. A positive reaction was elicited only with testis mRNA. The present findings suggest that the expression of hSMP-1 gene is testis-specific and occurs during the early stages of germ cell differentiation. In a comparative study, the location of the hSMP-1 protein in sperm and in germ cells of the seminiferous tubules of rats was determined. The target antigen was immunolocated on the head and tail of rat sperm and in late spermatids and spermatozoa of rat testis. These results suggest that, in the rat, the HSD-1 gene is expressed during spermiogenesis.     

Role of the Y-located putative gonadoblastoma gene in human spermatogenesis

The gonadoblastoma locus on the human Y chromosome (GBY) is postulated to serve normal functions in spermatogenesis, but could exert oncogenic properties in predisposing susceptible germ cells to tumorigenesis in incompatible niches such as streaked gonads in XY sex reversed patients or dysfunctional testis in males. The testis-specific protein Y-linked (TSPY) repeat gene has recently been demonstrated to be the putative gene for GBY, based on its location on the GBY critical region, expression patterns in early and late stages of gonadoblastoma and ability to induce gonadoblastoma-like structures in the ovaries of transgenic female mice. Over-expression of TSPY accelerates G₂/M progression in the cell cycle by enhancing the mitotic cyclin B-CDK1 kinase activities. Currently the normal functions of TSPY in spermatogenesis are uncertain. Expression studies of TSPY, and its X-homologue, TSPX, in normal human testis suggest that TSPY is co-expressed with cyclin B1 in spermatogonia and various stages of spermatocytes while TSPX is principally expressed in Sertoli cells in the human testis. The co-expression pattern of TSPY and cyclin B1 in spermatogonia and spermatocytes suggest respectively that 1) TSPY is important for male spermatogonial cell replication and renewal in the testis; and 2) TSPY could be a catalyst/meiotic factor essential for augmenting the activities of cyclin B-cyclin dependent kinases, important for the differentiation of the spermatocytes in prophase I and in preparation for consecutive rounds of meiotic divisions without an intermediate interphase during spermatogenesis.     

INDUCTION OF SPERMATOGENESIS BY INGUINAL VARICOCELE REPAIR IN AZOOSPERMIC MEN

13 infertile patients who had complete azoospermia and clinical varicocele underwent inguinal varicocele repair. Semen analyses were obtained starting 3 months after varicocele repair. Bilateral varicocele repair in 2 men and unilateral in 11 men were performed. Induction of spermatogenesis was achieved in 3 (23%) patients. Two of them had hypospermatogenesis and one had maturation arrest at spermatid stage. No pregnancies by natural intercourse resulted. Although one couple used fresh ejaculate for intracytoplasmic sperm injection, the result was unsuccessful. All men with Sertoli cell-only and early maturation arrest remained azoospermic after surgery. No association between successful outcome and patient age, sex hormone analysis, varicocele grade, testicular volume, unilateral or bilateral varicocele repair were apparent. Varicocele repair can result in the induction of spermatogenesis for men with hypospermatogenesis and late maturation arrest. No other related factor could be detected.     

RELATIONSHIP BETWEEN MAST CELL AND INOS EXPRESSION IN TESTICULAR TISSUE ASSOCIATED WITH INFERTILITY

The objective of this study was to investigate mast cells and iNOS expression in testis tissue, and to correlate these results with spermatogenetic disorders. A total of 136 testicular biopsies were obtained from the testes of 80 patients with infertility. Their age ranged from 21 to 45 years. The biopsy specimens were immunohistochemically stained with antihuman tryptase for mast cells. In each section, all interstitial fields were evaluated for the total number of mast cells as well as the total number of Leydig cells. The number of mast cells per Leydig cell was calculated and recorded as mast cell index. Immunohistochemical iNOS staining was evaluated semiquantitatively according to intensity and the proportion of the stained cells. There was a significant increase of the mast cell index in all groups with testicular disorder compared with normal spermatogenesis group (p < 0.05). Increase of the index was in the order of hypospermatogenesis, maturation arrest and SCO, and index of SCO group was especially higher, i.e., more than twice than other groups. iNOS score was significantly higher in the SCO group than in the men with normal spermatogenesis, hypospermatogenesis, and maturation arrest (p < 0.05). Finally, a significant statistical correlation was found between the iNOS score and mast cells index (r = 0,758, p = 0,001). Increase of mast cell index was observed in the groups of infertile testis, and high expression of iNOS in Leydig cells was associated with the highest mast cell index in SCO, the lesion with the most severe damage of the germ cell.     

