Wenxian Zeng

Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.

Spermatogonial stem cells from domestic animals: progress and prospects

Spermatogenesis, an elaborate and male-specific process in adult testes by which a number of spermatozoa are produced constantly for male fertility, relies on spermatogonial stem cells (SSCs). As a sub-population of undifferentiated spermatogonia, SSCs are capable of both self-renewal (to maintain sufficient quantities) and differentiation into mature spermatozoa. SSCs are able to convert to pluripotent stem cells during in vitro culture, thus they could function as substitutes for human embryonic stem cells without ethical issues. In addition, this process does not require exogenous transcription factors necessary to produce induced-pluripotent stem cells from somatic cells. Moreover, combining genetic engineering with germ cell transplantation would greatly facilitate the generation of transgenic animals. Since germ cell transplantation into infertile recipient testes was first established in 1994, in vivo and in vitro study and manipulation of SSCs in rodent testes have been progressing at a staggering rate. By contrast, their counterparts in domestic animals, despite the failure to reach a comparable level, still burgeoned and showed striking advances. This review outlines the recent progressions of characterization, isolation, in vitro propagation, and transplantation of spermatogonia/SSCs from domestic animals, thereby shedding light on future exploration of these cells with high value, as well as contributing to the development of reproductive technology for large animals.

THY1 is a surface marker of porcine gonocytes.

Gonocytes are important for the study of spermatogenesis. Identification and isolation of gonocytes has been reported in rodents but not in pigs due to a lack of molecular markers for gonocytes. The objective of this study was to identify THY1 expression in porcine testicular tissue and subsequently utilise THY1 as a marker to isolate and enrich porcine gonocytes from testes of newborn piglets. Immunohistochemical analysis showed that THY1 was expressed in gonocytes. Double-immunofluorescent analysis of THY1 and ZBTB16 indicated that THY1 and ZBTB16 were partially co-localised in gonocytes. Double-immunofluorescent analysis of both THY1 and GATA4 suggested that THY1(+) cells were not Sertoli cells. Magnetic-activated cell sorting of THY1(+) cells yielded a cell population with an enrichment of UCHL1(+) gonocytes 3.4-fold of that of the unsorted testicular cell population. Western blot and quantitative reverse transcription-polymerase chain reaction analyses confirmed that the selected THY1(+) fraction had a higher expression of UCHL1 than the unsorted cells. In conclusion, the study demonstrated that THY1 is a surface marker of gonocytes in testes of pre-pubertal boars and could be utilised to identify and isolate porcine gonocytes. The findings will also facilitate culture and manipulation of male germline stem cells.

Long-term propagation of porcine undifferentiated spermatogonia

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and fertility throughout the adult life of a male. Genetic manipulations of SSCs combined with germ cell transplantation present a novel approach for gene therapy and production of genetically modified animals. However, the rarity of SSCs within mammalian testes remains an impediment to related applications, making in vitro expansion of SSCs a prerequisite. Nevertheless, long-term culture systems of SSCs from large animals have not been established yet. In this study, we developed an optimized in vitro culture condition for porcine undifferentiated spermatogonia. The germ cells were isolated and enriched from 7-day-old porcine testes by an optimized differential planting. We tested different feeder layers and found that neonatal autologous Sertoli cells acted better than the SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cell line and adult Sertoli cells. The effects of several growth factors were also investigated. Using neonatal Sertoli cells as feeder and Dulbeccos modified eagle medium: nutrient mixture F-12 (DMEM/F12) culture medium supplemented with 10% KSR and four cytokines, the undifferentiated spermatogonia can proliferate in vitro for at least 2 months without loss of stemness. The expression of SSC markers indicated that the cultured cells maintained SSC expression profiles. Moreover, xenotransplantation and in vitro induction showed that the long-term cultured cells preserved the capacity to colonize in vivo and differentiate in vitro, respectively, demonstrating the presence of SSCs in the cultured cells. In conclusion, the conditions described in this study can support the normal proliferation of porcine undifferentiated spermatogonia with stemness and normal karyotype for at least 2 months. This culture system will serve as a basic refinement in the future studies and facilitate studies on SSC biology and genetic manipulation of male germ cells.

Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120–240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epthetial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer.

5’-AMP-Activated Protein Kinase Regulates Goat Sperm Functions via Energy Metabolism In Vitro

