Metastasis-associated protein 1 participates in regulating luminal acidification of the epididymis via repressing estrogen receptor alpha transcription.

BACKGROUND: As a component of the nucleosome remodeling and deacetylating (NuRD) complex, metastasis-associated protein 1 (MTA1) has been reported to be abundant in male reproductive system and might participate in spermatogenesis and sperm maturation, whereas the precise functional role of MTA1 in these processes is still undetermined. OBJECTIVE: To investigate the effect and potential function of MTA1 in male fertility. MATERIALS AND METHODS: Mta1 knockout mice (Mta1-/- ) were employed to detect their reproductive phenotype. The pH value of Mta1-/- epididymal luminal fluid was measured, and the potential mechanism of MTA1 involved in regulating luminal acidification was detected in vivo and in vitro. A vasectomy model with abnormal pH of epididymal lumen was established to further detect the effect of MTA1 on epididymal luminal microenvironment. RESULTS: Mta1-/- mice were fertile without any detectable defects in spermatogenesis or sperm motility while the deficiency of MTA1 could acidify the initial segment of epididymis to a certain extent. MTA1 could interact with estrogen receptor alpha (ERα) and inhibit the transcription of ERα target gene, hydrogen exchanger 3 (NHE3), and ultimately affect the epididymal luminal milieu. After vasectomy, the Mta1-/- mice presented a more acidic epididymal lumen which was closer to the normal state compared to the wild-type model. DISCUSSION AND CONCLUSION: MTA1 is dispensable for male fertility in mice, but plays a potentially important function in regulating luminal acidification of the epididymis.

TGF-ß1 induced activations of Smad2 and miRNAs inhibit SF-1- and LRH-1-dependent CYP19 expression in rat Leydig cells†.

P450 aromatase, encoded by the Cyp19 gene, catalyzes the synthesis of estrogen, which is crucial for mammalian germ cell differentiation. We have previously shown that transforming growth factor beta 1 (TGF-ß1) attenuated the accumulation of steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) and eventually reduced the transcription of Cyp19 in rat Leydig cells (LCs). Here, we report that TGF-ß1 treatment-induced phosphorylation of Smad2 and decreased the expression levels of SF-1 and LRH-1 by elevating the expression levels of microRNA-21-3p and microRNA-339-5p in vivo and in vitro. Furthermore, both TGF-ß1 treatment and over-expression of Smad2 inhibited the SF-1 or LRH-1-regulated promoter activity of the Cyp19 gene, and p-Smad2 physically interacted with SF-1 and LRH-1. Our findings collectively suggest that TGF-ß1 may inhibit the expression of CYP19 in LCs mainly through two ways. On the one hand, TGF-ß1 acts through Smad2 to repress the accumulation of SF-1 and LRH-1 at post-transcriptional level by upregulating specific microRNAs. On the other hand, TGF-ß1 inhibits the transcriptional activity of Cyp19 through the interaction of p-Smad2 with SF-1/LRH-1.

PA1 participates in the maintenance of blood-testis barrier integrity via cooperation with JUN in the Sertoli cells of mice.

BACKGROUND: The blood-testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown. RESULTS: Here, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1. CONCLUSION: Overall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.

The Involvement of the Chemokine RANTES in Regulating Luminal Acidification in Rat Epididymis.

