Functional screening of congenital heart disease risk loci identifies 5 genes essential for heart development in zebrafish.

Congenital heart disease (CHD) is the most common birth defect worldwide and a main cause of perinatal and infant mortality. Our previous genome-wide association study identified 53 SNPs that associated with CHD in the Han Chinese population. Here, we performed functional screening of 27 orthologous genes in zebrafish using injection of antisense morpholino oligos. From this screen, 5 genes were identified as essential for heart development, including iqgap2, ptprt, ptpn22, tbck and maml3. Presumptive roles of the novel CHD-related genes include heart chamber formation (iqgap2 and ptprt) and atrioventricular canal formation (ptpn22 and tbck). While deficiency of maml3 led to defective cardiac trabeculation and consequent heart failure in zebrafish embryos. Furthermore, we found that maml3 mutants showed decreased cardiomyocyte proliferation which caused a reduction in cardiac trabeculae due to inhibition of Notch signaling. Together, our study identifies 5 novel CHD-related genes that are essential for heart development in zebrafish and first demonstrates that maml3 is required for Notch signaling in vivo.

Key proteins of proteome underlying sperm malformation of rats exposed to low fenvalerate doses are highly related to P53.

Fenvalerate (Fen) is an endocrine disruptor, capable of interfering with the activity of estrogen and androgen. Our objective was to explore the molecular mechanisms of Fen on sperm in vivo. Adult male Sprague-Dawley rats were orally exposed to 0, 0.00625, 0.125, 2.5, 30 mg/kg/day Fen for 8 weeks. Sperm morphology, differential proteomics of sperm and testes, bioinformatic analysis, western blotting (WB), and RT-PCR were used to explore the mechanism of Fen on sperm. Data showed that low Fen doses significantly induced sperm malformations. In sperm proteomics, 47 differentially expressed (DE) proteins were enriched in biological processes (BPs) related to energy metabolism, response to estrogen, spermatogenesis; and enriched in cellular components (CCs) relating to energy-metabolism, sperm fibrous sheath and their outer dense fibers. In testicular proteomics, 56 DE proteins were highly associated with mRNA splicing, energy metabolism; and enriched in CCs relating to vesicles, myelin sheath, microtubules, mitochondria. WB showed that the expression of selected proteins was identical to their tendency in 2D gels. Literature indicates that key DE proteins in proteomic profiles (such as Trap1, Hnrnpa2b1, Hnrnpk, Hspa8, and Gapdh) are involved in P53-related processes or morphogenesis or spermatogenesis. Also, P53 mRNA and protein levels were significantly increased by Fen; bioinformatic re-analysis showed that 88.5% DE proteins and P53 formed a complex interacting network, and the key DE proteins were coenriched with P53-related BPs. Results indicate that key DE proteins of proteome underlying sperm malformations of rats exposed to low Fen doses are highly related to P53.

Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis.

BACKGROUND: Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood. METHODS: We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system. RESULTS: Four low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure. CONCLUSION: Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.

Spermatogenesis is normal in Tex33 knockout mice.

Testis expressed gene 33 (Tex33) is a recently reported testis-specific gene and it is evolutionarily conserved in vertebrates. The Tex33 expression is found in cytoplasm of round spermatids in Mus musculus. However, the in vivo function of Tex33 remains unknown. In this study, we made a 62bp in frame deletion on Exon2 of Tex33 gene by CRISPR/Cas9 in C57B/L6 mouse, which cause frame shift mutation of Tex33 gene. Tex33 -/-adult male were fertile, and there is no significant change on litter size compared with male wildtype (Tex33 +/+) adult. Besides, no overt differences were found in testis/body weight ratios, testicular/epididymal tissue morphology, sperm counts, sperm morphology and spermatozoa motility in adult Tex33-/- male mice (N = 3), in comparison with Tex33 +/+ adult (N = 3). TUNEL assay also indicates the germ cells apoptosis ratio was not significantly changed in adult Tex33-/- adult male mouse testis (N = 3), compared with adult Tex33+/+ male (N = 3). Importantly, the first wave of elongating spermatids formation happens in 5w old mice. We find that the first wave of spermiogenesis is not disrupted in both 5-week-old Tex33+/+ and Tex33 -/-male mouse testes and three hallmarks of spermatogenesis, PLZF,γ-H2AX and TNP1, are all detectable in seminiferous tubule. All results indicate that Tex33 is a redundant gene to spermatogenesis. This study can help other researchers avoid repetitive works on redundant genes.

