The regulation of IGFBP3 by BMP2 has a role in human endometrial remodeling.

In mammals, bone morphogenetic protein 2 (BMP2) is a critical regulator of endometrial decidualization and early implantation. Insulin-like growth factor-binding protein 3 (IGFBP3) is highly expressed in the endometrium and at the maternal-fetal interface in multiple species, including humans. BMP2-induced IGFBP3 signaling has been confirmed to have a role in trophoblast cell invasion; however, the involvement of this signaling pathway in endometrial remodeling remains poorly understood. To determine the roles of BMP2 in regulating IGFBP3 expression during the transformation of endometrial stromal cells, we employed immortalized human endometrial stromal cells (HESCs) and primary human decidual stromal cells (HDSCs) as study models. We showed that BMP2 significantly increased the expression of IGFBP3 in a dose- and time-dependent manner in both HESCs and primary HDSCs. Additionally, the BMP2-induced upregulation of IGFBP3 is mediated by the inhibitor of DNA-binding 1 (ID1), and knockdown of ALK3 completely abolished BMP2-induced upregulation of ID1. Moreover, BMP2 increased the expression of matrix metalloproteinases 2 (MMP2) and promoted cell migration in HESCs and primary HDSCs. Knockdown of either IGFBP3 or ID1 significantly suppressed the basal and the BMP2-induced increase in MMP2 expression as well as the cell migration in both cell models. These data demonstrated that BMP2 upregulated the expression of ID1, which in turn induced the expression of IGFBP3, and these BMP2-induced cell activities were most likely mediated by the ALK3 type I receptor. The increased expression of IGFBP3 promoted the MMP2 expression and cell migration in both HESCs and HDSCs. These findings deepen our understanding of a newly identified mechanism by which BMP2 and IGFBP3 regulate endometrial remodeling in humans, which provides insight into potential therapies for endometrium-related diseases and pregnancy-induced complications.

TGF-ß1 promotes vitamin D-induced prostaglandin E2 synthesis by upregulating vitamin D receptor expression in human granulosa-lutein cells.

There is increasing evidence showing the importance of vitamin D (Vit D) and its nuclear receptor, the Vit D receptor (VDR), in female reproductive health. Transforming growth factor-ß1 (TGF-ß1) and its functional receptors are expressed in human oocytes and granulosa cells that participate in follicular development and ovulation. Recently, Sma- and Mad-related protein 3 (SMAD3; a downstream effector of TGF-ß1) has been proposed to mediate crosstalk between the Vit D and TGF-ß1 signaling pathways, but this relationship has not been fully explored and has yet to be tested in human granulosa-lutein (hGL) cells. In this study, we showed that TGF-ß1 significantly promoted the expression of VDR, and this stimulatory effect occurred through the activin receptor-like kinase 5 type I receptor-mediated SMAD3 and ERK1/2 signaling pathways in hGL cells. Additionally, we showed that Vit D increased the expression of cyclooxygenase 2 (COX-2) and the synthesis of prostaglandin E2 (PGE2) in a time- and dose-dependent manner. Furthermore, we demonstrated a synergistic effect of TGF-ß1 and Vit D on the expression of COX-2 and synthesis of PGE2, and this effect could be attenuated by silencing the expression of VDR. Our findings indicate that TGF-ß1 upregulates the expression of VDR, which promotes Vit D-induced COX-2 expression and subsequent PGE2 production by activating the SMAD3 and ERK1/2 signaling pathways in hGL cells.

FGF9 is a downstream target of SRY and sufficient to determine male sex fate in ex vivo XX gonad culture.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to -833 to -821 of human FGF9 and -1010 to -998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12-16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

Incorporating Simplified Fournier's Gangrene Severity Index with early surgical intervention can maximize survival in high-risk Fournier's gangrene patients.