Background: A complex interplay between different cell types in the epithelium leads to activation of the luminal acidifying capacity of the epididymis, a process that is crucial for sperm maturation and storage. Basal cells sense the luminal angiotensin II (ANG II) and stimulate proton secretion in clear cells through nitric oxide (NO). Our previous study has shown the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) was expressed in the F4/80 positive macrophages of human epididymis. The objective of this study was to explore the involvement of RANTES in regulating the luminal acidification in the rat epididymis. Methods: The role of RANTES was investigated by in vivo perfusion with recombinant RANTES, Met-RANTES, and PBS of different pH values. Furthermore, rats vasectomy was performed to alter the epididymal luminal pH. RIA was used to measure the tissue homogenate ANG II concentration. Real time-PCR and western blot were employed to examine the expression levels of AGTR2, RANTES, CCR1, CCR5, and iNOS in epididymis. Results: RANTES was restricted to the basal macrophages of epididymal ducts and co-localized with its receptors CCR1 and CCR5. Both V-ATPase and iNOS were up-regulated in the cauda epididymis after perfused with recombinant RANTES, while the antagonist Met-RANTES perfusion led to a complete abrogation of the increased expression of V-ATPase in the apical membrane of clear cells and iNOS in macrophages. Upon alkaline perfusion, RANTES expression was significantly increased and the apical accumulation of V-ATPase in the clear cells was induced in the cauda epididymis. The luminal pH in the cauda epididymis increased after vasectomy. The concentration of the ANG II and the expression levels of AGTR2, RANTES, CCR1, CCR5, and iNOS dropped in the cauda epididymis following vasectomy. Conclusion: Upon the activation of basal cells, RANTES might induce the NO release from macrophages by interacting with its receptors, which increases proton secretion by adjacent clear cells. Thus, RANTES is possible to participate in the crosstalk among basal cells, macrophages and clear cells for the fine control of an optimum acidic luminal environment that is critical for male fertility.

miR-192 enhances sensitivity of methotrexate drug to MG-63 osteosarcoma cancer cells.

Chemo-resistance remains a considerable obstacle encountered in osteosarcoma (OS) therapy. Evidence has implied that a reduction in the expression of microRNAs (miRs/miRNAs) leads to exacerbated chemo-resistance. Hence, to better understand the role of miR-192 in the pathogenesis of OS during methotrexate (MTX) treatment, we restore miR-192 in the MG-63 cells and investigate the mechanisms, which are associated with MTX-resistance in OS. Exogenetic overexpression of miR-192 was established by transfecting miR-192 mimics into MG-63 cells using Lipofectamine. Trypan blue dye exclusion test was performed to evaluate the proliferation of the MG-63 cells. Chemo-resistance to MTX was determined using the MTT method after 48 h. ELISA cell death assay was performed to evaluate the apoptosis rate. The quantitative RT-PCR (RT-qPCR) was applied to determine the mRNA expression levels before and after the transfection. Our results illustrated that miR-192 is down-regulated in OS tumor cells. Transfection of miR-192 noticeably alleviated the mRNA expression levels of MMP9, c-Myc, K-Ras, CXCR-4, and ADAMTS compared with the control groups (P-values< 0.05). MTX Combination treatment with miR-192 noticeably elevated the cytotoxic effect of MTX and alleviated its IC50 (P < 0.05). Moreover, miR-192 significantly increased the apoptotic effect of MTX. These results implied that miR-192 enhances the sensitivity of MG-63 cells to MTX. Collectively, our results elucidated that miR-192 contributes to chemo-sensitizing MG-63 cells to MTX, and could be considered as a promising agent to overcome MTX-resistance in OS.

Spatiotemporal expression of NDRG2 in the human fetal brain.

N-myc downstream-regulated gene 2 (NDRG2) has been implicated in the development of central nervous system and brain diseases such as brain tumors, ischemic stroke and neurodegenerative disorders. However, it remains unclear that the spatiotemporal distribution of NDRG2 in the human fetal brain. In this study, we examined the expression pattern of NDRG2 in different regions of human fetal brain at 16-28 gestational weeks (GWs) by using RT-PCR, western blot and immunohistochemistry. Firstly, RT-PCR revealed that mRNA of NDRG2 was detected in the human brain regions of fetuses at 16-28 GWs such as medulla oblongata (MdO), mesencephalon (MeE), cerebellum (Cbl), frontal lobe (Fr), ventricular (VZ)/subventricular zone (SVZ) and hippocampus (hip), and the expressions of NDRG2 mRNA in these human fetal brain regions were increased with gestational maturation. Furthermore, western blot and immunohistochemistry results revealed that at 28 GWs, the expression of NDRG2 protein was restricted to the MdO's olivary nucleus, MeE's aqueduct, cerebellar internal granular layers, cerebral cortex of the Fr, VZ/SVZ of lateral ventricle, and hippocampal dentate gyrus, and highest expression in the VZ/SVZ, and lowest in the MeE. Finally, double immunohistochemistry results showed that NDRG2 in the MdO, Cbl and VZ/SV at 28 GWS was mainly expressed in neurons (NeuN positive cells), and in some astrocytes (GFAP positive cells). Taken together, these results suggest that NDRG2 is mainly expressed in human fetal neurons of various brain regions during development, which may be involved in neuronal growth and maturation.