Cistanches herba induces testis cytotoxicity in male mice

We investigated the effects of Cistanches herba (CH) on the male reproductive system in mice, assessing CREM gene expression and spermatogenesis. Our results demonstrate that CH treatment lead to a significant decrease in sperm count dose-dependently, 298.3 ± 48.9 vs. 296.6 ± 102.4 (250 mg/kg), 236.7 ± 75.1 (500 mg/kg), 223.0 ± 48.7 × 10⁶ (1000 mg/kg), respectively. Additionally, serum testosterone levels decreased following CH treatment to as low as ~57% compared with the vehicle-treated group. CREM gene expression was also down-regulated following CH treatment and histological examination of the testicular seminiferous tubules showed severe damage on CH treatment. These results suggest that CH induces cytotoxicity in the male reproductive system, through the inhibition of spermatogenesis, testicular damage, and limited hormonal function.     

Association of interleukin-1beta C + 3953T gene polymorphism with human male infertility

Cytokines are involved in the regulation of spermatogenesis likely mediating the crosstalk among Sertoli and germ cells to facilitate germ cell movement across the seminiferous epithelium during cellular events such as germ cell differentiation. Members of the Interleukin-1 (IL-1) family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions. Interleukin-1 alpha (IL-1α), IL-1β, and the IL-1 antagonistic molecule (IL-1 Ra) are present in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of C?+?3953T polymorphism of the human IL-1B gene with human male infertility. The case control study comprised of two groups: 222 infertile patients and 230 fertile healthy control men. Genotyping for SNP C?+?3953T IL-1B was carried out by polymerase chain reaction followed by analysis with specific endonucleases (PCR-RFLP). DNA sequencing was used to validate the PCR-RFLP results. The genotype frequencies of the IL-1B Taq C/T polymorphism were compared between infertile men and controls. The frequency was significantly higher in asthenozoospermic patients compared to fertile control men (odds ratio?=?10.4, CI: 2.50- 43.96, p?=?0.001). The C?+?3953T of the IL-1B gene is associated with male infertility risk in the asthenozoospermic patients from an Indian population.     

Citrus limon extract: possible inhibitory mechanisms affecting testicular functions and fertility in male mice

The effect of oral administration of 50% ethanolic leaf extract of Citrus limon (500 and 1,000?mg/kg body weight/day) for 35 days on fertility and various male reproductive endpoints was evaluated in Parkes strain of mice. Testicular indices such as histology, 3β- and 17β-HSD enzymes activity, immunoblot expression of StAR and P450scc, and germ cell apoptosis by TUNEL and CASP- 3 expression were assessed. Motility, viability, and number of spermatozoa in the cauda epididymidis, level of serum testosterone, fertility indices, and toxicological parameters were also evaluated. Histologically, testes in extract-treated mice showed nonuniform degenerative changes in the seminiferous tubules. Treatment had adverse effects on steroidogenic markers in the testis and induced germ cell apoptosis. Significant reductions were noted in epididymal sperm parameters and serum level of testosterone in Citrus-treated mice compared to controls. Fertility of the extract-treated males was also suppressed, but libido remained unaffected. By 56 days of treatment withdrawal, alterations induced in the above parameters returned to control levels suggesting that Citrus treatment causes reversible suppression of spermatogenesis and fertility in Parkes mice. Suppression of spermatogenesis may result from germ cell apoptosis because of decreased production of testosterone. The present work indicated that Citrus leaves can affect male reproduction.     