OBJECTIVES: To determine the optimal surgical timing in high-risk patients with Fournier's gangrene by the Simplified Fournier's Gangrene Severity Index. METHODS: From 1989 to 2018, 118 male patients diagnosed with Fournier's gangrene with complete medical records were retrospectively reviewed. Patients' demographics, laboratory parameters at initial diagnosis, Fournier's Gangrene Severity Index and Simplified Fournier's Gangrene Severity Index, and the time interval from emergency room arrival to surgical intervention were collected. The Fournier's gangrene patients were categorized into low-risk (Simplified Fournier's Gangrene Severity Index ≤2) and high-risk groups (Simplified Fournier's Gangrene Severity Index >2). Differences between the variables within the two groups were analyzed. The optimal surgical timing was analyzed with the receiver operating characteristic curve in high-risk Fournier's gangrene patients. RESULTS: The overall mortality of 118 Fournier's gangrene patients was 14.4%. After risk stratification with the Simplified Fournier's Gangrene Severity Index scoring system, the mortality of low-risk and high-risk Fournier's gangrene patients was 1.3% and 41.0%, respectively. In the high-risk group, the time interval from emergency room arrival to surgical intervention was the only variable with a significant difference between survivors and non-survivors (P = 0.039). The optimal surgical timing was determined at 14.35 h, which allowed the highest sensitivity (0.688) and specificity (0.762) to affect mortality. The mortality was significantly lower in high-risk Fournier's gangrene patients with early surgical intervention compared with late intervention (23.8% vs 68.8%, P = 0.007). CONCLUSIONS: The Simplified Fournier's Gangrene Severity Index is a quick and reliable screening tool for first-line physicians to identify high-risk patients with Fournier's gangrene (Simplified Fournier's Gangrene Severity Index >2) who have poor survival outcomes. We recommended early surgical intervention within 14.35 h to maximize the survival of high-risk Fournier's gangrene patients.

LRWD1 Regulates Microtubule Nucleation and Proper Cell Cycle Progression in the Human Testicular Embryonic Carcinoma Cells.

Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is a testis-specific protein that mainly expressed in the sperm neck where centrosome is located. By using microarray analysis, LRWD1 is identified as a putative gene that involved in spermatogenesis. However, its role in human male germ cell development has not been extensively studied. When checking in the semen of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia, the level of LRWD1 in the sperm neck was significantly reduced with a defective neck or tail. When checking the sub-cellular localization of LRWD1 in the cells, we found that LRWD1 resided in the centrosome and its centrosomal residency was independent of microtubule transportation in NT2/D1, the human testicular embryonic carcinoma, cell line. Depletion of LRWD1 did not induce centrosome re-duplication but inhibited microtubule nucleation. In addition, the G1 arrest were observed in LRWD1 deficient NT2/D1 cells. Upon LRWD1 depletion, the levels of cyclin E, A, and phosphorylated CDK2, were reduced. Overexpression of LRWD1 promoted cell proliferation in NT2/D1, HeLa, and 239T cell lines. In addition, we also observed that autophagy was activated in LRWD1 deficient cells and inhibition of autophagy by chloroquine or bafilomycin A1 promoted cell death when LRWD1 was depleted. Thus, we found a novel function of LRWD1 in controlling microtubule nucleation and cell cycle progression in the human testicular embryonic carcinoma cells. J. Cell. Biochem. 119: 314-326, 2018. © 2017 Wiley Periodicals, Inc.

MAEL promoter hypermethylation is associated with de-repression of LINE-1 in human hypospermatogenesis.

