Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking.

BACKGROUND: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. METHODS: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. RESULTS: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. CONCLUSION: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.

[Expression characteristics of the 1700008O03Rik gene in the mouse testis during spermatogenesis and results of bioinformatic analysis].

OBJECTIVE: To investigate the characteristics of the expression of the RIKEN cDNA 1700008O03 (1700008O03Rik) gene in the testis of the mouse from birth to sexual maturity and its potential role in regulating spermatogenesis. METHODS: Using mouse gene expression profile microarray, we screened the testis-specific gene 1700008O03Rik from the mouse. We studied the expression characteristics of the gene in the development of the mouse testis by reverse transcription PCR, quantitative real-time PCR, Western-blot, immunohistochemistry and immunofluorescence, and analyzed the structure of the 1700008O03Rik protein and its homology with other species using the bioinformatic software. RESULTS: 1700008O03Rik gene was highly expressed in the testis of the mouse, increasing in an age-dependent manner, and mainly in the endochylema of oblong spermatozoa. Bioinformatic analysis revealed a high homology of the 1700008O03Rik protein between human and mice, and phylogenetic tree analysis showed it to be highly conserved in mammalian evolution. CONCLUSIONS: 1700008O03Rik is a highly expressed gene in the mouse testis, mainly in the endochylema of oblong spermatozoa, which may be involved in the regulation of spermatogenesis in mice.

[Expression characteristics of the Daxx gene in the mouse testis during spermatogenesis].

OBJECTIVE: To investigate the expression characteristic of the Daxx gene in the mouse testis and its role in spermatogenesis. METHODS: Real-time PCR, Western blot and immunofluorescence were used in examining the expression characteristics of DAXX in the testis tissue from wild-type, Sertoli cell-specific androgen receptor knockout (SCARKO) and androgen receptor knockout (ARKO) mice at different postnatal weeks . RESULTS: The Daxx gene was highly expressed in the testis tissue and mainly in the nuclei of the wild-type mice at 4 postnatal weeks. Compared with the wild-type, the ARKO mice showed a markedly decreased expression of DAXX (0.299±0.026), which displayed a polar distribution in the spermatogenic cells (0.853±0.058) and exhibited no significant difference in the SCARKO mice (1.000±0.015). CONCLUSIONS: The Daxx gene expression is the highest in the middle-stage development of the mouse testis, significantly decreased in ARKO mice as compared with the wild-type, and its location influenced by specific AR knockout in Sertoli cells. DAXX may be involved in the regulation of spermatogenesis in mice.

The Wilms tumor gene, Wt1, is critical for mouse spermatogenesis via regulation of sertoli cell polarity and is associated with non-obstructive azoospermia in humans.

Azoospermia is one of the major reproductive disorders which cause male infertility in humans; however, the etiology of this disease is largely unknown. In the present study, six missense mutations of WT1 gene were detected in 529 human patients with non-obstructive azoospermia (NOA), indicating a strong association between WT1 mutation and NOA. The Wilms tumor gene, Wt1, is specifically expressed in Sertoli cells (SCs) which support spermatogenesis. To examine the functions of this gene in spermatogenesis, Wt1 was deleted in adult testis using Wt1(flox) and Cre-ER(TM) mice strains. We found that inactivation of Wt1 resulted in massive germ cell death and only SCs were present in most of the seminiferous tubules which was very similar to NOA in humans. In investigating the potential mechanism for this, histological studies revealed that the blood-testis barrier (BTB) was disrupted in Wt1 deficient testes. In vitro studies demonstrated that Wt1 was essential for cell polarity maintenance in SCs. Further studies found that the expression of cell polarity associated genes (Par6b and E-cadherin) and Wnt signaling genes (Wnt4, Wnt11) were downregulated in Wt1 deficient SCs, and that the expression of Par6b and E-cadherin was regulated by Wnt4. Our findings suggest that Wt1 is important in spermatogenesis by regulating the polarity of SCs via Wnt signaling pathway and that WT1 mutation is one of the genetic causes of NOA in humans.

[Expression characteristics of the Ccdc70 gene in the mouse testis during spermatogenesis].

