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Peter J I Ellis, Yong Yu and Shujun Zhang

The ability to pre-select offspring sex via separation of X- and Y-bearing sperm would have profound ramifications for the animal husbandry industry. No fully satisfactory method is as yet available for any species, although flow sorting is commercially viable for cattle. The discovery of antigens that distinguish X- and Y-bearing sperm, i.e. offspring sex-specific antigens (OSSAs), would allow for batched immunological separation of sperm and thus enable a safer, more widely applicable and high-throughput means of sperm sorting. This review addresses the basic processes of spermatogenesis that have complicated the search for OSSAs, in particular the syncytial development of male germ cells, and the transcriptional dynamics of the sex chromosomes during and after meiosis. We survey the various approaches taken to discover OSSA and propose that a whole-genome transcriptional approach to the problem is the most promising avenue for future research in the field.

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Jishang Gong, Quanwei Zhang, Qi Wang, Youji Ma, Jiaxiang Du, Yong Zhang and Xingxu Zhao

PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA–protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18–40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18–30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM–receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity.

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Fengyin Li, Yong Tao, Yunhai Zhang, Yunsheng Li, Fugui Fang, Ya Liu, Hongguo Cao, Xiaorong Zhang and Shixian Zhou

Ovary grafting is not only a method of investigating follicle and oocyte development, but also a useful model to explore the possibility of the re-establishment of the reproductive axis in male-to-female sexual reversal. This study investigated ovary survival and follicle development after mouse ovaries were transplanted into immune-intact castrated male mice. Ten-day-old mouse ovaries were transplanted into the back muscle of adult outbred castrated male mice treated with immunosuppressants. Twenty-two days later, the ovary structure and the number of follicles present was examined by hematoxylin and eosin staining. The oocytes were harvested, and then used for in vitro maturation (IVM) and IVF. The results showed that primordial and antral follicles were mainly found in the grafts, and there were obvious differences compared with 32-day-old fresh ovaries (P<0.05). Embryos were derived from collected oocytes after IVM and IVF with a 72.4% cleavage rate and 7.9% blastocyst rate; 12 live pups were generated by embryo transfer. The hormone assay showed that plasma concentrations of both estrogen and progesterone increased after ovarian transplantation (P<0.01). In conclusion, immune-intact adult castrated male mice can support ovary survival and further development of follicles with endocrine function after ovarian transplantation.

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Guo-Min Zhang, Ming-Tian Deng, Zhi-Hai Lei, Yong-Jie Wan, Hai-Tao Nie, Zi-Yu Wang, Yi-Xuan Fan, Feng Wang and Yan-Li Zhang

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

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Ruizhi Deng, Chengquan Han, Lu Zhao, Qing Zhang, Beifen Yan, Rui Cheng, Biao Wei, Peng Meng, Tingchao Mao, Yong Zhang and Jun Liu

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value < 0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

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Yali Xu, Yong Fan, Weimin Fan, Jia Jing, Ke Xue, Xing Zhang, Bin Ye, Yingjie Ji, Yue Liu and Zhide Ding

Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

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Ziliang Ji, Ruijing Lu, Lisha Mou, Yong-Gang Duan, Qiang Zhang, Yadong Wang, Yaoting Gui and Zhiming Cai

Hyperthermia and oxidative stresses are the two central elements contributing to varicocele-related sperm damage. Growing evidence indicates that microRNAs (miRNAs) are involved in the regulation of the heat and oxidative stress responses. In this study, we analyzed the expressions of several stress-related miRNAs in the sperm and found that the expression of miR-15a was significantly decreased in patients with varicocele compared with the control. Furthermore, miR-15a repressed the expression of HSPA1B, which is a typical stress-induced chaperone protein, through directly binding its 3′-UTR. The expressions of miR-15a and HSPA1B exhibited an inverse correlation in sperm. Our results provide a valuable insight into the varicocele-related sperm impairment and male infertility, and may help to develop potential therapeutic targets and novel biomarkers for male infertility.

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Xiao-Qian Meng, Ke-Gang Zheng, Yong Yang, Man-Xi Jiang, Yan-Ling Zhang, Qing-Yuan Sun and Yun-Long Li

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

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Yong-Hai Li, Yi Hou, Wei Ma, Jin-Xiang Yuan, Dong Zhang, Qing-Yuan Sun and Wei-Hua Wang

CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm–oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm–oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 ± 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 ± 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm–oocyte interactions during fertilization.

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Yan Xu, Miao Liu, Yi-hua Gu, Xiao-feng Jia, Yong-Mei Chen, Michelle Santos, Ai-Zhen Wu, Xiao-dong Zhang, Hui-Juan Shi and Ching-Ling C Chen

With tetraspanning topology, members of the membrane-spanning four-domain subfamily A (MS4A) may facilitate signaling or ion channel functions in many tissues. In this study, we report the cloning of a full-length cDNA from rat testis, designated Ms4a14 (Sp3111), which encodes the MS4A protein with 1139 amino acid residues. In situ hybridization and immunohistochemical analyses indicate that Ms4a14 is predominantly expressed from round spermatids to spermatozoa at specific stages in the rat testis at both the mRNA and protein level. Immunofluorescence analysis revealed that MS4A14 (SP3111) is located in the acrosome and the midpiece of the flagellum in mature sperm. Previously, we explored and reported the involvement of MS4A14 in reproductive functions, using antibody blockage during IVF and a transgenic RNA interference method in a mouse model. Our results suggested that MS4A14 is involved in fertilization and zygote division. As MS4A14 protein exists in mammals, such as humans, cows, dogs, and rodents, MS4A14 may play a ubiquitous role in mammalian reproduction.