STUDY QUESTION: Does the hypermethylation of the maelstrom spermatogenic transposon silencer (MAEL) promoter and subsequent de-repression of transposable elements represent one of the causes of spermatogenic failure in infertile men? SUMMARY ANSWER: Experimental hypermethylation of a specific region (-131 to +177) of the MAEL promoter leads to decreased expression of MAEL with increased expression of the transposable element LINE-1 (L1) and in infertile men methylation of the MAEL promoter is associated with the severity of spermatogenic failure. WHAT IS KNOWN ALREADY: MAEL induces transposon repression in the male germline and is required for mammalian meiotic progression and post-meiotic spermiogenesis. Patients with non-obstructive azoospermia (NOA), defined as no sperm in the ejaculate due to spermatogenic failure, and histopathologically proven hypospermatogenesis (HS) is not uncommon and its etiology is largely unknown. STUDY DESIGN, SIZE, DURATION: Luciferase reporter assay and a targeted DNA methylation model were used to explore the effects of hypermethylation of MAEL promoter on gene expression. Germ cell-enriched testicular cells from infertile patients were used to determine the methylation levels of MAEL and expressions of MAEL and L1. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty-six patients with histopathologically proven NOA and HS and 12 patients with obstructive azoospermia and normal spermatogenesis (NS) were enrolled in this study. Demographic and clinical information were obtained. The severity of HS was determined by a spermatogenic scoring system. The methylation levels of 26 CpGs in the MAEL promoter was measured, and quantitative real-time RT-PCR was used to determine the expressional levels of MAEL and L1. MAIN RESULTS AND THE ROLE OF CHANCE: Targeted DNA methylation of MAEL promoter suppressed MAEL expression and de-repressed L1 activity in vitro. Patients with HS had significantly higher mean methylation levels of 26 consecutive CpGs in the MAEL promoter, compared to patients with NS. The MAEL methylation levels were negatively correlated with MAEL transcript levels and higher methylation level of MAEL was associated with severe spermatogenic defect. L1 transcript level was significantly higher in patients with HS. No differences in age, frequency of testicular insults and genetic anomalies was noted between patients with high or low MAEL methylation levels. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Because of the difficulty in the use of human germ cells for study, the in vitro targeted DNA methylation model was performed by using human NCI-H358 cells to explore the effects of MAEL methylation on transposable elements activity. Because the germ cell-enriched testicular cells isolated from a testicular sample were relatively few, the purity of cell populations was not determined. WIDER IMPLICATIONS OF THE FINDINGS: Measurement of the methylation level of MAEL gene may be feasible to predict the severity of spermatogenic failure or the outcome of testicular sperm retrieval. STUDY FUNDING/COMPETING INTERESTS: This work was supported through grants from the Ministry of Science and Technology of Taiwan (100-2314-B-006-017) and National Cheng Kung University Hospital, Tainan, Taiwan (NCKUH 20120266). The authors declare no conflicts of interest.

Validation and simplification of Fournier's gangrene severity index.

OBJECTIVES: To validate the predictive value of Fournier's Gangrene Severity Index in patients with Fournier gangrene and to facilitate patient mortality risk-stratification by simplifying the Fournier's Gangrene Severity Index. METHODS: From January 1989 to December 2011, 85 male patients with clinically-documented Fournier's gangrene undergoing intensive treatment and with complete medical records were recruited. The demographic information and nine parameters of Fournier's Gangrene Severity Index were compared between survivors and non-survivors. The parameters that showed a significant difference between the two groups were selected to generate a simplified scoring index. RESULTS: Of the 85 patients recruited, 16 patients died of the disease with mortality rate of 18.8%. The Fournier's Gangrene Severity Index score at initial diagnosis was significantly higher in non-survivors than in survivors. Of the nine parameters of Fournier's Gangrene Severity Index, the scores of serum creatinine level, hematocrit level and serum potassium level were significantly different between the two groups. However, the mean body temperatures, heart rate, respiration rate, white blood cell count, serum sodium and bicarbonate levels were non-significantly different. Of the 12 patients with chronic kidney disease or end-stage renal disease, 10 died of severe sepsis. A simplified scoring index including parameters of creatinine, hematocrit and potassium was generated, which provided sensitivity and specificity of 87% and 77% in predicting patient mortality, respectively. The predictive values of this simplified Fournier's Gangrene Severity Index were shown to be non-inferior to Fournier's Gangrene Severity Index in our patients. CONCLUSIONS: The simplified Fournier's Gangrene Severity Index is easy to use at initial diagnosis, and offers a way to compare outcomes in different clinical populations.

Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Δ) mice.

In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-codon-improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; gene(lox) (/)(Δ) genotype should be used to evaluate phenotypes instead of Cre; gene(lox/lox) owing to the fact that the latter usually bears incomplete deletion of the flox allele(s).

Diurnal temperature change is associated with testicular torsion: a nationwide, population based study in Taiwan.