OBJECTIVE: To investigate the expression characteristics of the gene of coiled-coil domain-containing protein 70 (Ccdc70) in the mouse testis and its potential role in spermatogenesis. METHODS: Using expression profile microarray, we screened the mouse testis-specific gene Ccdc70, studied its expression characteristics in the mouse testis by RT-PCR, real-time PCR, Western blot and immunohistochemistry, followed by bioinformatic analysis of the Ccdc70 protein. RESULTS: The Ccdc70 gene was expressed highly in the testis but lowly in the epididymis of the mice. The Ccdc70 protein was expressed mainly in the spermatocytes and round spermatids of the testis and in the epithelial cells of the epididymis. Bioinformatic analysis showed a structural domain in the Ccdc70 protein, which was highly conserved in mammalian evolution. CONCLUSION: The Ccdc70 gene is highly expressed in the mouse testis and mainly in the spermatocytes, round spermatids, and epididymal epithelial cells, which indicates that it is involved in the regulation of spermatogenesis and epididymal sperm maturation.

[Expression of a testis-specific gene 1700001022RIK in mice and its bioinformatic analysis].

OBJECTIVE: To identify the expression characteristics of the 1700001022RIK (RIKEN cDNA 1700001022) gene in mice and explore its function by bioinformatic analysis. METHODS: Using the expression profile of gene microarray, we detected the expression of a new testis-specific gene, 1700001022RIK, in mice. We analyzed its expression characteristics in the testis tissue and their changes in different developmental stages of the testis by RT-PCR, real-time RT-PCR, Western blot, and immunohistochemistry. We performed bioinformatic analysis using a bioinformatic software. RESULTS: The 1700001022RIK gene was specifically expressed in the mouse testis in an age-dependent manner, most highly in the adult mice. The 1700001022RIK protein was mainly expressed in the spermatogonia, spermatocytes, and round spermatids of the adult mice. Bioinformatic analysis showed that the 1700001022RIK protein amino acid sequence had a high similarity in human and mice, which indicated that this gene was highly conserved in mammals. CONCLUSION: 1700001022RIK is a testis-specific gene mainly expressed in the spermatogonia, spermatocytes, and round spermatids of seminiferous tubules, which might be involved in the regulation of spermatogenesis.

[UBE2B gene and male infertility: an update].

Male infertility is a worldwide problem, and about 15% of the cases are associated with spermatogenesis-related gene mutation. The mammalian gene UBE2B is the homolog of the RAD6 gene of yeast, belonging to the ubiquitin proteasome system and playing an important role in spermatogenesis. Mice lacking the UBE2B gene are infertile, with reduced sperm motility, increased morphologically abnormal sperm, and inhibited meiosis of spermatogonia. Accumulated evidence shows that UBE2B gene mutants and single nucleotide polymorphisms are associated with male infertility. This article reviews the relation between the UBE2B gene and male infertility, offering some theoretical evidence for the diagnosis and treatment of male infertility.

Effect of all-trans-retinoic acid on the expression of primordial germ cell differentiation-associated genes in mESC-derived EBs.

atRA (all-trans-retinoic acid) is known to induce the differentiation of mESCs (mouse embryonic stem cells) into PGCs (primordial germ cells) in vitro. However, it is not clear as to what changes occur in PGC differentiation-associated genes or what mechanisms are involved when EBs (embryoid bodies) derived from mESCs are induced by atRA. EBs derived from mESCs were treated with 1, 2 or 5 µM atRA for 16 h, 2 days or 5 days. Real-time PCR and Western blot analysis were performed to detect the relative levels of PGC differentiation-associated genes (Lin28, Blimp1, Stra8 and Mvh) and the corresponding proteins respectively. Immunofluorescence was used to detect the protein location and distribution in EBs. The expression characteristics of genes could be divided into three categories: rapidly reached the peak value in 16 h and then decreased (Stra8, Lin28), initially low and then increased to reach the peak value in 5 days (Mvh) and relatively unchanged (Blimp1). A low level of Lin28 was expressed in EBs treated with atRA for 2 days or 5 days. The variation in the level of Lin28 mRNA did not influence the change in the level of Blimp1 mRNA. The changes in Stra8/Lin28 were consistent with the corresponding changes in the levels of their respective mRNAs, but the changes for Mvh/Blimp1 were not consistent with the corresponding changes in the levels of their respective mRNAs. Blimp1 expression may be independent of the effect of atRA on PGC differentiation. atRA may promote the start of a period in which there is a low level of Lin28 expression during PGC differentiation.

[Abnormal expression and significance of MIR-184 in human renal carcinoma].