The heat shock protein family gene Hspa1l in male mice is dispensable for fertility.

BACKGROUND: Heat shock protein family A member 1 like (Hspa1l) is a member of the 70kD heat shock protein (Hsp70) family. HSPA1L is an ancient, evolutionarily conserved gene with a highly conserved domain structure. The gene is highly abundant and constitutively expressed in the mice testes. However, the role of Hspa1l in the testes has still not been elucidated. METHODS: Hspa1l-mutant mice were generated using the CRISPR/Cas9 system. Histological and immunofluorescence staining were used to analyze the phenotypes of testis and epididymis. Apoptotic cells were detected through TUNEL assays. Fertility and sperm motilities were also tested. Quantitative RT-PCR was used for analyzing of candidate genes expression. Heat treatment was used to induce heat stress of the testis. RESULTS: We successfully generated Hspa1l knockout mice. Hspa1l -/- mice exhibited normal development and fertility. Further, Hspa1l -/- mice shown no significant difference in spermatogenesis, the number of apoptotic cells in testes epididymal histology, sperm count and sperm motility from Hspa1l +/+ mice. Moreover, heat stress does not exacerbate the cell apoptosis in Hspa1l -/- testes. These results revealed that HSPA1L is not essential for physiological spermatogenesis, nor is it involved in heat-induced stress responses, which provides a basis for further studies.

In vitro testicular organogenesis from human fetal gonads produces fertilization-competent spermatids.

Unlike most organs that mature during the fetal period, the male reproductive system reaches maturity only at puberty with the commencement of spermatogenesis. Robust modelling of human testicular organogenesis in vitro would facilitate research into mechanisms of and factors affecting human spermatogenic failure and male fertility preservation in prepubertal tumor patients. Here, we report successful recapitulation of human testicular organogenesis in vitro from fetal gonadal ridge. Our model displayed the formation of mature seminiferous epithelium and self-renewing spermatogonia. Remarkably, in vitro-derived haploid spermatids have undergone meiotic recombination, and showed increased genetic diversity as indicated by genetic analysis. Moreover, these spermatids were able to fertilize oocytes and support subsequent blastocyst formation. The in vitro testicular organogenesis system described here will play an important role in elucidating the regulation of human testis development and maintaining male fertility in prepubertal cancer patients.

Clinical and transcriptional signatures of human CD204 reveal an applicable marker for the protumor phenotype of tumor-associated macrophages in breast cancer.

BACKGROUND: Tumor-associated macrophages in human breast cancer are poorly understood. Specific tumor-associated macrophage-related molecular mechanisms among different intrinsic molecular subtypes remain unclear. Here, we have identified and explored the roles of the tumor-associated macrophages novel marker: CD204 in different subtypes of breast cancer. RESULTS: CD204 was upregulated in four subtypes of breast cancer, and this was associated with poor survival outcomes. CD204 could promote tumor cell proliferation, migration, and invasion and was involved in immune system-related pathways among all subtypes. Special pathways in each subtype were also found. High CD204 mRNA expressions were associated with high proportions of protumor immune cell populations, and most immunoinhibitors positive correlated with CD204 expression in all subtypes. CONCLUSIONS: These findings contribute to a better understanding and managing the protumor phenotype of tumor-associated macrophages in different subtypes of breast cancer. METHODS: The expression of CD204 and its clinical outcome were analyzed. The roles of CD204 in the regulation of tumor cell proliferation, migration, and invasion were studied. Potential pathways influenced by CD204 were displayed. Immune cell infiltration in different CD204 mRNA expression status and correlations between CD204 and immunoinhibitors were also analyzed.

