CCDC189 depletion leads to oligo-astheno-teratozoospermia and male infertility in mice.

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the poly(A)-binding protein (PABP) family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs.

Coiled-coil domain containing 159 is required for spermatid head and tail assembly in mice†.

The centrosome is critical for maintaining the sperm head-tail connection and the formation of flagellar microtubules. In this study, we found that in mouse testes, CCDC159 (coiled-coil domain-containing protein 159) is specifically localized to the head-tail coupling apparatus (HTCA) of spermatids, a structure that ensures sperm head-tail tight conjunction. CCDC159 contains a C-terminal coiled-coil domain that functions as the centrosomal localization signal. Gene knockout (KO) of Ccdc159 in mice resulted in acephalic spermatozoa, abnormal flagella, and male infertility. To explore the mechanism behind CCDC159 regulating spermatogenesis, we identified CCDC159-binding proteins using a yeast two-hybrid screen and speculated that CCDC159 participates in HTCA assembly by regulating protein phosphatase PP1 activity. Further RNA-sequencing analyses of Ccdc159 KO testes revealed numerous genes involved in male gamete generation that were downregulated. Together, our results show that CCDC159 in spermatids is a novel centrosomal protein anchoring the sperm head to the tail. Considering the limitation of KO mouse model in clarifying the biological function of CCDC159 in spermatogenesis, a gene-rescue experiment will be performed in the future.

University students' fertility awareness and its influencing factors: a systematic review.

INTRODUCTION: In recent years, a growing number of researchers have begun to study fertility awareness (FA). Evidence suggests that college students in their reproductive years have a common understanding of fertility, risk factors for infertility, and assisted reproductive technologies. Therefore, this systematic review summarizes these studies and explores the factors affecting college students' fertility awareness. METHODS: A systematic literature search of databases (PUBMED/MEDLINE, Cochrane, Web of Science, Embase, and EBSCO) was conducted from inception to September 2022. Studies that assessed the levels of fertility awareness and factors influencing college students were considered for the review. The qualities of the included studies were evaluated using the Strengthening the Reporting of Observational Studies in Epidemiology guidelines. This systematic review is reported according to the preferred reporting items for systematic review (PRISMA) guidelines. RESULTS: Twenty-one articles met the eligibility criteria and were included. The preliminary results showed that participants reported low to moderate FA. Female medical students demonstrated higher levels of fertility awareness. The association between age, years of education, and FA was insufficient. CONCLUSION: The results of the current study suggest that increased FA interventions are warranted, especially for the male, non-medical student population. Governments and educational institutions should strengthen education programs for young students on reproductive health to help them raise awareness about childbirth, and society should provide family support for young people.

Mus musculus Barrier-To-Autointegration Factor 2 (Banf2) is Not Essential for Spermatogenesis or Fertility.

The barrier-to-autointegration factor (BAF) is widely expressed in most human tissues and plays a critical role in chromatin organization, nuclear envelope assembly, gonadal development, and embryonic stem cell self-renewal. Complete loss of BAF has been shown to lead to embryonic lethality and gonadal defects. The BAF paralog, namely, barrier-to-autointegration factor 2 (BANF2), exhibits a testis-predominant expression pattern in both humans and mice. Unlike BAF, it may cause isolated male infertility. Therefore, we used the CRISPR/Cas9 system to generate Banf2-knockout mice to further study its function in spermatogenesis. Unexpectedly, knockout mice did not show any detectable abnormalities in histological structure of the testis, epididymis, ovary, and other tissues, and exhibited normal fertility, indicating that Banf2 is not essential for mouse spermatogenesis and fertility.

Meiotic gatekeeper STRA8 regulates cell cycle by interacting with SETD8 during spermatogenesis.

