Proper phosphorylation of septin 12 regulates septin 4 and soluble adenylyl cyclase expression to induce sperm capacitation.

Septin-based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12- and Sept4-null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4-null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation-deficient (S196A), and SEPT4-depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E- and Sept12 S196A-knock-in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E- and S196A-mutant mouse sperm. In the sperm of the SEPT4-knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation.

Childhood neurodevelopmental disorders and maternal diabetes: A population-based cohort study.

AIM: To assess the risk of a wide spectrum of neurodevelopmental disorders (NDDs) in offspring of mothers with type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM). METHOD: This retrospective cohort study included 877 233 singletons born between 2004 and 2008 in Taiwan. Children were followed up to 2015 for diagnoses of NDDs, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), developmental delay, intellectual disability, cerebral palsy, and epilepsy/infantile spasms using health insurance claims data. We performed Cox regression models to estimate the relative risks of NDDs associated with maternal diabetes. Covariates included parental age, year of birth, child sex, family income, urbanization level, hypertensive disorder, and preterm delivery status. RESULTS: In utero there were 338 (0.04%) children exposed to T1DM, 8749 (1.00%) to T2DM, and 90 200 (10.28%) to GDM. The effect of T1DM on NDDs was the largest, followed by T2DM, then GDM. T1DM was associated with an increased risk of developmental delay, intellectual disability, and epilepsy/intellectual spasms in offspring. T2DM was associated with an increased risk of ASD, ADHD, developmental delay, intellectual disability, cerebral palsy, and epilepsy/intellectual spasms. GDM was associated with an increased risk of ASD, ADHD, and developmental delay. INTERPRETATION: Maternal diabetes during pregnancy, including T1DM, T2DM, and GDM, is associated with an increased risk of some NDDs in offspring.

Preterm birth and weight-for-gestational age for risks of autism spectrum disorder and intellectual disability: A nationwide population-based cohort study.

PURPOSE: To evaluate gestational age (GA) and small-for-gestational age (SGA) as continuums and gender on the incidences of autism spectrum disorder (ASD) and co-occurring intellectual disability (ID). METHODS: This is a population-based cohort study using the 2004-2008 Taiwan Maternal and Child Health Database. The diagnosis of ASD was determined by International Classification of Diseases, 9th Revision (ICD-9). Generalized estimating equations models were fit to evaluate associations between perinatal variables and ASD. RESULTS: This study included 916,315 individuals. A total of 9474 (1.0%) children were diagnosed with ASD, among whom 1594 (16.8%) had co-occurring ID. Lower GA carried higher odds of ASD with ID (GA < 28 weeks, aOR: 4.26, 95% CI: 2.13, 8.50; GA 28-30 weeks, aOR: 2.80, 95% CI: 1.57, 4.97; GA 31-33 weeks, aOR: 1.63, 95% CI: 1.05, 2.55; GA 34-36 weeks, aOR: 1.39, 95% CI: 1.16, 1.67) and ASD without ID (GA < 28 weeks, aOR:2.05, 95% CI: 1.25, 3.36; GA 28-30 weeks, aOR: 2.02, 95% CI: 1.46, 2.79; GA 31-33 weeks, aOR: 1.42, 95% CI: 1.13, 1.77; GA 34-36 weeks, aOR: 1.18, 95% CI: 1.08, 1.29). Male preterm infants had ASD risks negatively correlated to GA, while ASD risks were significantly increased only among female infants born late preterm. The degree of SGA showed a stepwise increased risk for ASD with and without ID in both male and female infants. CONCLUSION: Lower GA and the degree of SGA are both associated with ASD susceptibility, either with or without co-occurring ID, and remarkably increased the risk of ID.

Effectiveness of mental health website intervention on stress and depression for women with recurrent miscarriage: A randomized controlled trial.