Male germ cell transplantation: promise and problems

Male germ cell transplantation is a novel technique in which donor male stem germ cells are surgically transferred to the seminiferous tubules of a recipient testis by direct injection or via the rete testis or efferent duct. All germ cells that are destined to become stem spermatogonia are defined as male stem germ cells, including primordial germ cells from the gonadal ridges, and gonocytes and stem spermatogonia from the testis, all of which are transplantable and capable of undergoing normal spermatogenesis. Xenotransplantation of male germ cells from one species into the testis of another species, including human testicular cells in the mouse, has so far proved to be unsuccessful. However, the immunodeficient mouse testis can support rat spermatogenesis and produce apparently normal rat spermatozoa. The underlying mechanisms remain elusive. The present mini-review will focus on the importance of stem spermatogonial transplantation for testicular stem cell biology and discuss the likelihood of immune rejection after transplantation, which may limit the success of all male germ cell transplantation.     

Trials for development of once-a-month injectable, hormonal male contraceptive using dienogest plus testosterone undecanoate: dose standardization, efficacy and reversibility studies in rats

Background

The study was conducted to test the potential of using dienogest (DNG) plus testosterone undecanoate (TU) in rats for development of a once-a-month injectable male hormonal contraceptive.

Study Design

Dose selection studies were initiated with administration of DNG in three different doses of 20, 30 and 40 mg/kg body weight (bw) per week plus TU 25 mg/kg bw once in every 6 weeks. Status of spermatogenesis and sperm count in epididymis was evaluated. The frequency of DNG intervention was later extended to every 2- and 4-week intervals. Mating studies, toxicity and reversibility of spermatogenesis following stoppage of treatment were carried out with DNG 40 mg/kg bw at 4-week intervals.

Results

Complete arrest of spermatogenesis was observed after 60 days of treatment at all doses of DNG (20, 30 and 40 mg/kg bw per week)+TU. However, weights of testis and accessory sex organs (epididymis, prostate and seminal vesicle) declined significantly 60 days post treatment compared to vehicle-treated controls. Epididymis in the treated animals was completely devoid of sperm. When the frequency of DNG injection (20 mg/kg bw) was extended to once every 15 days, a few immotile and decapitated sperm were observed in the epididymis. With TU treatment unchanged, animals receiving DNG (40 mg/kg bw) once either every 2- or 4-week intervals demonstrated good and uniform arrest of spermatogenesis. DNG 40 mg/kg per 4 weeks+TU also demonstrated a significant rise in germ cell apoptosis in the seminiferous epithelium. There was no significant increase in the serum high-density lipoprotein and low-density lipoprotein levels at the end of 120 days of treatment. Following withdrawal of treatment after 60 or 120 days, qualitative restoration of spermatogenesis was rapid in the former compared to the latter.

Conclusion

Dienogest plus TU has the potential for development as a monthly injectable showing reversible hormonal male contraception with good efficacy.     

EXPRESSION OF CANNABINOID RECEPTOR 1 IN MOUSE TESTES

Marijuana smoke and cannabinoids adversely affect male reproductive function in human and rodent through the cannabinoid receptors. To understand the possible function of cannabinoid receptor 1 (CB1) in spermatogenesis, expression of CB1 in testis during the postnatal development was examined in mice. Semiquantitative RT-PCR analysis revealed that testicular CB1 mRNA level was relatively high at 1 week post partum (p.p.). Following decrease during prepubertal development (2 weeks p.p.) and CB1 mRNA level re-increased during puberty (4 weeks p.p.) and reached the peak in adult testis. At 1 week p.p., some spermatogonia and Leydig cells showed strong immunoreactivity of CB1. At 2 weeks p.p., CB1 immunoreactivity was largely found in the primary spermatocytes as well as spermatogonia, and Leydig cells showed a weak signal. In adult testis, strong immunoreactivity was found in Leydig cells and luminal epithelia of seminiferous tubule. Germ cells including spermatozoa were positive for CB1 immunoreactivity. On Western blot, multiple forms of CB1 proteins were detected in testes, suggesting oligomerization of CB1. Ubiquitous, but spatiotemporal difference in expression of CB1 in soma and germ line during postnatal development of testis suggests functional involvement of CB1 signaling in steroidogenesis, spermatogenesis and fertilization.     