STRA8 (Stimulated By Retinoic Acid Gene 8) is a retinoic acid (RA) induced gene that plays vital roles in spermatogonial proliferation, differentiation and meiosis. The SETD8 and STRA8 protein interaction was discovered using the yeast two-hybrid technique using a mouse spermatogonial stem cell (SSC) cDNA library. The interaction of these two proteins was confirmed using co-immunoprecipitation and identification of key domains governing the protein: protein complex. STRA8 and SETD8 showed a mutual transcriptional regulation pattern that provided evidence that SETD8 negatively regulated transcriptional activity of the STRA8 promoter. The SETD8 protein directly bound to the proximal promoter of the STRA8 gene. STRA8 increased the transcriptional activity of SETD8 promoter in a dose-dependent manner. For the first time, we have discovered that STRA8 and SETD8 display a cell cycle-dependent expression pattern in germline cells. Expression levels of SETD8 and H4K20me1 in S phase of STRA8 overexpression GC1 cells were different from that previously observed in tumour cell lines. In wild-type mice testis, SETD8, H4K20me1 and PCNA co-localized with STRA8 in spermatogonia. Further, our studies quantitated abnormal expression levels of cell cycle and ubiquitination-related factors in STRA8 dynamic models. STRA8 and SETD8 may regulate spermatogenesis via Cdl4-Clu4A-Ddb1 ubiquitinated degradation axis in a PCNA-dependent manner.

[Sperm acrosome formation-associated genes in mice: Advances in studies].

Spermiogenesis is a complex process of differentiation and morphologic alteration, in which sperm acrosome formation is an important stage. Acrosome is an essential component of the sperm head, which develops in four distinct phases: Golgi, cap, acro- somal, and maturation, each supported by precise and orderly regulation of various genes. The regulatory genes which act on Golgi ap- paratus include GOPC, Hrb, SPATA16, PICK1, and CK2α', those involved in the cap phase are Fads2, syntaxin 2, Kdm3a, and UBR7, and participating in acrosomal and maturation phases are KIFC1, Rnf19a, and DPY19L2. The abnormalities of these genes may affect male fertility by influencing the connection of the nuclear dense lamina and acroplaxome with the nuclear membrane and then the fusion and transportation of vesicles. This review focuses on the genes involved in different phases of acrosome formation.

KLF9­regulated FBXO31 inhibits the progression of endometrial cancer and enhances the sensitivity of endometrial cancer cells to cisplatin.

Endometrial cancer (EC) is one of the most common malignancies with an increasing annual incidence. F-box only protein 31 (FBXO31) plays a significant regulatory role in several types of cancer. The transcription factor Krüppel-like factor 9 (KLF9) of FBXO31 is reduced in EC as a tumor suppressor. However, their particular regulatory role and mechanism in EC have not been previously reported. Therefore, the UALCAN database was used to predict the expression levels of FBXO31 in EC. In addition, the regulatory effect of FBXO31 on EC cell proliferation, invasion, migration, apoptosis and cisplatin (DDP) sensitivity was investigated at the cellular level. The association between KLF9 and FBXO31 was predicted using the JASPAR database and verified by chromatin immunoprecipitation and luciferase reporter assays. Finally, the regulatory effects of KLF9 and FBXO31 overexpression or silencing were also explored. The results demonstrated that FBXO31 was poorly expressed in EC. Additionally, FBXO31 overexpression inhibited the malignant progression of EC cells and enhanced their sensitivity to DDP. Furthermore, KLF9 promoted FBXO31 transcription. Overall, the present study suggested that the KLF9-mediated regulation of FBXO31 could inhibit the progression of EC and enhance the sensitivity of EC cells to DDP.

Effect of social support on fetal movement self-monitoring behavior in Chinese women: a moderated mediation model of health beliefs.