PURPOSE: We investigated the association between climatic variables and testicular torsion in Taiwanese males. MATERIALS AND METHODS: Using the Taiwan Longitudinal Health Insurance Database, we reviewed the files of patients who were diagnosed with testicular torsion and underwent orchiectomy or orchiopexy between January 1996 and December 2008. Children younger than 1 year were excluded from the study. Climatic data were provided by the Taiwan Central Weather Bureau and included ambient temperature, relative humidity, diurnal temperature change and barometric pressure. Patients with acute appendicitis who underwent appendectomy were chosen as the control group. Climatic variables in relation to testicular torsion were analyzed using the Mann-Whitney U test and chi-square test, and seasonal climatic variations using the Kruskal-Wallis H test. Relative risk was calculated to compare the incidence of testicular torsion for diurnal temperature changes. RESULTS: A total of 65 patients with a mean age of 16.2 years presented with testicular torsion and were treated surgically. Four children younger than 1 year were excluded, and thus the study population consisted of 61 patients. The estimated incidence of testicular torsion was 2.58 per 100,000 person-years. There were no special climatic conditions on days of admission. However, 73.7% of the patients had testicular torsion when the diurnal temperature change was 6C or greater. Compared to the torsion rate for diurnal temperature changes less than 6C, the relative risk of testicular torsion at 6C or greater was 1.8 (p = 0.05). Average seasonal diurnal temperature change in the 2 days before hospitalization showed increases in all seasons except spring, which fluctuated. CONCLUSIONS: Diurnal temperature change was associated with testicular torsion and may be an etiological climatic factor affecting this condition. This is the first known study to demonstrate an association between diurnal temperature change and testicular torsion.

The MAEL expression in mitochondria of human spermatozoa and the association with asthenozoospermia.

PURPOSE: The maelstrom spermatogenic transposon silencer (MAEL) function in postmeiotic germ cells remains unclear, and its protein localization in human testis and spermatozoa awaits determination. This study aims to clarify the MAEL expression in human spermatogenesis and to explore its role in sperm function. MATERIALS AND METHODS: Twenty-seven asthenozoospermic men, 40 normozoospermic controls, and three obstructive azoospermic men were enrolled. The transcripts of MAEL in the seminiferous epithelium and MAEL downstream targets were identified by bioinformatics analysis. MAEL protein expression in human testis and ejaculated sperms were examined by immunohistochemical and immunogold staining, respectively. The roles of MAEL in mitochondria function were investigated by siRNA knockdown in human H358 cells. The association between MAEL protein levels and clinical sperm features was evaluated. RESULTS: Abundant MAEL was expressed in spermatid and spermatozoa of the human testis. Remarkably, MAEL was located in the mitochondria of ejaculated sperm, and bioinformatics analysis identified GPX4 and UBL4B as MAEL's downstream targets. Knockdown of MAEL sabotaged mitochondria function and reduced adenosine triphosphate (ATP) production in H358 cells. MAEL, GPX4, and UBL4B expression levels were significantly decreased in asthenozoospermic sperms than in controls. The MAEL protein levels were positively correlated with GPX4 and UBL4B in human sperm. Total motile sperm count (TMSC) was positively correlated with protein levels of MAEL, GPX4, and UBL4B in ejaculated sperms. CONCLUSIONS: We highlight prominent MAEL expression in the intratesticular spermatid and the mitochondria of ejaculated spermatozoa. MAEL directly binds to GPX4 and UBL4B, and loss of MAEL induces mitochondrial dysfunction. MAEL-mitochondrial function-motility relationship might advance our understanding of the causes of asthenozoospermia.

TGF-ß1 inhibits microvascular-like formation by decreasing VCAM1 and ICAM1 via the upregulation of SNAIL in human granulosa cells.

Three major endothelial cell junctional adhesion molecules (VCAM1, ICAM1 and E-SELECTIN) play important roles in the process of angiogenesis, a progression of extensive physiological vascularization that occurs during the formation of the corpus luteum. Our previous studies demonstrated that TGF-ß1 is a negative regulator of luteinization and progesterone production in luteinized human granulosa (hGL) cells. Whether TGF-ß1 can regulate the expression of these endothelial cell adhesion molecules and subsequent angiogenesis in hGL cells remains to be elucidated. Using dual inhibition approaches (small molecular inhibitors and siRNA-based knockdown), we provided the first data showing that TGF-ß1 significantly upregulates the expression of the SNAIL transcription factor, which in turn suppresses the expression of VCAM1 and ICAM1 in hGL cells. Additionally, we demonstrate that the suppressive effects on the expression of VCAM1 and ICAM1 induced by TGF-ß1 treatment were most likely via an ALK5-mediated SMAD-dependent signaling pathway. Furthermore, functional studies showed that hGL cells cultured on Matrigel exhibited two typical endothelial cell phenotypes, microvascular-like formation and a sprouting microvascular pattern. Notably, these phenotypes were significantly suppressed by either TGF-ß1 treatment or knockdown of VCAM1 and ICAM1. Our findings suggest that TGF-ß1 plays a potential role in the inhibition of granulosa cell angiogenesis by downregulating the expression of VCAM1 and ICAM1 during follicular development and corpus luteum formation.