We aimed to investigate the effect of a mental health website intervention on perceived stress, depression, sleep quality, and social support in women with recurrent miscarriage (RM). Performing a randomized controlled trial, the participants in the experimental group (n = 31) received a 12-week mental health website intervention; the participants in the control group (n = 31) received RM standard medical care only. The paired t-tests results for the mean posttest scores for depression (p = .023) and perceived stress (p = .041) in the experimental group showed a significant decrease, but did not in the control group.

Incontinentia pigmenti in a male infant and a proposed diagnostic algorithm.

It is extremely rare for males with incontinentia pigmenti to survive. We summarize a diagnostic evaluation protocol for such individuals to provide an explanation for male survival.

Septin 7 is a centrosomal protein that ensures S phase entry and microtubule nucleation by maintaining the abundance of p150glued.

Septins play important roles in regulating development and differentiation. Septin 7 (SEPT7) is a crucial component in orchestrating the septin core complex into highly ordered filamentous structures. Here, we showed that genetic depletion of SEPT7 or treatment with forchlorfenuron (FCF; a compound known to affect septin filament assembly) led to reduced the S phase entry in cell models and zebrafish embryos. In addition to colocalizing with actin filaments, SEPT7 resided in the centrosome, and SEPT7 depletion led to aberrant mitotic spindle pole formation. This mitotic defect was rescued in SEPT7-deficient cells by wild-type SEPT7, suggesting that SEPT7 maintained mitotic spindle poles. In addition, we observed disorganized microtubule nucleation and reduced cell migration with SEPT7 depletion. Furthermore, SEPT7 formed a complex with and maintained the abundance of p150glued , the component of centriole subdistal appendages. Depletion of p150glued resulted in a phenotype reminiscent of SEPT7-deficient cells, and overexpression of p150glued reversed the defective phenotypes. Thus, SEPT7 is a centrosomal protein that maintains proper cell proliferation and microtubule array formation via maintaining the abundance of p150glued .

Parental Socioeconomic Status and Autism Spectrum Disorder in Offspring: A Population-Based Cohort Study in Taiwan.

Studies from the United States have shown increasing incidence of autism spectrum disorder (ASD) with increasing socioeconomic status (SES), whereas in Scandinavian countries, no such relation was identified. We investigated how ASD risk in offspring varied according to parental SES in Taiwan, where there is universal health care. Through linking birth reporting data and data from Taiwan's national health insurance program, we studied 706,111 singleton births from 2004 to 2007 and followed them until 2015. Parental SES was determined by monthly salary at the time of childbirth, and child neuropsychiatric outcomes were defined using International Classification of Diseases codes. We identified 7,323 ASD cases and 7,438 intellectual disability (ID) cases; 17% of ASD cases had co-occurring ID. In multivariable Cox regression analysis, higher SES was independently associated with higher risk of ASD after we took into account urbanization levels, child sex, parental age, and other covariates. By contrast, higher SES was independently associated with lower risk of ID. Besides the SES disparity in ASD case ascertainment and in the access to health care, findings from Taiwan suggest that other social, environmental, biological, and immunological factors linked with parental SES levels may contribute to the positive relation of SES and ASD risk.

MutS protein-based fiber optic particle plasmon resonance biosensor for detecting single nucleotide polymorphisms.

A new biosensing method is presented to detect gene mutation by integrating the MutS protein from bacteria with a fiber optic particle plasmon resonance (FOPPR) sensing system. In this method, the MutS protein is conjugated with gold nanoparticles (AuNPs) deposited on an optical fiber core surface. The target double-stranded DNA containing an A and C mismatched base pair in a sample can be captured by the MutS protein, causing increased absorption of green light launching into the fiber and hence a decrease in transmitted light intensity through the fiber. As the signal change is enhanced through consecutive total internal reflections along the fiber, the limit of detection for an AC mismatch heteroduplex DNA can be as low as 0.49 nM. Because a microfluidic chip is used to contain the optical fiber, the narrow channel width allows an analysis time as short as 15 min. Furthermore, the label-free and real-time nature of the FOPPR sensing system enables determination of binding affinity and kinetics between MutS and single-base mismatched DNA. The method has been validated using a heterozygous PCR sample from a patient to determine the allelic fraction. The obtained allelic fraction of 0.474 reasonably agrees with the expected allelic fraction of 0.5. Therefore, the MutS-functionalized FOPPR sensor may potentially provide a convenient quantitative tool to detect single nucleotide polymorphisms in biological samples with a short analysis time at the point-of-care sites.