OBJECTIVE: Strengthening the management of women's self-monitoring during pregnancy is important to reduce fetal death in utero and improve maternal and infant outcomes. However, due to the lack of awareness among pregnant women about the importance of self-monitoring fetal movement, resulting in low behavioral compliance, adverse pregnancy outcomes remain common in China. This study aimed to investigate the relationship between social support and health beliefs and the self-monitoring behavior of fetal movement. In addition, we examined the moderating and mediating effects of health beliefs on fetal movement self-monitoring. METHODS: This cross-sectional study was conducted on 200 postpartum mothers in a tertiary hospital in China. The mothers were asked to complete a socio-demographic questionnaire, the fetal movement self-monitoring behavior questionnaire, the fetal movement self-monitoring health beliefs questionnaire, and the social support rating scale. Data from the questionnaires were analyzed and compared using SPSS 24.0 and PROCESS 3.2. RESULTS: The results of this study showed that the total scores of social supports, health beliefs, fetal movement self-monitoring were 42.98 ± 11.65, 78.605 ± 13.73, and 11.635 ± 2.86, respectively. The study found that when social support and health beliefs were included in the regression equation, both social support and health beliefs showed a positive correlation with fetal movement self-monitoring. Health beliefs partially mediated the effect of social support on fetal movement self-monitoring, accounting for 37.5% of the total effect. CONCLUSION: Social support and health beliefs play a crucial role in influencing the self-monitoring behavior of fetal movements. Therefore, strengthening social support and health beliefs during pregnancy has the potential to improve compliance with fetal movement self-monitoring behaviors for pregnant women.

[Preparation and identification of rabbit anti-mouse coiled-coil domain containing 189(Ccdc189)polyclonal antibody].

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.

[Preparation and application of rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB)].

Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.

[Production and application of rabbit polyclonal antibody against mouse testis expressed 38 (TEX38)].

Objective To produce rabbit polyclonal antibody against mouse testis expressed 38 (TEX38). Methods Full-length open reading frame sequence of TEX38 was amplified and inserted into the pET-30a-(+) vector to construct pET-30a-TEX38 prokaryotic plasmid. The recombinant plasmid was transformed into E.coli BL21, and expression was induced with isopropyl ß-D-thiogalactopyranoside (IPTG). New Zealand white rabbits were immunized with TEX38 protein after purification and denaturation, then TEX38 polyclonal antibodies were collected from rabbit serum samples. ELISA was performed to detect the antibody titer. Western blot and immunofluorescence staining were performed to determine the specificity of TEX38 polyclonal antibodies. Results The pET-30a-TEX38 recombinant plasmid was constructed, and TEX38 prokaryotic protein was expressed and purified successfully. After immunization, the titer of TEX38 antibody reached 1:1 000 000. Western blot analysis and immunofluorescence staining showed that TEX38 was localized in the mouse spermatogenic cells and sperms with a good specificity. Conclusion The rabbit polyclonal antibody against mouse TEX38 is successfully produced, and the expression of TEX38 in mouse spermatogenic cells and sperms is validated.

Relationship between social support and parenting sense of competence in puerperal women: Multiple mediators of resilience and postpartum depression.

Background: Maternal role competence is an important marker of achievement in the role of the mother, but parenting sense of competence (PSOC) among puerperal women is not high. Psychosocial factors, particularly social support, postnatal depression and resilience, have been identified as significant predictors of maternal role competence. However, information is limited regarding the mechanisms through which these psychosocial factors affect maternal role competence. Objective: To evaluate the multiple mediators of resilience and postpartum depression (PPD) in the relationship between social support and PSOC in puerperal women. Methods: A cross-sectional study was performed in a tertiary general hospital in Yangzhou, China. A total of 234 puerperal women at 6-8 weeks after birth completed the socio-demographic questionnaires, Social Support Rating Scale, Connor-Davidson Resilience Scale, Edinburgh Postnatal Depression Scale, and PSOC Scale. Results: Resilience and PPD mediated the relationship between social support and PSOC. The mediation effect of resilience and PPD and the total mediation effect were significant, individually accounting for 22.96, 21.70, and 44.65%, respectively, of the total effect. Moreover, pairwise contrast between the two indirect effects was not significant. The difference between the two pathways suggests that resilience and PPD play different roles in the relationship between social support and PSOC. Conclusions: This study showed that social support may exert its effects on PSOC in puerperal women with multiple mediators of resilience and PPD. This therefore highlights potential intervention targets to improve PSOC.