Colocalization of metastasis-associated proteins 1/2 and estrogen receptor alpha in rat epididymis.

It has been suggested that metastasis-associated proteins 1 and 2 (MTA1 and MTA2) are capable of suppressing estrogen receptor alpha (ERα) transactivation activity in breast cancer cells. ERα, which is present in the epididymis, is a crucial mediator of maintaining the luminal environment necessary for proper sperm maturation and function. The present study was undertaken to analyze the expression profile of both MTA1 and MTA2 in the epididymis of rats and to ascertain whether MTA1/2 colocalizes with ERα in the epididymis and primary cultured epididymal epithelial cells. Reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry analyses were utilized to demonstrate that MTA1 and MTA2 are expressed in the epididymis. Furthermore, these analyses revealed that MTA1 and MTA2 are predominantly localized in the nuclei of almost all epididymal epithelial cells. Immunofluorescence staining revealed that MTA1/2 colocalizes with ERα in epididymal epithelial cells. In conclusion, MTA1 and MTA2 are expressed in the epididymis of rats; these proteins colocalize with ERα in epididymal epithelial cells, suggesting that MTA1 and MTA2 may be involved in the regulation of ERα transactivation activity in the epididymis of rats to facilitate a stable environment in the lumen.

TGFB1 represses the expression of SF1 and LRH1 to inhibit E2 production in rat LCs.

Leydig cells (LCs) in the adult testis have been identified as the major sites of oestrogen production, which is crucial for mammalian germ cell differentiation. Our previous work showed that transforming growth factor beta 1 (TGFB1) inhibits estradiol (E2) secretion via down-regulating Cyp19 gene expression in mature rat LCs. However, the mechanism remains unclear. In the present study, the effects of TGFB1 on the expression levels of steroidogenic factor 1 (SF1), liver receptor homolog 1 (LRH1), cAMP response element-binding protein (CREB) and cAMP responsive element modulator (CREM) were evaluated both in primary cultured LCs and in rat testis. The involvement of TGFB1 signalling in the regulation of SF1 and LRH1 expression was then validated by applying the inhibitor of the TGFB type 1 receptor (TGFBR1) SB431542. Moreover, the expression of CYP19 in testicular LCs was investigated and the production of E2 in testicular interstitial fluid (TIF) was measured. The results showed that TGFB1 especially down-regulated the expression levels of SF1 and LRH1 both in primary cultured LCs and in rat testis. The down-regulations of TGFB1 in the production of E2 in TIF and the expression of CYP19 in testicular LCs were also observed in vivo These inhibitory effects could be reversed by TGFBR1 inhibitor SB431542. Our findings suggest that TGFB1 may act through the canonical signalling pathway involving ALK5 to restrain SF1 and LRH1 accumulation and eventually attenuate Cyp19 transcription and oestrogen production in LCs.

[C1q and tumor necrosis factor related protein 4 (CTRP4) suppresses caspase-1/IL-1ß inflammatory pathway in trophoblasts of rat models with preeclampsia].

Objective To explore the effect of C1q/tumor necrosis factor related protein 4 (CTRP4) on the placental trophoblasts of preeclampsia model rats. Methods Placental trophoblastic tissues were respectively collected from normal pregnant rats and model rats with preeclampsia, and then mRNA and protein expression levels of CTRP4, interleukin 1ß (IL-1ß), and caspase-1 were detected with quantitative real-time PCR (qRT-PCR) and Western blotting. Primary placental trophoblasts were isolated from normal pregnant rats and model rats; at different time points, flow cytometry was used to detect the number of PI+caspase-1+ pyroptotic cells; and qRT-PCR and Western blotting were used to detect expression levels of IL-1ß and caspase-1. Finally, recombinant CTRP4 protein (at the doses of 0.5, 5, 15, 25 or 50 ng/mL) or neutralizing CTRP4 antibody (at the doses of 10 or 20 ng/mL) were added into the medium of trophoblasts from model rats; after incubation for 72 h, the number of pyroptotic cells and the expression levels of IL-1ß and caspase-1 were detected. ResultsCaspase-1/IL-1ß inflammatory pathway was activated and CTRP4 expression was downregulated in placenta trophoblastic tissue from rats with preeclampsia. CTRP4 recombinant protein treatment significantly inhibited pyroptosis and the caspase-1/IL-1ß pathway in trophoblasts derived from rats with preeclampsia, while CTRP4 neutralizing antibody treatment had an opposite effect on pyroptosis and inflammation. Conclusion CTRP4 can significantly inhibit the activation of caspase-1/IL-1ß inflammatory pathway, and suppress the pyroptosis of trophoblasts derived from rats with preeclampsia.