Childhood neurodevelopmental disorders and maternal hypertensive disorder of pregnancy.

AIM: To examine the association of maternal chronic hypertension and pregnancy-induced hypertension (PIH)/preeclampsia with childhood neurodevelopmental disorders (NDDs) in a large-scale population-based cohort. METHOD: We conducted a linked Taiwan National Health Insurance Research Database cohort study of children born between 2004 and 2008 (n=877 233). Diagnoses of autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), developmental delay, intellectual disability, cerebral palsy (CP), and epilepsy/infantile spasms were identified from birth to the end of 2015. Cox proportional hazards models were fitted with adjustment for potential confounders to estimate the effect of maternal hypertensive disorder of pregnancy on childhood outcomes. RESULTS: Compared with the offspring of unexposed mothers, offspring of mothers with chronic hypertension or PIH/preeclampsia exhibited increased risk of developing a wide spectrum of NDDs. Chronic hypertension was associated with increased risks of ADHD (hazard ratio 1.22, 95% confidence interval [CI] 1.13-1.​31), developmental delay (1.29, 1.21-1.38), intellectual disability (1.67, 1.43-1.95), CP (1.45, 1.14-1.85), and epilepsy/infantile spasms (1.31, 1.10-1.56) in the offspring, whereas PIH/preeclampsia was associated with increased risks of ASD (1.27, 1.12-1.43), ADHD (1.23, 1.17-1.29), developmental delay (1.29, 1.24-1.35), intellectual disability (1.53, 1.37-1.71), CP (1.44, 1.22-1.70), and epilepsy/infantile spasms (1.36, 1.22-1.52) in the offspring after adjustment for potential confounders. The co-occurrence of maternal diabetes, preterm deliveries, or fetal growth restriction further increased the risk. INTERPRETATION: Chronic hypertension or PIH/preeclampsia seems to be sufficient to increase the risk of childhood NDDs. What this paper adds Children exposed to maternal hypertensive disorders have a higher cumulative incidence of neurodevelopmental disorders (NDDs) than unexposed children. Chronic hypertension or pregnancy-induced hypertension/preeclampsia seems to be sufficient to increase the risk of childhood NDDs. Co-occurrence of maternal diabetes, preterm deliveries, or fetal growth restriction further increases the risk.

The SEPT12 complex is required for the establishment of a functional sperm head-tail junction.

The connecting pieces of the sperm neck link the flagellum and the sperm head, and they are important for initiating flagellar beating. The connecting pieces are important building blocks for the sperm neck; however, the mechanism of connecting piece assembly is poorly understood. In the present study, we explored the role of septins in sperm motility and found that Sept12D197N knock-in (KI) mice produce acephalic and immotile spermatozoa. Electron microscopy analysis showed defective connecting pieces in sperm from KI mice, indicating that SEPT12 is required for the establishment of connecting pieces. We also found that SEPT12 formed a complex with SEPT1, SEPT2, SEPT10 and SEPT11 at the sperm neck and that the D197N mutation disrupted the complex, suggesting that the SEPT12 complex is involved in the assembly of connecting pieces. Additionally, we found that SEPT12 interacted and colocalized with γ-tubulin in elongating spermatids, implying that SEPT12 and pericentriolar materials jointly contribute to the formation of connecting pieces. Collectively, our findings suggest that SEPT12 is required for the formation of striated columns, and the capitulum and for maintaining the stability of the sperm head-tail junction.

Urinary phthalate metabolites are associated with biomarkers of DNA damage and lipid peroxidation in pregnant women - Tainan Birth Cohort Study (TBCS).