[Preparation and application of rabbit polyclonal antibody against mouse Tubby-like protein 2 (TULP2)].

Objective To generate rabbit polyclonal antibody against mouse Tubby(Tub)-like protein 2 (TULP2) and detect the expression of TULP2 in mouse testis. Methods pET30a (+)-TULP2 and pET30(+)-TULP2-C recombinant plasmids were constructed by inserting TULP2 full-length gene fragment and TULP2-C gene fragment containing Tub domain into pET30a (+). pET30a (+)-TULP2 and pET30(+)-TULP2-C were transformed into E. coli BL21, and the prokaryotic protein expressions were induced with the supplementation of IPTG. The prokaryotic recombinant proteins were purified with His-Binding-resin, and denaturation was performed by adding urea with gradient concentration. Adult male New Zealand white rabbits were inoculated with recombinant TULP2 and TULP2-C proteins as immunogens to generate two kinds of TULP2 polyclonal antibodies. Titers of antibodies were detected by ELISA. The efficiency and specificity of antibodies were determined by Western blot and immunofluorescence (IF) staining. Results pET30a (+)-TULP2 and pET30a (+)-TULP2-C recombinant plasmids were constructed successfully, and the protein expressions of TULP2 and TULP2-C could be induced by adding IPTG. The titers of polyclonal antibodies were 1:1 000 000. Western blot and IF staining showed poor specificity of TULP2-C antibody. TULP2 antibody could specifically recognize the endogenous TULP2 protein in the testes of adult wild-type mice, and IF staining showed that TULP2 was expressed specifically in the round spermatids and elongating spermatids of mice. Conclusion A rabbit anti-mouse TULP2 polyclonal antibody is generated successfully using TULP2 full-length protein, which can be used for detecting TULP2 expression by Western blot and IF staining.

The impact of stigma on mental health and quality of life of infertile women: A systematic review.

Introduction: The stigma of not giving birth to children affects approximately 53. 08~64% of female infertility patients worldwide. This stigma not only causes harm to the mental health of these infertility patients, but also affects their quality of life, making them bear the adverse social consequences such as domestic violence, marriage breakdown, or even delay in receiving the treatment. Therefore, it is crucial to have a deep understanding of the patients' stigma and effective intervention in alleviating it. Aims/Question: This study aims to discuss and summarize the stigma in infertile women and its impact on patients, and to provide a theoretical basis for the clinical treatment and nursing intervention of disease stigma in infertile female patients. Methods: The literature search used four English databases (Cochrane Library, EMBASE, Web of Science, and PubMed) and two Chinese databases (CNKI and Wanfang). The search time of the literature ranges from the establishment of the library to 2022, with no language restriction. Results: The review included 28 studies, with 20 cross-sectional studies and 8 qualitative studies. This study found that social support, living environment, education level, occupation, and fertility awareness were the major influencing factors of infertility stigma. Conclusions: Infertility stigma can bring heavy mental pressure and psychological burden to female infertility patients and affect their quality of life. Therefore, effective and targeted psychological interventions should be developed to reduce the patients' stigma and improve their quality of life. Implications for practice: Healthcare workers must develop targeted nursing interventions, provide professional counseling services to reduce the level of stigma in female infertility patients, alleviate fertility stress, and improve their quality of life.

Testis-expressed protein 33 is not essential for spermiogenesis and fertility in mice.