Background/Aims: ATP is essential for mammalian sperm to survive and maintain fertilizing capacity. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. The aims of the present study were to explore the localization of AMPK in goat sperm and to investigate whether and how AMPK regulates sperm functions in vitro. Methods: Sperm were treated with AMPK modulators (AICAR, metformin and Compound C) during incubation. Sperm motility was assessed with a computer-assisted spermatozoa analysis system (CASA). Membrane integrity, acrosome reaction and mitochondrial membrane potentials were detected by SYBR-14/PI, FITC-PNA and JC-1 staining, respectively. And the lactate content, ATP content, AMPK activity, activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) were also measured with the commercial assay kits. Immunofluorescence staining was used to analyze the distribution of PK, LDH, AMPK and phospho-Thr172-AMPK in sperm. The role of AMPK was further studied during induction of capacitation and acrosome reaction. Results: We found that AMPKα was localized in the entire acrosomal region, the midpiece and the flagellum, while the phospho-Thr172-AMPK was distributed in the head, the midpiece and flagellum. Activation of AMPK by AICAR and metformin significantly improved sperm motility, membrane integrity and acrosome reaction, largely maintained sperm mitochondrial membrane potentials, lactate content and ATP content, and enhanced the activity of AMPK, PK and LDH, whereas inhibition by Compound C triggered the converse effects. Moreover, PK was localized in the acrosomal area and the midpiece, while LDH was distributed in the tail. Induction of capacitation and acrosome reaction led to AMPK phosphorylation. AMPK phosphorylation regulated the activity of energetic enzymes. Conclusion: This study for the first time provides evidence that AMPK governs goat sperm functions through energy metabolism in vitro. This finding will help to improve assisted reproductive techniques in goats and the other species.

Histone lysine methylation exhibits a distinct distribution during spermatogenesis in pigs

Spermatogenesis is a continual process throughout the adult life of a male, which is governed by unique transcriptional regulation and massive alterations of chromatin. Histone modification was one of the underlying epigenetic mechanisms during spermatogenesis. It has been shown that methylation of histone lysine exhibits a distinct distribution in mice during spermatogenesis and some histone lysine methylation is essential for male fertility. However, the dynamic change of methylated histone in porcine testis tissue was largely unknown. Here, we studied the dynamic modulation of three types of methylation (monomethylation, dimethylation, and trimethylation) of H3K4, H3K27, and H4K20 during spermatogenesis in pigs. The results showed that H3K4me2/3, H3K27me3, and H4K20me1/2/3 were extensively localized in adult pig testis. Interestingly, we found that undifferentiated spermatogonia contained strongly H4K20me2 and H4K20me3, but little H4K20me1, whereas the differentiated spermatogonia possessed H4K20me1 and H4K20me2 and little H4K20me3. The findings of this study help for the understanding of epigenetic modifications during spermatogenesis in pigs and provide information for further studies.

Melatonin protects mouse spermatogonial stem cells against hexavalent chromium-induced apoptosis and epigenetic histone modification

ABSTRACT Given the potential biological functions of spermatogonial stem cells (SSCs) in spermatogenesis and in delivering parental genetic information to the next generation, how these cells respond to environmental toxins and carcinogens should be investigated. We examined the toxic effect of hexavalent chromium (Cr(VI)) on global histone modifications and apoptotic signaling pathways in SSCs. We determined the effect of melatonin, one of the most powerful endogenous free radical scavengers and wide‐spectrum antioxidants, in protecting SSCs from Cr(VI)‐induced apoptosis and global histone modification by Western blot analysis. In addition, we examined the in vivo effect of melatonin on Cr(VI)‐induced histological changes of seminiferous tubules in mouse testes. We also evaluated the fertility of male mice by monitoring litter size following intraperitoneal injection of these chemicals. Our study demonstrated the Cr(VI)‐induced global increases in H3K9me3 and H3K27me3 and activated the apoptotic signaling pathway. Pretreatment of SSCs with melatonin alleviated Cr(VI)‐induced apoptosis and the global increase of H3K9me3. Exposure to melatonin also attenuated the Cr(VI)‐induced increase of the abundance of histone methyltransferase ESET. Furthermore, exogenous administration of melatonin protected mice against Cr(VI)‐induced changes in testicular histology and germ cell apoptosis, which helped maintain normal spermatogenesis and male fertility. Our study revealed a potential new therapeutic approach for male reproductive injury caused by Cr(VI). HighlightsMelatonin alleviated Cr(VI)‐induced SSCs apoptosis in vitro.Melatonin alleviated Cr(VI)‐induced testicular histological damage in vivo.Melatonin alleviated Cr(VI)‐increased ESET expression and H3K9me3 level.

Glutamine protects rabbit spermatozoa against oxidative stress via glutathione synthesis during cryopreservation

Mammalian spermatozoa are extremely susceptible to high doses of reactive oxygen species (ROS). The aim of the present study was to investigate the potential role of glutamine in protecting rabbit spermatozoa against ROS stress during cryopreservation and post-thaw incubation. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with glutamine. The addition of 20mM glutamine significantly improved sperm motility, acrosome integrity, membrane integrity and mitochondrial activity. Meanwhile, 20mM glutamine addition decreased lipid peroxidation and DNA damage in frozen-thawed spermatozoa. Interestingly, supplementation with 20mM glutamine led to increases in glutathione content and γ-glutamyl cysteine synthetase and glutathione peroxidase activity, with concomitant decreases in ROS levels during cryopreservation and post-thaw incubation. In conclusion, the addition of glutamine to extender solutions protects rabbit spermatozoa from ROS attack by enhancing glutathione synthesis.

Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages

The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.