Phthalate exposure and oxidative stress have been linked to adverse reproductive outcomes in experimental studies, whereas no clear line has been drawn for human, especially in pregnant women. This study explored relationships between urinary phthalate metabolites and biomarkers of lipid peroxidation and oxidative and nitrosative DNA damage. Measurements from 97 Taiwanese pregnant women were taken at three different times during second and third trimesters. Five oxidative/nitrosative stress biomarkers - 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-nitroguanine (8-NO2Gua), 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA), 8-isoprostaglandin F2α (8-isoPF2α), and malondialdehyde (MDA), and 11 phthalate metabolites were measured in urine samples. Linear regressions in each visit and linear mixed-model regressions were fitted to estimate percent changes in oxidative/nitrosative stress biomarkers resulting from inter-tertile increase of phthalate metabolite level and the cumulative concentrations of di (2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate. The highest urine concentrations of phthalate metabolites and the greatest number of significant positive associations between phthalate metabolites and oxidative/nitrosative stress biomarkers were observed in the third visit and in repeated measurements analysis, respectively. Of the biomarkers related to DNA damage, 8-OHdG (25.4% inter-tertile increase for mono-iso-butyl phthalate) was more sensitive to phthalate exposure than 8-NO2Gua. Among the biomarkers of lipid peroxidation, HNE-MA (61.2% inter-tertile increase for sum of DEHP metabolites) was more sensitive than 8-isoPF2α and MDA. Our findings support the hypothesis that pregnant phthalate exposure increases the oxidative stress biomarkers of DNA damage and lipid peroxidation. Future research may elucidate the mediating roles of oxidative/nitrosative stress biomarkers in the link between phthalate exposure and adverse reproductive outcomes.

Does sex matter? Association of fetal sex and parental age with pregnancy outcomes in Taiwan: a cohort study.

BACKGROUND: Worldwide several studies have examined the associations of fetal sex, paternal age and maternal age with pregnancy outcomes, with the evidence regarding paternal age being less consistent. Although in Taiwan we keep good records on birth certificates, these issues have been seldom researched. Our objective was to assess the association of fetal sex and parental age with gestational hypertension/preeclampsia, eclampsia and preterm delivery in the Taiwanese population. METHODS: We conducted a nationwide study and included 1,347,672 live births born between 2004 and 2011 in Taiwan. Gestational hypertension/preeclampsia and eclampsia were ascertained based on the International Classification of Diseases codes; preterm delivery (< 37 weeks) was defined according to the gestational age documented by healthcare providers. We implemented logistic regression models with covariates adjusted to assess the association of fetal sex and parental age with pregnancy outcomes. RESULTS: The prevalence was 2.27% for gestational hypertension/preeclampsia, 0.07% for eclampsia and 6.88% for preterm delivery. After considering other parent's age and covariates, we observed a significantly stepped increase in the risk of both gestational hypertension/preeclampsia and preterm delivery as paternal and maternal age increased. For example, fathers aged ≥50 years were associated with a significantly higher risk of gestational hypertension/preeclampsia (odds ratio [OR]: 1.60, 95% CI: 1.39, 1.84) and preterm delivery (OR: 1.38, 95% CI: 1.27, 1.51) than fathers aged 25-29 years. Analysis on fetal sex showed that relatively more female births were linked to gestational hypertension/preeclampsia and more male births linked to preterm delivery, compared to the whole population. CONCLUSIONS: We found both paternal and maternal age, as well as fetal sex, were associated with the risk of pregnancy outcomes. Some findings on fetal sex contradicted with previous research using non-Asian samples, suggesting that ethnicity may play a role in the association of fetal sex and pregnancy outcomes. Besides, there is a need to counsel couples who are planning their family to be aware of the influence of both advanced maternal and paternal age on their pregnancy outcomes.

SEPT12 phosphorylation results in loss of the septin ring/sperm annulus, defective sperm motility and poor male fertility.

Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.

Stress, anxiety and depression perceived by couples with recurrent miscarriage.