Gene expression analyses have revealed that there are >2,300 testis-enriched genes in mice, and gene knockout models have shown that a number of them are responsible for male fertility. However, the functions of numerous genes have yet to be clarified. The aim of the present study was to identify the expression pattern of testis-expressed protein 33 (TEX33) in mice and explore the role of TEX33 in male reproduction. Reverse transcription-polymerase chain reaction and western blot assays were used to investigate the mRNA and protein levels of TEX33 in mouse testes during the first wave of spermatogenesis. Immunofluorescence analysis was also performed to identify the cellular and structural localization of TEX33 protein in the testes. Tex33 knockout mice were generated by CRISPR/Cas9 gene-editing. Histological analysis with hematoxylin and eosin or periodic acid-Schiff (PAS) staining, computer-assisted sperm analysis (CASA) and fertility testing, were also carried out to evaluate the effect of TEX33 on mouse spermiogenesis and male reproduction. The results showed that Tex33 mRNA and protein were exclusively expressed in mouse testes and were first detected on postnatal days 21-28 (spermiogenesis phase); their expression then remained into adulthood. Immunofluorescence analysis revealed that TEX33 protein was located in the spermatids and sperm within the seminiferous tubules of the mouse testes, and exhibited specific localization to the acrosome, flagellum and manchette during spermiogenesis. These results suggested that TEX33 may play a role in mouse spermiogenesis. However, Tex33 knockout mice presented no detectable difference in testis-to-body weight ratios when compared with wild-type mice. PAS staining and CASA revealed that spermatogenesis and sperm quality were normal in mice lacking Tex33. In addition, fertility testing suggested that the Tex33 knockout mice had normal reproductive functions. In summary, the findings of the present study indicate that TEX33 is associated with spermiogenesis but is not essential for sperm development and male fertility.

[Prokaryotic expression of mouse Stra8 and preparation and identification of rabbit anti-mouse Stra8 polyclonal antibody].

Objective To prokaryotically express mouse stimulated by retinoic acid gene 8 (Stra8) and prepare rabbit anti-mouse Stra8 polyclonal antibody. Methods The recombinant plasmid pET28a-Stra8 was constructed by cloning technology and identified by double enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3). The expression of Stra8 recombinant protein was induced by IPTG. The prokaryotic protein was purified by His-TAG affinity chromatograph and then used to immune New Zealand white rabbits to obtain Stra8 polyclonal antibody. ELISA, Western blot and immunofluorescence assays were used to determine the antibody titer, validity and specificity, respectively. Results The recombinant plasmid pET28a-Stra8 was constructed successfully as double enzyme digestion and sequencing showed, and the prokaryotic protein was expressed and purified effectively. The titer of the polyclonal antibody reached 1:106. Western blotting showed that the polyclonal antibody could specifically recognize native Stra8 protein in the testis. Immunofluorescence assay revealed that the polyclonal antibody had good reactivity, and could recognize Stra8 protein in mouse testis. Conclusion Stra8 prokaryotic protein can be effectively induced in E. coli and specific rabbit anti-Stra8 polyclonal antibody has been prepared.

[Preparation and application of rabbit polyclonal antibody against mouse Tex33].

Objective To prepare rabbit-antimouse testis expressed 33 (Tex33) polyclonal antibody and detect the expression of Tex33 during mouse spermatogenesis. Methods Tex33 open reading frame was amplified by PCR from mouse testis cDNA. The PCR product was finally sub-cloned into pET-30a vector. The prokaryotic expression plasmid pET-30a-Tex33 was constructed and then transformed into E. coli BL21. Tex33 prokaryotic protein was induced by IPTG and then purified by Ni-NTA affinity chromatography. Tex33 polyclonal antibody was obtained from adult male New Zealand white rabbits immunized with purified Tex33 protein. ELISA, Western blotting and immunofluorescence assays were used to determine the potency and specificity. Results pET-30a-Tex33 recombinant vector was successfully constructed. After induced by IPTG, the recombinant Tex33 protein could be expressed effectively. The titer of polyclonal antibody was 1:1 000 000. Western blotting showed that the antibody could recognize Tex33 protein in mouse testis. Immunofluorescence assay revealed that Tex33 gene was expressed in spermatids and sperms of adult mouse testis. And Tex33 was located on the acrosome and flagellum of spermatozoa. Conclusion We have prepared rabbit-antimouse Tex33 polyclonal antibody with high specificity and found that Tex33 gene was expressed in mouse testis.