OBJECTIVE: To explore the role of microRNA-184(MIR-184) in the development of renal cell carcinoma(RCC). METHODS: The expressions of MIR-184 in 51 patients with RCC Investigated, normal adjacent tissues (ADTs) matched by fluorescence quantitative PCR technology (RT-qPCR) and the correlations analyzed between MIR-184 expression and the age, gender and clinical stage of RCC patients. RESULTS: The average expression of MIR-184 in RCC was -14.664 6 ± 5.362 4, while that in ADTs was -10.408 7 ± 3.482 7(P<0.01). Bounded with the MIR-184 expression in RCC, patients were divided into lower-expression group and higher-expression group. Meanwhile, the RCC patients were divided into three groups according to the age, gender and clinical stage of the patients. Chi-square statistical analysis showed that the expression level of MIR-184 was not significantly correlated with the patient's age, gender and clinical stage (respectively: P>0.03, P>0.99, P>0.03). CONCLUSION: MIR-184 in RCC was significantly lower than that in ADTs, which may have potential significance in the occurrence and development of RCC.

[The progress of TMPRSS2-ETS gene fusions and their mechanism in prostate cancer].

The gene fusions between transmembrane protease serine 2 (TMPRSS2) and E26 (ETS) transcription factors are present in over 50% of patients with prostate cancer. TMPRSS2-ERG is the most common gene fusion type. The ERG overexpression induced by TMPRSS2-ERG gene fusion contributes to the development of prostate cancer. Both androgen receptor binding and genotoxic stress induce chromosomal proximity and TMPRSS2-ETS gene fusions. TMPRSS2-ERG gene fusion functions as a biomarker for prostate cancer, which can be easily detected in urine. This review focuses on the characteristics, oncogenic and rearranged mechanism, and clinical application of TMPRSS2-ETS gene fusions.

[Mouse-induced pluripotent stem cells have the potential to differentiate into induced primordial germ cells].

OBJECTIVE: To investigate whether mouse-induced pluripotent stem (iPS) cell line IP14D-1 has the potential to differentiate into induced primordial germ cells (iPGCs), and to explore the changes in the expression of iPGCs-differentiation associated genes and their possible mechanisms. METHODS: Undifferentiated IP14D-1 was cultured to proliferate and then differentiated to form 4-, 7- and 9-day-old induced embryoid bodies (iEBs) in vitro, respectively. RT-PCR and immunofluorescence were used to detect the expressions of Lin28, Blimpl, Stra8 and Mvh, as well as the localization of the corresponding protein in iEBs. RESULTS: The expression of Blimpl was higher than that of Lin28 in the undifferentiated IP14D-1 and mouse embryonic stem cells (mESCs). Mvh and Stra8 as well as mESCs and EBs were also expressed in IP14D-1 and iEBs, but with no significant differences. The expression of Lin28 was gradually increased in the IP14D-1-derived iEBs from 4 to 7 days, but decreased at 9 days, and the expression of Blimp1 was gradually reduced with the prolonged growing time of iEBs. CONCLUSION: A stable system was established for the culture and differentiation of IP14D-1 and IP14D-1-derived iEBs. The expressions of Lin28, Blimp1, Mvh and Stra8 were not significantly different between the undifferentiated IP14D-1 and mESCs, nor were the expressions of Mvh and Stra8 between iEBs and EBs. IP14D-1 and iEBs had the potential to differentiate into iPGCs, which increased in number in the 7-day-old iEBs, and the expression of iPGC-differentiation associated Lin28 became lower in the older iEBs.

[Advances in the researches of spermatogenic protein, Ropporin].

Ropporin has been identified as a spermatogenic cell-specific protein and may be involved in sperm maturation, motility, capacitation, hyperactivation and acrosome reaction. However, latest studies have shown that Ropporin is expressed weakly in normal non-testis tissues and highly in hematologic malignancies. Its highly conservative expression in mammalians demonstrates its importance to life. This paper updates the characterization, expression and its distribution, and biological function of Ropporin, and the advances in the clinical researches of the protein.

[Differential expression of ODF1 in human ejaculated spermatozoa and its clinical significance].