AIM: Recurrent miscarriage is considered a major life event. The main purposes of this study were to compare the differences in stress, anxiety, social support, sleep quality and depressive symptoms in couples experiencing recurrent miscarriage compared to peers who experience full-term normal childbirth in southern Taiwan. METHODS: Convenience sampling and snowball sampling were used respectively to recruit 78 couples with and 80 couples without recurrent miscarriage from October 2014 to July 2015. Five structured questionnaires including Perceived Stress Scale, State- Anxiety Inventory, Interpersonal Support Evaluation List, Pittsburgh Sleep Quality Index and Edinburgh Depression Scale were administered. RESULTS: Women who experienced recurrent miscarriage perceived significantly higher levels of stress, anxiety and depressive symptoms than their husbands. Women in the recurrent miscarriage group reported significantly greater depressive symptoms than women of the other group. A stepwise multiple regression analysis indicated key predictors of depressive symptoms among women of childbearing age, accounting for 62.9% of the variance, were anxiety, stress, social support and history of recurrent miscarriage. CONCLUSION: Women with recurrent miscarriage suffer mild to moderate depressive symptoms and a greater incidence of depression than their peers who experienced normal childbirth. Health professionals can use the knowledge gained from these findings to evaluate women with recurrent miscarriage for stress, anxiety and depressive symptoms and develop supportive interventions.

Elevated miR-200a and miR-141 inhibit endocrine gland-derived vascular endothelial growth factor expression and ciliogenesis in preeclampsia.

KEY POINTS: Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is a critical factor that facilitates trophoblast invasion in placenta. Plasma miR-141 and miR-200a levels were elevated, while EG-VEGF was decreased in peripheral blood and placenta of preeclamptic patients. Furthermore, numbers of cilia in the placenta from preeclamptic women were significantly decreased. Elevated miR-141 and miR-200a inhibited the expression of EG-VEGF, downstream extracellular signal-regulated kinase (ERK)/matrix metalloproteinase 9 signalling and cilia formation, thus leading to defective trophoblast invasion. The growth of the primary cilium, which transduced ERK signalling upon EG-VEGF induction for proper trophoblast invasion, was also inhibited by miR-141 and miR-200a upregulation. ABSTRACT: Preeclampsia is a severe gestational complication, and inadequate trophoblast invasion during placental development is an important pathoaetiology. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is a critical factor that facilitates trophoblast invasion in placenta. By binding to the primary cilium, EG-VEGF initiates the signalling cascade for proper embryo implantation and placental development. The miR-200 family was predicted to target the EG-VEGF 5'-untranslated region, and its specific binding site was confirmed using a dual luciferase and a co-transfection assay. In the peripheral blood and placenta of preeclamptic patients, EG-VEGF showed significantly lower expression, whereas plasma miR-141 and miR-200a had higher expression compared with the controls. The biological significance of miR-141 and miR-200a was verified using an overexpression method in a trophoblast cell line (HTR-8/SVneo). Elevated miR-141 and miR-200a inhibited the expression of EG-VEGF, matrix metalloproteinase 9 (MMP9) and downstream extracellular signal-regulated kinase (ERK) signalling, thus leading to defective trophoblast invasion. Additionally, the growth of the primary cilium, which transduces ERK/MMP9 signalling upon EG-VEGF induction, was inhibited by miR-141 and miR-200a upregulation. Furthermore, the number of cilia in the human placenta of preeclamptic women was significantly decreased compared to normal placenta. In conclusion, the study uncovers the clinical correlations among the miR-200 family, EG-VEGF and the primary cilium in preeclampsia and the underlying molecular mechanisms. The results indicate that miR-141 and miR-200a directly targeted EG-VEGF, suppressed primary cilia formation and inhibited trophoblast invasion. Thus, miR-141 and miR-200a could be explored as promising miRNA biomarkers and therapeutic targets in preeclampsia.

NLRP7 Is Involved in the Differentiation of the Decidual Macrophages.