The fertility preservation decision-making and testicular sperm retrieval outcome in older adolescents with nonmosaic Klinefelter syndrome and azoospermia.

BACKGROUND: This study aims to analyze the fertility preservation decision-making and the sperm retrieval rate (SRR) in older adolescents (age 15-19 years) with nonmosaic Klinefelter syndrome (KS) and azoospermia in a male reproductive clinic, and to determine the accumulated SRR in older adolescents by literature review. METHODS: Older adolescents with nonmosaic KS and azoospermia referred for hypogonadism and fertility concerns were enrolled. Reproductive counseling and fertility preservation options were offered to patients/parents. The acceptability and the reasons affecting the reproductive decision-making were analyzed. Patients/parents who agreed on fertility preservation received microdissection testicular sperm extraction (mTESE) and cryopreservation. A comprehensive literature review regarding the SRRs in older adolescents with KS was conducted. RESULTS: A total of eight older adolescents were enrolled. After fertility preservation counseling, three patients/parents (37.5%) agreed to receive mTESE, and spermatozoa were successfully retrieved in two. "Lack of interest" and "inconsistent sperm retrieval result" were the main reasons for refusal. A total of 89 older adolescents from nine articles, and ours were collected for SRR analysis. Most of the reports had a limited number of cases, and none of them described the acceptance rate of sperm retrieval in adolescents. Forty-three out of 89 older adolescents (48.3%) had successful sperm retrieval, and there was no significant difference in the SRR between the mTESE and conventional TESE. CONCLUSION: Successful testicular sperm retrieval in older adolescents with KS is not superior to those reported in adults. Adolescents and their parents should undergo a detailed reproductive consultation process and shared decision-making discussion before considering testicular sperm retrieval.

LRWD1 expression is regulated through DNA methylation in human testicular embryonal carcinoma cells.

BACKGROUND: Sperm growth and maturation are correlated with the expression levels of Leucine-rich repeat and WD repeat-containing protein 1 (LRWD1), a widely expressed protein in the human testicles. The decrease in LRWD1 cellular level was linked to the reduction in cell growth and mitosis and the rise in cell microtubule atrophy rates. Since DNA methylation has a major regulatory role in gene expression, this study aimed at exploring the effect of the modulation of DNA methylation on LRWD1 expression levels. RESULTS: The results revealed the presence of a CpG island up of 298 bps (- 253 ~ + 45) upon LRWD1 promoter in NT2/D1 cells. The hypermethylation of the LRWD1 promoter was linked to a reduction in the transcription activity in NT2/D1 cells, as indicated by luciferase reporter assay. The methylation activator, floxuridine, confirmed the decrease in the LRWD1 promoter transcriptional activity. On the other hand, 5-Aza-2'-deoxycytidine (5-Aza-dc, methylation inhibitor), significantly augmented LRWD1 promoter activity and the expression levels of mRNA and proteins. Furthermore, DNA methylation status of LRWD1 promoter in human sperm genomic DNA samples was analyzed. The results indicated that methylation of LRWD1 promoter was correlated to sperm activity. CONCLUSIONS: Thus, the regulation of LRWD1 expression is correlated with the methylation status of LRWD1 promoter, which played a significant role in the modulation of spermatogenesis, sperm motility, and vitality. Based on these results, the methylation status of LRWD1 promoter may serve as a novel molecular diagnostic marker or a therapeutic target in males' infertility.