Expression of P16(INK4a) in testis of rhesus monkey during heat stress and testosterone undecanoate induced azoospermia or oligozoospermia

Previous studies on azoospermia or oligozoospermia induced by heat stress or high doses of testosterone mainly focused on germ cell apoptosis; no data regarding their possible effect on spermatogonia mitosis are available. We have established unilateral cryptorchid and testosterone undecanoate (TU)-treated monkey models and examined expression of P16(INK4a) in the testis to look at its possible role in azoospermia or oligozoospermia induced by the heat stress or the TU treatment. The results showed that both heat stress and TU were capable of inducing expression of P16(INK4a) mainly in spermatogonia and other types of germ cells as well as Sertoli cells at the later stage of germ cell apoptosis, namely on Day 10 after operation or on Day 60 after TU injection. It is, therefore, suggested for the first time that P16(INK4a) protein may inhibit the spermatogonia mitosis in the testis at the later stage of the germ cell apoptosis, resulting in arrest of spermatogenesis.     

抑癌基因Deleted in Liver Cancer1在人胶质瘤细胞株中的作用及机制

目的探讨抑癌基因DLC1表达的isoform1及isoform2两个亚型在人胶质瘤细胞株中的表达、功能及机制。方法RT—PCR方法检测体外培养的胶质瘤细胞株U87-MG和SHG-44中DLC1 isoform1，2的mRNA水平；10μM5-aza-2’de-oxycytidine（5＇-AZA）或者共含有500nM trichostatinA（TSA）处理细胞36h，检测DLC1 isoform1，2的表达；MTT方法检测转染DLC1 isoform2及没有GAP活性的isoform2突变体DLC1-K714E表达质粒对胶质瘤细胞株U87-MG和SHG-44增殖的影响．结果在U87-MG细胞株中没有检测到DLC1 isoform1；在SHG-44中检测到微量的DLC1 isoform1，5＇-AZA或者共含有TSA处理使其表达上调;DLC1 isoform2在上述细胞株都能检测到，5＇-AZA或者共含有TSA处理细胞未改变其表达水平；过表达DLC1 isoform2及其突变体DCL1—K714E促进U87-MG和SHG-44细胞株增殖。结论DLC1 isoform1在胶质瘤细胞株中不表达或低表达．可能原因是由于不转录或者启动子发生甲基化。DLC1 isoform2有一定的表达水平，高表达的DLC1 isoform2促进胶质瘤细胞增殖．且不依赖GAP活性．     

精索静脉曲张大鼠睾丸HSP60表达的改变与生精功能损害的相关性

目的:探索精索静脉曲张大鼠睾丸热休克蛋白(HSP60)表达的改变对生精功能损害的影响。方法:选择30只SD大鼠,20只作为手术组,建立精索静脉曲张模型,10例作为假手术对照组。3个月后,取下睾丸常规染色切片,用Makler积分法随机选择曲细精管,手术组200个,对照组100个,并测量内径、管周膜厚度、细胞层数及生精细胞成熟程度,计算平均得分;免疫组化方法测定HSP60的表达量,并比较二组之间的差异。结果:手术组的曲细精管Makler评分显著低于对照组,二者有非常显著性差异(P<0.01)。在手术组曲细精管HSP60表达量为3.85±0.37,对照组表达量为2.10±0.32,二者有非常显著性差异(P<0.01),且表达均定位于精子细胞,其表达量与评分值呈负相关(r=-0.9405,P<0.01)。结论:精索静脉曲张睾丸组织HSP60表达增加可导致生精功能损害,并影响精子细胞的成熟,可能是不育的原因之一。     