[C1q tumor necrosis factor-related protein 6 (CTRP6) inhibits the proliferation and migration of ovarian cancer cells].

OBJECTIVE: To explore the role of C1q tumor necrosis factor-related protein 6 (CTRP6) in the proliferation and migration of ovarian cancer cells. METHODS: ELISA was used to detect the serum CTRP6 contents in ovarian cancer patients and healthy volunteers, and CTRP6 levels in the supernatants of SKOV3, 3AO and HO8910 epithelial ovarian cancer cell lines and IOSE80 normal ovarian epithelial cells. Recombinant human CTRP6 protein was applied to treat HO8910 cells. After incubation for 48 hours, ELISA was used to detect the levels of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) in the supernatants. The proliferation of HO8910 cells were detected by CCK-8 assay and the ability of invasion was determined by Transwell(TM) invasion assay. CTRP6 siRNA or anti-CTRP6 antibody was used to treat HO8910 cells, and then the ability of proliferation was also detected by CCK-8 assay and the ability of migration was evaluated by wound healing experiment. RESULTS: The level of CTRP6 decreased in the sera of the patients with ovarian cancer and the supernatants of epithelial ovarian cancer cells. The levels of IL-8 and VEGF were reduced by the recombinant CTRP6 protein treatment, and the cell proliferation and invasion were also inhibited. CTRP6 siRNA or anti-CTRP6 antibody treatment promoted cell proliferation and migration. CONCLUSION: The level of CTRP6 dropped in the sera of the patients with ovarian cancer as well as in the supernatants of epithelial ovarian cancer cells. CTRP6 could inhibit the proliferation and migration of ovarian cancer cells via blocking IL-8/VEGF pathway.

Different locations of RANTES and its receptors on mouse epididymal spermatozoa.

Our previous study showed that the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) originating from the mouse epididymis bound to the midpiece of luminal spermatozoa. The present study was undertaken to investigate the association between RANTES and epididymal spermatozoa and to determine whether the association is mediated by the RANTES receptors CCR1, CCR3 or CCR5. The use of reverse transcription polymerase chain reaction (RT-PCR), immunohistochemical staining and immunofluorescent staining demonstrated that RANTES secreted by apical and narrow cells of mouse epididymal ducts was associated with luminal spermatozoa. Flow cytometric analysis and immunofluorescent labelling revealed that the association between RANTES and spermatozoa of different regions weakened gradually as the spermatozoa moved along the epididymis. Moreover, CCR1, CCR3 and CCR5 were expressed in epididymal spermatozoa and located on the head of epididymal spermatozoa, while RANTES was generally located at the midpiece. In conclusion, RANTES and its receptors were not in the same sperm location, suggesting that RANTES binding to mouse epididymal spermatozoa is independent of CCR1, CCR3 and CCR5.

[Analysis of 72 affective disorder cases in forensic psychiatric expertise].

OBJECTIVE: To explore criminal characteristics of patients with affective disorder. METHODS: Analysis was conducted in 72 cases of affective disorder diagnosed in Ankang Hospital, Public Security Bureau of Hangzhou, from 2000 to 2004. RESULTS: There was a correlation between outbreak of the affective disordered and frequency of committing crime. There was a significant difference between the mania and the depression (P<0.01) with respect to harmful behavior. The criminal behavior characteristics of patients with affective disorder were different from that of the schizophrenia, with more realistic and less pathologic intention. CONCLUSION: Recurrent attacks are warning signs for affective disorder patients committing crime. The criminal behavior characteristics of the affective disorder are different from that of the schizophrenia, probably because of the differences in etiological factor, development, symptom, and severity of the disorders.