RéSUMé: CONTEXTE: La croissance et la maturation des spermatozoïdes sont corrélées avec les niveaux d'expression de la protéine 1 riche en répétitions Leucine et contenant des répétitions WD (LRWD1), une protéine largement exprimée dans les testicules humains. La diminution du niveau cellulaire en LRWD1 a été liée à une réduction de la croissance et des mitoses cellulaires, et à une augmentation des taux d'atrophie des microtubules cellulaires. Puisque la méthylation de l'ADN joue un rôle régulateur majeur dans l'expression des gènes, cette étude visait à explorer l'effet de la modulation de la méthylation de l'ADN sur les niveaux d'expression de LRWD1. RéSULTATS: Les résultats ont révélé la présence d'un îlot CpP de 298 pbs (-253~+45) sur le promoteur de LRWD1dans les cellules NT2/D1. L'hyperméthylation du promoteur de LRWD1 était liée à une réduction de l'activité de transcription dans les cellules NT2/D1, comme indiqué par l'analyse de l'expression d'un gène rapporteur codant pour la luciférase. L'activateur de méthylation, la floxuridine, a confirmé la diminution de l'activité transcriptionnelle du promoteur de LRWD1. D'autre part, la 5-Aza-2'-déoxycytidine (5-Aza-dc, inhibiteur de méthylation), a significativement augmenté l'activité du promoteur de LRWD1 et les niveaux d'expression de l'ARNm et des protéines. En outre, le statut de méthylation de l'ADN du promoteur de LRWD1 dans les échantillons d'ADN génomique de sperme humain a été analysé. Les résultats ont indiqué que la méthylation du promoteur de LRWD1 était corrélée à l'activité des spermatozoïdes. CONCLUSIONS: Ainsi, la régulation de l'expression LRWD1 est corrélée avec le statut de méthylation du promoteur de LRWD1, qui a joué un rôle important dans la modulation de la spermatogenèse, de la mobilité et de la vitalité des spermatozoïdes. Sur la base de ces résultats, le statut de méthylation du promoteur de LRWD1 peut servir de nouveau marqueur diagnostic moléculaire ou de cible thérapeutique dans l'infertilité masculine.

The expression level of septin12 is critical for spermiogenesis.

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Fibroblast growth factor 9 stimulates steroidogenesis in postnatal Leydig cells.

Fibroblast growth factor 9 (FGF9) is a potent mitogen and survival factor required for morphogenesis during embryonic development and numerous biological functions at adulthood. The reproductive phenotype of mice lacking Fgf9 gene exhibits male to female sexual reversal, suggesting a crucial role of Fgf9 in male sex determination. Our previous study showed that polymorphic microsatellite of FGF9 genes is associated with 46XY female with ambiguous genitalia, implying that the aberrant expression of FGF9 might affect androgen secretion. In this study, we aimed to investigate the effect of FGF9 on testosterone production in mouse Leydig cell and to study the signalling pathways by which FGF9 modulate steroidogenesis. Our results show that mRNAs of Fgf9 and Fgfr isoforms (Fgfr2IIIc, Fgfr3 and Fgfr4) were all expressed in mouse Leydig cells. FGF9 significantly stimulates mouse Leydig cell testosterone production in a dose- and time-dependent manner. Ras-MAPK, PI3K and PKA signalling pathways are involved in the FGF9-induced steroidogenesis. These results provide supportive evidence linking the aberrant expression of FGF9 to human gonadal dysgenesis and suggest a role of FGF9 in postnatal testicular development.

Bone morphogenetic protein 6 affects cell-cell communication by altering the expression of Connexin43 in human granulosa-lutein cells.

Connexin 43 (Cx43)-coupled gap junctions in granulosa cells play an important role in follicular development, oocyte maturation, and corpus luteum maintenance. Bone morphogenetic protein 6 (BMP6) is highly expressed in human oocytes and granulosa cells and is involved in the regulation of female reproduction. Currently, whether oocyte- and granulosa cell-derived BMP6 affects the expression of Cx43 and its related gap junction intercellular communication (GJIC) activity in human granulosa cells remains unknown. In this study, we demonstrate that BMP6 treatment significantly suppressed the expression of Cx43 in both primary and immortalized (SVOG) human granulosa-lutein cells. Using both pharmacological inhibitors and small interfering RNA-mediated knockdown approaches, we demonstrate that ALK2 and ALK3 BMP type I receptors are involved in BMP6-induced suppressive effects on Cx43 expression and GJIC activity in SVOG cells. Furthermore, these cellular activities are most likely mediated by the SMAD1/SMAD5-SMAD4-dependent signaling pathway. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human Cx43 promoter. Our findings provide new insight into the molecular mechanisms by which an intrafollicular growth factor regulates cell-cell communication in human granulosa cells.

Gonadotrophin-releasing hormone-I and -II stimulate steroidogenesis in prepubertal murine Leydig cells in vitro.