OBJECTIVE: To compare the expressions of ODF1 (outer dense fiber of the sperm tail 1) in ejaculated spermatozoa from normozoospermic and asthenozoospermic men with low sperm motility. METHODS: Semen analyses were performed on the semen samples obtained from normozoospermic (n=20) and asthenozoospermic (n=20) volunteers according to the WHO criteria. To rule out the contamination of germ cells and leucocytes, the human ejaculated spermatozoa were purified by a discontinuous Percoll density gradient centrifugation. RT-PCR and Western blot were used to detect the expressions of ODF1 in the spermatozoa from the two groups. RESULTS: RT-PCR showed that the expression of ODF1 mRNA was significantly lower in the spermatozoa from the asthenozoospermic patients than in those from the normozoospermic men (1.35 +/- 0.25 vs. 2.79 +/- 0.28, P < 0.05). Western blot confirmed the results from RT-PCR and revealed an obviously decreased expression of ODF1 in the spermatozoa of the asthenozoospermic patients, with statistically significant difference from the normozoospermic group (1.44 +/- 0.26 vs. 3.64 +/- 0.34, P < 0.05). CONCLUSION: The expression of ODF1 was significantly decreased in the ejaculated spermatozoa of asthenozoospermic men, which might be responsible for low sperm motility.

[Expression and location of SPAG9 in human ejaculated spermatozoa].

OBJECTIVE: SPAG9, as a member of the MAPK family, plays an important role in sperm-egg fusion. This study aimed to detect the expression of SPAG9 in human ejaculated spermatozoa. METHODS: Different human tissues (as from the muscle, liver, esophagus, lung, stomach, kidney, prostate, uterus, testis and epididymis) and semen samples were obtained from healthy volunteers, and semen analyses were performed according to the WHO criteria. Human ejaculated spermatozoa were purified by discontinuous Percoll density gradient centrifugation to rule out the contamination of germ cells and leucocytes. RT-PCR and indirect immunofluorescence were used to detect the expression of SPAG9 in human spermatozoa. RESULTS: RT-PCR showed that SPAG9 mRNA was expressed in different tissues and human ejaculated spermatozoa. Indirect immunofluorescence studies revealed the location of SPAG9 protein in the equatorial plate and flagella of human spermatozoa. CONCLUSION: SPAG9 is expressed in ejaculated spermatozoa and may play a role in sperm capacitation and motility.

[Extracellular matrix regulates expressions of germ cell differentiation associated genes in mouse embryonic stem cells].

OBJECTIVE: Interactions of cells with the extracellular matrix (ECM) are essential for cell differentiation. The authors sought to determine the roles of different ECMs in the expressions of germ cell differentiation associated genes after mouse embryonic stem cells (mESCs) differentiated into embryoid bodies (EBs). METHODS: EBs derived from mESCs were maintained in suspension for 3 days and then cultured on the plates coated with various ECMs, including fibronectin (F), laminin (L), matrigel (M), collagen (C) and nonadhensive agarose (A), respectively, for 1, 2, 3 or 4 days, followed by evaluation of the expressions of the genes associated with germ cell differentiation by RT-PCR. RESULTS: The EBs of the F and L groups exhibited facilitated adherent differentiation. The expressions of the Blimp-1, Stella, Mvh and Stra8 genes were increased gradually in the F and L but not obviously in the M and C groups. The overall gene expressions were low in the A group, but high and then gradually decreased in the blank control group. Endogenous fibronectin, laminin and integrin beta1 were obviously expressed in the L and control groups. CONCLUSION: Laminin /integrin beta1 signaling may play a role in regulating the differentiation of mESCs into primordial germ cells (PGCs). Exogenous laminin can facilitate the differentiation of mESC-derived EBs into PGCs by acting on the integrin beta1 subunit, while exogenous fibronectin may be involved in the regulation of the differentiation through other integrin subunit. Endogenous laminin and fibronectin secreted by EBs may also facilitate cell differentiation in the absence of exogenous ECMs.

[Expression of the voltage-dependent anion channel gene in human ejaculated spermatozoa].

OBJECTIVE: To identify the genes involved in sperm motility. METHODS: We hybridized asthenospermia and normal motile sperm cDNA samples with the human whole genome Affymetrix chip to screen differentially expressed genes. Then we detected the mRNA expressions of the voltage-dependent anion channel genes (VDACs) in human organs and spermatozoa by RT-PCR and compared their expressions in the poor and normal motility spermatozoa. RESULTS: Differentially expressed genes VDACs were identified by analysis of the hybridization signals, including the 3 subtypes VDAC1, VDAC2 and VDAC3. The expression of VDAC2 mRNA was significantly decreased in the poor motility sperm (0.568 +/- 0.036), as compared with the healthy men (0.803 +/- 0.043, P < 0.01). CONCLUSION: The decreased expression of VDAC2 in the ejaculated spermatozoa is possibly associated with the reduction of sperm motility.