Expression of CDV-1R Gene in Mouse Epididymis as Revealed by in Situ Hybridization

We have previously studied mouse Cdv (carnitine deficiency-associated gene expressed in ventricle)-1 related gene Cdv-1R and its human counterpart CDV-1R, and revealed that mouse Cdv-1R was predominantly expressed in testis by multiple tissue northern analysis. To further localize the Cdv-1R mRNA in mouse testis and epididymis tissue, in situ hybridization study was reported in this article. In the adult mice, the Cdv-1R expression was intensively found in the epithelial cells of the caput and corpus epididymis, whereas it was moderately detected in the initial segment, and weakly in the cauda epididymis. In the seminiferous tubles of the testis, no obvious hybridization signals were observed above the background level. This Cdv-1R region-specific expression pattern in the epididimis suggests Cdv-1R may play an important role in sperm maturation. Moreover, considering the Cdv-1R has a similar expression distribution in epididymis to the OCTN2, it would appear that Cdv-1R might be involved in the carnitine pathway in the epididimis.     

抑癌基因Deleted in Liver Cancer1在人胶质瘤细胞株中的作用及机制

目的探讨抑癌基因DLC1表达的isoform1及isoform2两个亚型在人胶质瘤细胞株中的表达、功能及机制。方法RT-PCR方法检测体外培养的胶质瘤细胞株U87-MG和SHG-44中DLC1isoform1,2的mRNA水平;10μM5-aza-2'-de-oxycytidine(5'-AZA)或者共含有500nMtrichostatinA(TSA)处理细胞36h,检测DLC1isoform1,2的表达;MTT方法检测转染DLC1isoform2及没有GAP活性的isoform2突变体DLC1-K714E表达质粒对胶质瘤细胞株U87-MG和SHG-44增殖的影响。结果在U87-MG细胞株中没有检测到DLC1isoform1;在SHG-44中检测到微量的DLC1isoform1,5'-AZA或者共含有TSA处理使其表达上调;DLC1isoform2在上述细胞株都能检测到,5'-AZA或者共含有TSA处理细胞未改变其表达水平;过表达DLC1isoform2及其突变体DCL1-K714E促进U87-MG和SHG-44细胞株增殖。结论DLC1isoform1在胶质瘤细胞株中不表达或低表达,可能原因是由于不转录或者启动子发生甲基化。DLC1isoform2有一定的表达水平,高表达的DLC1isoform2促进胶质瘤细胞增殖,且不依赖GAP活性。     

CLONING AND CHARACTERIZATION OF TEMPERATURE-RELATED GENE TRS1

To investigate the mechanism of spermatogenesis arrest derived from heat treatment and to screen temperature-related genes involved in spermatogenesis, the authors analyzed the differences in gene expression between cryptorchid and scrotal testes in rats, and cloned a full-length cDNA named TRS1. In situ hybridization showed that TRS1 mRNA was mainly expressed in spermatocyte and round spermatids in testis. The expression level decreased in cryptorchid testis, suggesting that the lower scrotal temperature is a key factor in keeping the normal expression of TRS1. At the N-terminal of TRS1, there was a plecstrin homology (PH) domain signature. This PH domain has high similarity to that in PEPP2, a homosapien protein, which has a characteristic of binding phosphatidylinositol 3-phosphate via its PH domain in vitro. These findings suggest that TRS1 may be important in spermatogenesis and give clues for further research on the function of TRS1.     

The Biology of Human Male Reproduction: An Overview

In this overview, the focus is on the biology of human male reproduction. The structure, and Function of the testis are described, and the endocrine control of spermatogenesis is discussed. The formation of spermatozoa during spermatogenesis and their maturation in the epididymis are reviewed. The physiology I ejaculation and sperm transport in the Female tract are discussed. Sperm capacitatiors, the acrosome reaction, and Fertilization of human gametes are also reviewed.