AIM: To study the effect and mechanism of gonadotrophin-releasing hormone (GnRH) on murine Leydig cell steroidogenesis. METHODS: Purified murine Leydig cells were treated with GnRH-I and -II agonists, and testosterone production and steroidogenic enzyme expressions were determined. RESULTS: GnRH-I and -II agonists significantly stimulated murine Leydig cell steroidogenesis 60%-80% in a dose- and time-dependent manner (P < 0.05). The mRNA expressions of steroidogenic acute regulatory (StAR) protein, P450scc, 3beta-hydroxysteroid dehydrogenase (HSD), but not 17alpha-hydroxylase or 17beta-HSD, were significantly stimulated by both GnRH agonists with a 1.5- to 3-fold increase (P < 0.05). However, only 3beta-HSD protein expression was induced by both GnRH agonists, with a 1.6- to 2-fold increase (P < 0.05). CONCLUSION: GnRH directly stimulated murine Leydig cell steroidogenesis by activating 3b-HSD enzyme expression.

The prophylactic protective effect of sesamol against ferric-nitrilotriacetate-induced acute renal injury in mice.

The aim of this study was to examine the prophylactic protective effects of 3,4-methylenedioxyphenol (sesamol) on ferric-nitrilotriacetate (Fe-NTA)-induced acute renal damage in mice. We induced acute renal injury in mice by treating them with 4 mg/kg of Fe-NTA for 3h. We used blood biochemistry, creatinine clearance, and histological examinations to assess renal function. With a high-performance chemiluminescence analyzer, we also determined the hydroxyl radical and superoxide anion levels (free radicals) generated. Renal xanthine oxidase activities were also assessed. Sesamol inhibited Fe-NTA-induced acute renal injury, renal lipid peroxidation, the levels of renal hydroxyl radical and superoxide anion generated, and the activity of xanthine oxidase in mice. Therefore, we concluded that sesamol protected mice against Fe-NTA-induced oxidative-stress-associated acute renal injury by at least partially inhibiting the production of reactive oxygen species.

Post-coital gross hematuria: an unusual presentation of benign prostatic hyperplasia.

AIM: To describe an unusual symptom of benign prostatic hyperplasia (BPH). METHODS: A patient presented to our urology clinic having experienced post-coital gross hematuria for 2 years. He had not experienced lower urinary tract symptoms (LUTS). A series of examinations were performed to determine the source of bleeding. RESULTS: The prostate was defined as the active bleeding source responsible for the patient's post-coital hematuria. Endoscopic fulguration did not alleviate the symptom. The use of dutasteride, a dual inhibitor of 5alpha-reductase, solved the problem. CONCLUSION: This study reports for the first time that post-coital gross hematuria is one of the clinical presentations of BPH, which can be successfully treated with 5alpha-reductase inhibitor.

Causes and Clinical Features of Infertile Men With Nonobstructive Azoospermia and Histopathologic Diagnosis of Hypospermatogenesis.

OBJECTIVE: To analyze the causes and the clinical features of infertile men with nonobstructive azoospermia and hypospermatogenesis (HS). MATERIALS AND METHODS: This retrospective cohort study included 100 patients with nonobstructive azoospermia and HS and 8 patients with obstructive azoospermia and normal spermatogenesis. The severity of HS was subdivided into 3 groups (mild, moderate, and severe) based on spermatogenic score. Data of history, physical findings, serum hormone profiles, genetic studies, and sperm retrieval rate were collected. Whole genome DNA methylation analysis and microarray mRNA expression analysis were used to identify the candidate genes of methylation dysregulation in HS. RESULTS: Thirty-two (32%) patients had at least 1 prior/current testicular insults and 13 (13%) patients had genetic anomalies. Fifty-five (55%) patients were categorized as idiopathic HS. Patients with mild HS had a higher frequency of testicular insults, and patients with severe HS had a significantly higher frequency of genetic anomalies. Sperm retrieval rate was 100%, 100%, and 88.4% for patients with mild, moderate, and severe HS, respectively. Four sterility-related genes, including BOLL, DDX4, HORMAD1, and MAEL, were found to have increased methylation at CpGs of the promoter regions and decreased mRNA expressions in HS testis. CONCLUSION: The causes of HS are complex and multifactorial. The main causes of HS were prior or current testicular insults and chromosomal or genetic anomalies. More than half of the patients were categorized as idiopathic HS. With high throughput analysis, methylation dysregulations of BOLL, DDX4, HORMAD1, and MAEL are believed to be associated with HS.