Gossypol repressed the gap junctional intercellular communication between Sertoli cells by decreasing the expression of Connexin43.

Previous studies showed that gossypol could block the gap junctional intercellular communication (GJIC) between cultured cells. The present study was designed to investigate the effects of gossypol on the GJIC and the expression of connexin43 (Cx43) in the cultured cells. A Sertoli cell line, TM4, was treated with different concentrations of gossypol 1.25, 2.5, 5, and 10micromol/L for 6, 12, 24, and 48h. Cell viability was assessed with CCK-8 assay. GJIC in the cells was determined using the scrape loading and dye transfer (SLDT) assay; the expression of Cx43 was detected by RT-PCR, immunofluorescence and Western blot analysis. The SLDT assay showed gossypol significantly decreased GJIC between adjacent cells. RT-PCR, immunofluorescence and Western blot analyses demonstrated the expression of Cx43 in TM4 cells. The expression of Cx43 was gradually decreased with the increasing concentrations of gossypol, and the effect occurred as early as 6h after the treatment and continued until 48h. These results suggested that gossypol impaired GJIC by decrease of Cx43 expression in the cells, which is important for Sertoli cells to regulate spermatogenesis.

[Expression of amphiregulin in human endometrium during the menstrual cycle].

OBJECTIVE: To investigate the expression of amphiregulin in human endometrium during the menstrual cycle. METHODS: Endometrial tissues were collected from the patients undergoing hysterectomy or endometrial biopsy. Real-time RT-PCR, in situ hybridization and immunohistochemistry were used to detect the expression characteristics of amphiregulin in human endometrium in proliferative and secretory phases. RESULTS: Real-time RT-PCR showed the expression of amphiregulin mRNA in secretory phase was 32 times that in proliferative phase. The results from in situ hybridization and immunohistochemistry showed that amphiregulin was located in the cytoplasm and mainly expressed in the gland of endometrium. The expressions of amphiregulin mRNA in proliferative and secretory phases were 0.54+/-0.22 and 2.96+/-0.47 (P<0.01), and the expressions of amphiregulin protein in proliferative and secretory phases were 0.77+/-0.47 and 2.60+/-0.43 (P<0.01), respectively. The data demonstrated that the amphiregulin expression was significantly increased in secretory phase, which was consistent with the results from RT-PCR. CONCLUSION: The expression of amphiregulin was increased in the secretory phase of human endometrium, which suggests that amphiregulin may play an important role in human implantation.

[Roles of calcium ion channels and its clinical significance in sperm motility].

As an important intracellular messenger, Ca2+ plays a major role in sperm motility. In spermatozoa, multiple Ca2(+)-permeable channels have been identified in the plasma membrane of mammalian sperm, including voltage-gated Ca2+ channels (Cav channels), cyclic nucleotide-gated channels (CNGC), cation channels of sperm (CatSper) and the transient receptor potential (TRP) family. As calcium regulation of sperm motility is mainly mediated by these calcium channels, any aberration of the channels can lead to the decline of sperm activities. Recent progress in the researches on the relationship between sperm motility and calcium-related ion channels is briefly reviewed in this article.

Differential expression of VASA gene in ejaculated spermatozoa from normozoospermic men and patients with oligozoospermia.

AIM: To detect the expression of VASA in human ejaculated spermatozoa, and to compare the expression of VASA between normozoospermic men and patients with oligozoospermia. METHODS: Ejaculated spermatozoa were collected from normozoospermic men and patients with oligozoospermia by masturbation, and subsequently segregated through a discontinuous gradient of Percoll to obtain the spermatozoa. Reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (QRT-PCR), immunoflurescence and Western blotting were used to detect the expression of VASA in mRNA and protein levels. RESULTS: VASA mRNA was expressed in the ejaculated spermatozoa. QRT-PCR analysis showed that VASA mRNA level was approximately 5-fold higher in normozoospermic men than that in oligozoospermic men. Immunofluorescence and Western blotting analysis showed that VASA protein was located on the cytoplasmic membrane of heads and tails of spermatozoa, and its expression was significantly decreased in oligozoospermic men, which is similar to the result of QRT-PCR. CONCLUSION: The expression of VASA mRNA and protein was significantly decreased in the sperm of oligozoospermic men, which suggested the lower expression of the VASA gene might be associated with pathogenesis in some subtypes of male infertility and VASA could be used as a molecular marker for the diagnosis of male infertility.