Proteomic Analysis of Dpy19l2-Deficient Human Globozoospermia Reveals Multiple Molecular Defects.

PURPOSE: To investigate the differences in protein expression between Dpy19l2-deficient human globozoospermia and normozoospermia. EXPERIMENTAL DESIGN: Human sperm samples from three globozoospermic donors with Dpy19l2 deletion and three normal controls are subjected to TMT quantitative technology. SPESP1, HIST1H4A, and LYZL1 are randomly selected for western blotting analysis. GO annotations are performed using the Database for Annotation, Visualization, and Integrated Discovery. RESULTS: A total of 2567 proteins are identified, of which 2510 proteins are quantified, and 491 are differentially expressed (fold-change > 2), with 370 upregulated and 121 downregulated in globozoospermic patients. The levels of several important proteins, including SPACA 1, IZUMO1, ZPBP1, and PLCZ1, are decreased in globozoospermic sperm. Bioinformatics analysis indicates the Dpy19l2-deficient sperm presented molecular defects in acrosome, chromatin, sperm-egg interaction, and fertilization. CONCLUSIONS AND CLINICAL RELEVANCE: The present study is the first to analyze total globozoospermia with Dpy19l2 deletion using high-throughput proteomics. This study may provide insights into the mechanism of globozoospermia.

Proteomics analysis of asthenozoospermia and identification of glucose-6-phosphate isomerase as an important enzyme for sperm motility.

Asthenozoospermia, in which sperm motility is affected, is one of the primary causes of male infertility. However, the exact mechanism responsible for the defective motility remains unknown. It is important to identify the precise proteins or pathways involved in sperm motility. The present study analyzed five asthenozoospermic sperm samples and five healthy controls using TMT-based quantitative method and identified 152 differentially expressed proteins, with 84 upregulated and 68 downregulated in asthenozoospermia. Four proteins (GPI, MDH1, PGAM1 and PGAM2) were found in several over-represented energy metabolism pathways using bioinformatics analysis. Glucose-6-phosphate isomerase (GPI), a rate-limiting enzyme converting glucose-6-phosphate to fructose-6-phosphate, was found to be significantly decreased in asthenozoospermia by Western blotting and ELISA on an extended sample size. Furthermore, substitution of glucose with fructose-6-phosphate significantly promoted asthenozoospermic sperm motility in vitro. Taken together, our results suggest that the poor motility of sperm in asthenozoospermia may partly result from defects in GPI-associated energy metabolism. SIGNIFICANCE: To identify the key proteins or pathways involved in sperm motility, the accurate TMT-based quantitative method was applied to characterize protein profiles of asthenozoospermic sperm. GPI, an enzyme involved in energy metabolism, was found to be differentially abundant, and validated by extended sample analysis. The supplement of the product of GPI, fructose-6-phosphate, could significantly improve sperm motility. Our study could provide new insights into the molecular basis of sperm motility and the improvement of motility in asthenozoospermia.

Analysis of miRNA-mRNA network reveals miR-140-5p as a suppressor of breast cancer glycolysis via targeting GLUT1.

Aim: Aerobic glycolysis is characteristic of breast cancer. Comprehensive expression profiles of key proteins, their prognosis and detailed relationships between miRNAs and mRNAs remain unclear. Materials & methods: Oncomine database, Kaplan-Meier overall survival and miRNA-mRNA network analysis were performed. A key miRNA was identified and explored in vitro and in vivo. Results & conclusion: Eleven key glycolytic proteins were found with higher expression and poor prognosis: GLUT1, SLC2A5, HK1, PFKP, ALDOA, TPI1, GAPDH, PGK1, ENO1, GOT1 and GOT2. Seven miRNAs were predicted targeting 11 key glycolytic proteins: miR-140-5p, miR-3064-5p, miR-152-3p, miR-449b-5p, miR-449a, miR-194-5p and miR-34a-5p. Among them, miR-140-5p was found to be downregulated in breast cancer and directly targeted GLUT1, resulting in an antiglycolytic and antiproliferative effect.