Stra8 may inhibit apoptosis during mouse spermatogenesis via the AKT signaling pathway.

Stimulated by retinoic acid 8 (Stra8), one of genes induced by retinoic acid (RA), is required for the meiotic initiation of male spermatogenesis. The present study found that Stra8 inhibited apoptosis in male Stra8­knockout mice, and in mice with vitamin A deficiency and vitamin A recovery in vivo. This phenotype was also verified in GC1 spermatogonia (spg) cells overexpressing Stra8. In addition, microarray analysis identified that there were nine differentially expressed genes (DEGs) in the Stra8­overexpressed GC1 spg cells compared with the control groups; the expression of these nine genes was verified via mRNA expression levels. The DEGs were as follows: Phosphatidylinositol­dependent kinase 1 (PDK1), a key gene upstream of protein kinase B (AKT); angiopoietin 2, a B­cell lymphoma 2 (Bcl­2)­inhibited gene; transcription factor 4, glutathione S­transferase P91 and ubiquitin­specific protease 33, mitogen­activated protein kinase (MAPK)­related genes; oxidative stress induced growth inhibitor 1, related to the P53 pathway; Bcl­2, P53, ERK (MAPK1/3), c­Jun N­terminal kinase (MAPK8/9), and P38 (MAPK14), all of which are key genes involved in the AKT signaling pathway. Therefore, the present study further verified these genes and found that the mRNA and protein expression levels of PDK1, AKT, Bcl­2 and ERK were increased. Although the mRNA expression level of P53 was decreased, there was no significant difference in the protein expression level in Stra8­overexpressing GC1 spg cells compared with controls. In addition, Caspase 3, one of the executioner caspases, was decreased in Stra8­overexpressing GC1 spg cells compared with the control groups. Therefore, it was suggested that Stra8 may directly or indirectly inhibit caspases through the AKT signaling pathway and ultimately exert an anti­apoptotic effect in the male reproductive system.

Stimulated by retinoic acid gene 8 (Stra8) plays important roles in many stages of spermatogenesis.

To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.

[Preparation and application of rabbit anti-mouse Setd8 polyclonal antibody].

Objective: To purify the recombinant Setd8 protein and prepare rabbit anti-mouse Setd8 polyclonal antibody. Methods: The recombinant plasmid p ET-30a-Setd8 was constructed by double enzyme digestion and linkage,and then transformed into E. coli BL21. The expression of the target protein was induced by IPTG and the expression product was purified by Ni-NTA affinity chromatograph. The purified protein was used to immunize New Zealand white rabbits to produce polyclonal antibody. The titer and specificity of the antibody were identified by ELISA,Western blotting and immunohistochemistry. Results: The prokaryotic expression vector p ET-30a-Setd8 was constructed successfully. After induced by IPTG,the recombinant Setd8 protein was expressed effectively in E. coli BL21. Polyclonal antibody against Setd8 was generated by immunizing rabbits with the routine method. ELISA showed that the titer of rabbit anti-Setd8 antiserum was 1 ∶ 1 000 000.Western blotting demonstrated that the polyclonal antibody could recognize the native mouse Setd8 protein. Immunohistochemistry revealed that Setd8 protein recognized by the polyclonal antibody was mainly distributed in the nucleus of spermatogonia in adult mouse testis. Conclusion: Using the prokaryotic expression vector p ET-30a-Setd8,we have prepared successfully the polyclonal antibody with high affinity and specificity.