Macrophage polarization, regulated appropriately, may play important roles in successful pregnancy. In the face of the vital roles of decidua macrophages in pregnancy, it is insufficient to recognize the trigger of macrophage differentiation and polarization. We aimed to explore the link between the NLRP7 gene and macrophage polarization in human deciduas. Here, we enrolled the endometrial tissues from eight pregnant women in the first trimester. We found that NLRP7 was abundant in endometrial tissues and that NLRP7 was expressed in decidual macrophages of the first-trimester pregnancy. NLRP7 was predominately expressed in the decidual M2 macrophages, as compared with the M1 macrophages. Furthermore, our results suggest that NLRP7 is associated with decidual macrophage differentiation. NLRP7 over-expression suppresses the expression of M1 markers and enhances the expression of the M2 markers. Considering that NLRP7 relates to decidualization and macrophage differentiation, we propose that NLRP7 is a primate-specific multitasking gene to maintain endometrial hemostasis and reproductive success. This finding may pave the way for therapies of pathological pregnancies.

Fetuin-A Inhibits Placental Cell Growth and Ciliogenesis in Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a type of unbalanced glucose tolerance that occurs during pregnancy, which affects approximately 10% of pregnancies worldwide. Fetuin-A is associated with insulin resistance, and the concentration of circulating fetuin-A increases in women with GDM, however, the role of fetuin-A in the placenta remains unclear. In this study, we enrolled placental samples from twenty pregnant women with GDM and twenty non-GDM pregnant women and found that the abundance of fetuin-A was upregulated in terms of mRNA and protein levels. Fetuin-A inhibited placental cell growth by inducing apoptosis and inhibiting S phase entry. Irregular alignment of mitotic chromosomes and aberrant mitotic spindle poles were observed. In addition, centrosome amplification was induced by fetuin-A treatment, and these amplified centrosomes nucleated microtubules with disorganized microtubule arrays in placental cells. Furthermore, fetuin-A inhibited autophagy, and thus blocked the growth of the primary cilium, a cellular antenna that regulates placenta development and differentiation. Thus, our study uncovered the novel function of fetuin-A in regulating placental cell growth and ciliogenesis.

Testis-Specific SEPT12 Expression Affects SUN Protein Localization and is Involved in Mammalian Spermiogenesis.

Male infertility is observed in approximately 50% of all couples with infertility. Intracytoplasmic sperm injection (ICSI), a conventional artificial reproductive technique for treating male infertility, may fail because of a severe low sperm count, immotile sperm, immature sperm, and sperm with structural defects and DNA damage. Our previous studies have revealed that mutations in the septin (SEPT)-coding gene SEPT12 cause teratozoospermia and severe oligozoospermia. These spermatozoa exhibit morphological defects in the head and tail, premature chromosomal condensation, and nuclear damage. Sperm from Sept12 knockout mice also cause the developmental arrest of preimplantation embryos generated through in vitro fertilization and ICSI. Furthermore, we found that SEPT12 interacts with SPAG4, a spermatid nuclear membrane protein that is also named SUN4. Loss of the Spag4 allele in mice also disrupts the integration nuclear envelope and reveals sperm head defects. However, whether SEPT12 affects SPAG4 during mammalian spermiogenesis remains unclear. We thus conducted this study to explore this question. First, we found that SPAG4 and SEPT12 exhibited similar localizations in the postacrosomal region of elongating spermatids and at the neck of mature sperm through isolated murine male germ cells. Second, SEPT12 expression altered the nuclear membrane localization of SPAG4, as observed through confocal microscopy, in a human testicular cancer cell line. Third, SEPT12 expression also altered the localizations of nuclear membrane proteins: LAMINA/C in the cells. This effect was specifically due to the expression of SEPT12 and not that of SEPT1, SEPT6, SEPT7, or SEPT11. Based on these results, we suggest that SEPT12 is among the moderators of SPAG4/LAMIN complexes and is involved in the morphological formation of sperm during mammalian spermiogenesis.

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.