The Conditioned Medium of Calcined Tooth Powder Promotes the Osteogenic and Odontogenic Differentiation of Human Dental Pulp Stem Cells via MAPK Signaling Pathways.

The calcined tooth powder (CTP), a type of allogeneic biomimetic mineralized material, has been confirmed that can promote new bone formation when obtained at high temperature. The aim of this study was to investigate effects of the conditioned medium of calcined tooth powder (CTP-CM) on the osteogenic and odontogenic differentiation of human dental pulp stem cells (hDPSCs) and the underlying mechanisms involved. First, ALP activity assay determined that 200 µg/mL was the optimal concentration of CTP-CM for the following experiments. CTP-CM had no significant effect on the proliferation of hDPSCs as indicated by CCK-8 and FCM analysis. Both the gene and protein (DSPP/DSPP, RUNX2/RUNX2, OCN/OCN, OSX/OSX, OPN/OPN, ALP/ALP, and COL-1/COL-1) expression levels increased in the CTP-CM-induced hDPSC group as compared with those in the control group at day 3 or 7, showing the positive regulation of CTP-CM on the osteo/odontogenic differentiation of hDPSCs. Mechanistically, MAPK signaling pathways were activated after the CTP-CM treatment, and the inhibitors targeting MAPK were identified which weakened the effects of CTM-CM on the committed differentiation of hDPSCs. These findings could lead to the creation of stem cell therapies for dental regeneration.

The Protein Phosphorylation Landscape of Mouse Spermatids during Spermiogenesis.

The characteristic tadpole shape of sperm is formed from round spermatids via spermiogenesis, a process which results in dramatic morphological changes in the final stage of spermatogenesis in the testis. Protein phosphorylation, as one of the most important post-translational modifications, can regulate spermiogenesis; however, the phosphorylation events taking place during this process have not been systematically analyzed. In order to better understand the role of phosphorylation in spermiogenesis, large-scale phosphoproteome profiling is performed using IMAC and TiO2 enrichment. In total, 13 835 phosphorylation sites, in 4196 phosphoproteins, are identified in purified mouse spermatids undergoing spermiogenesis in two biological replicates. Overall, 735 testis-specific proteins are identified to be phosphorylated, and are expressed at high levels during spermiogenesis. Gene ontology analysis shows enrichment of the identified phosphoproteins in terms of histone modification, cilium organization, centrosome and the adherens junction. Further characterization of the kinase-substrate phosphorylation network demonstrates enrichment of phosphorylation substrates related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis shows wide phosphoregulation across a diverse range of processes during spermiogenesis and can help to further characterize the process of sperm generation. All MS data are available via ProteomeXchange with the identifier PXD011890.

Effects of chemotherapeutic agent bendamustine for non-hodgkin lymphoma on spermatogenesis in mice.

Non-Hodgkin lymphoma (NHL) is one of the most common cancers affecting men of reproductive age. The high response rate of bendamustine as first-line treatment for NHL, coupled with young age of patients, makes elucidation of the impact of treatment on male reproduction important. Our aim was to determine the effects of bendamustine on male reproduction by animal model. Male mice were treated with bendamustine (40 mg/kg) through tail vein injection while cisplatin was given as a standard (3 mg/kg) through intraperitoneal injection. After 3 weeks, bendamustine induced weight loss and sperm morphology abnormalities were compared to the control. Additionally, sperm with folded tails were the most frequent abnormality in bendamustine-treated mice. But the mechanism of sperm abnormality induced by bendamustine remains to be elucidated. These results indicate bendamustine may affect spermatozoa of patients who have been treated for NHL.

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa.

Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from Chlamydomonas (DRC5) to mammals that localizes to the N-DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of Tcte1 results in male sterility because Tcte1-null spermatozoa show aberrant motility. Although the axoneme is structurally normal in Tcte1 mutant spermatozoa, Tcte1-null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.

Obesity aggravates toxic effect of BPA on spermatogenesis.

Both bisphenol A (BPA) and obesity affect male reproductive system. However, whether there is an interaction between them remains poorly understood. The aim of the present study was to evaluate the interaction between BPA exposure and obesity on semen quality and elucidate the mechanism in humans and animals. We firstly analyzed the interaction on semen volume, sperm count per ejaculate, sperm concentration and sperm motility in 357 men, and found that urinary BPA concentration was significantly correlated with sperm count per ejaculate in obese men (ß=-34.62; 95% CI: -60.75, -8.48; P=0.01). Then we validated the interaction using lean and obese mice with administration of BPA. Significant interactions between BPA exposure and obesity on sperm count and sperm concentration was observed in mice. Finally, we conducted metabolomics analyses to identify metabolites related to the interaction. Metabolites related to the interaction, including capric acid, dodecanoic acid, l-palmitoylcarnitine, niacinamide, etc., are known to play critical roles in fatty acid oxidation and tricarboxylic acid cycle indicating increased oxidative stress associated with male reproductive dysfunction. Thus, our study finds an interaction between BPA exposure and obesity on sperm count and reveals potential metabolic mechanisms. It emphasizes the importance to study interactions between endocrine disrupting chemicals and obesity, and opens avenues for the possible use of animal models in identifying the interactions.

Cytoskeletons of Two Reproductive Germ Cell Lines Response Differently to Titanium Dioxide Nanoparticles Mediating Vary Reproductive Toxicity.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used for many years. Their toxic effects on the male reproductive system had been reported, but the underlying mechanisms were still not clear. Here we utilized two germ cell lines (GC-2 and TM4) to explore the possible toxic effects of TiO2 NPs on male reproductive system. Our results showed that TiO2 NPs did not affect cell viability but induced cell apoptosis of both GC-2 and TM4 cells up to 100 µg/ml. Microtubule networks and microtubule dynamics of GC-2 but not TM4 cells were changed. The microfilaments arrangement of TM4 cells altered after treated with TiO2 NPs, and the phagocytosis activity of TM4 cells decreased significantly. Although the microfilament network of GC-2 cells seemed normal, the migration ability of GC-2 cells was significantly impaired. Totally TiO2 NPs is toxic to GC-2 cells by inducing cell apoptosis, disturbing microtubule arrangement and microtubule dynamic, and impairing cell migration ability. In addition, they altered the microfilament network and reduced the phagocytic activity of TM4 cells. We firstly reported that cytoskeletons (microtubules and microfilaments) in different cells showed different responses to TiO2 NPs, which might mediate different toxic mechanisms.

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 µg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development (mbp and syn2a) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

CHES-1-like, the ortholog of a non-obstructive azoospermia-associated gene, blocks germline stem cell differentiation by upregulating Dpp expression in Drosophila testis.

Azoospermia is a high risk factor for testicular germ cell tumors, whose underlying molecular mechanisms remain unknown. In a genome-wide association study to identify novel loci associated with human non-obstructive azoospermia (NOA), we uncovered a single nucleotide polymorphism (rs1887102, P=2.60 ×10-7) in a human gene FOXN3. FOXN3 is an evolutionarily conserved gene. We used Drosophila melanogaster as a model system to test whether CHES-1-like, the Drosophila FOXN3 ortholog, is required for male fertility. CHES-1-like knockout flies are viable and fertile, and show no defects in spermatogenesis. However, ectopic expression of CHES-1-like in germ cells significantly reduced male fertility. With CHES-1-like overexpression, spermatogonia fail to differentiate after four rounds of mitotic division, but continue to divide to form tumor like structures. In these testes, expression levels of differentiation factor, Bam, were reduced, but the expression region of Bam was expanded. Further reduced Bam expression in CHES-1-like expressing testes exhibited enhanced tumor-like structure formation. The expression of daughters against dpp (dad), a downstream gene of dpp signaling, was upregulated by CHES-1-like expression in testes. We found that CHES-1-like could directly bind to the dpp promoter. We propose a model that CHES-1-like overexpression in germ cells activates dpp expression, inhibits spermatocyte differentiation, and finally leads to germ cell tumors.

Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility.