Improved the in vitro blastocyst development in oocytes recovered from cyclic buffaloes.

This study examined the impact of cyclicity (with or without cycle corpus luteum; CL) on oocyte quality and embryonic development in buffaloes. We collected oocytes from the ovaries of slaughtered buffaloes (N = 158 cyclic; n = 316 ovaries and N = 177 acyclic; n = 353 ovaries). Blood progesterone concentration and number of oocytes per ovary were higher in cyclic buffaloes. Cyclic buffalo ovaries produce higher oocytes with I + II and fewer III + IV grades. Oocytes from cyclic buffaloes had a higher maturation rate based on cumulus expansion, cleavage rate and embryo development to the 8-cell, morula and blastocyst stages than acyclic buffaloes. In conclusion, oocytes recovered from the ovaries of the cyclic buffaloes showed improved oocyte competence and subsequent in vitro blastocyst development.

PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

Transcriptome analysis revealed differences in the microenvironment of spermatogonial stem cells in seminiferous tubules between pre-pubertal and adult buffaloes.

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs.

Seasonality and photoperiod influence in vitro production of buffalo embryos.

Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January-March, April-June, July-September and October-December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April-June) and maximum during fourth quarter (October-December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.

Melatonin alleviates the deterioration of oocytes from mice subjected to repeated superovulation.

Induction of repeated superovulation with exogenous hormones is widely used in assisted reproductive technology (ART). Though it is generally safe, emerging evidence has indicated that repeated superovulation may compromise oocyte quality. However, few studies have explored how to ameliorate such impairment. Because melatonin has beneficial influences on oocytes in various detrimental environments, we aimed to explore whether melatonin could protect mouse oocytes after repeated superovulation. We found that repeated superovulation markedly reduced meiotic maturation and disrupted spindle organization and chromosome alignment. Furthermore, we observed reduced mitochondrial content and enhanced early apoptosis in oocytes from mice subjected to repeated superovulation. In addition, 5-methylcytosine (5mc) fluorescence intensity was lower in oocytes from experimental mice than in those from control mice, indicating that repeated superovulation disrupts genomic DNA methylation, and elevations in reactive oxygen species levels indicated that repeated superovulation also induces oxidative stress. Conversely, melatonin administration improved oocyte maturation and attenuated the observed defects. Interestingly, supplementation with melatonin during in vitro maturation had the same protective effects on oocytes as in vivo melatonin administration. In summary, our results show that melatonin can improve oocyte quality after repeated superovulation and thus provide a potential strategy to improve ART efficiency.

Maturation of buffalo oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in mitochondrial function and the membrane lipid profile.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P<0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P<0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P<0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes.

Effects of resveratrol on in vitro maturation of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer.

As a natural plant-derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 µM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti-apoptotic B-cell lymphoma 2 (BCL-2) gene and significant downregulation of the pro-apoptotic BCL2-associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 µM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 µM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

Generation of transgenic-cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein.

Huanjiang Xiang pig is a unique native minipig breed originating in Guangxi, China, and has great utility value in agriculture and biomedicine. Reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) and SCNT-mediated genetic modification show great potential value in genetic preservation and utilization of Huanjiang Xiang pigs. Our previous work has successfully produced cloned and transgenic-cloned embryos using somatic cells from a Huanjiang Xiang pig. In this study, we firstly report the generation of transgenic-cloned Huanjiang Xiang pigs carrying an enhanced green fluorescent protein (eGFP) gene. A total of 504 SCNT-derived embryos were transferred to two surrogate recipients, one of which became pregnant and gave birth to three live piglets. Exogenous eGFP transgene had integrated in all of the three Huanjiang Xiang piglets identified by genotyping. Furthermore, expression of eGFP was also detected from in vitro cultured skin fibroblast cells and various organs or tissues from positive transgenic-cloned Huanjiang Xiang pigs. The present work provides a practical method to preserve this unique genetic resource and also lays a foundation for genetic modification of Huanjiang Xiang pigs with improved values in agriculture and biomedicine.

[Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna].

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺4/NO⁻3) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺4/NO⁻3). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺4/NO⁻3) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.

Role of linker histone H1c during the reprogramming of Chinese swamp buffalo (Bubalus Bubalis) embryos produced by somatic cell nuclear transfer.

During reprogramming, there is exchange of histone H1c and the oocyte-specific linker histone, and H1c may play a critically important role in the reprogramming process of somatic cell nuclear transfer (SCNT). The aim of the present study was to investigate the role of the H1c gene in SCNT reprogramming in Chinese swamp buffalo (Bubalus bubalis) using RNA interference (RNAi). Chinese swamp buffalo H1c gene sequences were obtained and H1c-RNAi vectors were designed, synthesised and then transfected into a buffalo fetal skin fibroblast cell line. Expression of H1c was determined by real-time polymerase chain reaction to examine the efficiency of vector interference. These cells were then used as a nuclear donor for SCNT so as to observe the further development of SCNT embryos. Inhibition of H1c gene expression in donor cells significantly improved the developmental speed of embryos from the 1-cell to 8-cell stage. Furthermore, compared with the control group, inhibition of H1c gene expression significantly reduced the blastocyst formation rate. It is concluded that linker histone H1c is very important in SCNT reprogramming in Chinese swamp buffalo. Correct expression of the H1c gene plays a significant role in preimplantation embryonic development in B. bubalis.

ZPAC is required for normal spermatogenesis in mice.

The ubiquitin-proteasome pathway, involved in genetic recombination and sex-chromosome silencing during meiosis, plays critical roles in the specification of germ-line stem cells and the differentiation of gametes from gonocytes. Zygote-specific proteasome assembly chaperone (ZPAC) is expressed in the early mouse embryo, where it is important for progression of the mouse maternal-to-zygotic transition. The role of ZPAC during spermatogenesis in the adult gonads, however, remains unknown. In this study, rapid amplification of cDNA ends was used to determine the Zpac cDNA sequence, a 1584-bp transcript that includes a putative 1122-bp open reading frame coding for a 373 amino acid protein. Western blot and immunohistochemistry revealed that ZPAC was specifically expressed in gonads. To further dissect the function of ZPAC during spermatogenesis, we employed PiggyBac-based RNA interference vectors for transgenesis combined with cell transplantation to deplete Zpac during spermatogenesis. This RNAi-mediate depletion in Zpac expression disrupted normal spermatogenesis from spermatogonial stem cells. Two independent yeast two-hybrid screens further revealed an interaction between ZPAC and SYCE1. Together, these data suggest that ZPAC is required for normal spermatogenesis in mice.

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2'-deoxycytidine.

Summary Reprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2'-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.

[Effect of TSA and VPA treatment on long-tailed macaque (Macaca fascicularis)-pig interspecies somatic cell nuclear transfer].

Long-tailed macaque-pig interspecies somatic cell nuclear transfer (iSCNT) is beneficial to yield embryonic stem cells from iSCNT embryos with similar genetic background as human, which can be used as materials for medical and basic research. The primary objective of this study was to investigate the effects of concentrations and treatment duration of two histone deacetylase inhibitors-Trichostatin A (TSA) and Valproic acid (VPA) and two different embryo culture media (PZM-3 and HECM-10) on the in vitro development of iSCNT embryos. The results suggested that when PZM-3 was used as the embryo culture medium, the blastocyst rate of 10 nmol/L TSA treatment for 48 h was significantly higher than the control group (22.78% vs 9.86%, P< 0.05). However, neither in PZM-3 nor in HECM-10, 2-10 mmol/L VPA treatment did not increase the in vitro developmental potential of iSCNT embryos. It was concluded that TSA treatment could enhance the in vitro developmental potential of long-tailed macaque-pig iSCNT embryos.

Effects of feeder cells on proliferation of inducible pluripotent stem cells in chicken.

Although inducible pluripotent stem cells (iPSC) have been identified in poultry, the induction efficiency is low, because different culture media, feeder cells and feeder layer treatments affect the efficiency of somatic cell reprogramming. We investigated improvement of the feeder culture system for induction of chicken iPSC by comparing the effects of different types and treatments of feeder cells on the growth and proliferation of chicken iPSC. Mouse embryo fibroblasts (MEF), but not Sandoz inbred mouse-derived thioguanine-resistant and ouabain-buffalo rat cells, were suitable feeder cells that supported proliferation of chicken iPSC. Institute of Cancer Research (ICR) mice, but not Kunming mice, were suitable for preparing MEF that support cell proliferation. Also, MEF feeder cells that had been inactivated by mitomycin C were effective. Leukemia inhibitory factor was not required for chicken iPSC culture when MEF feeder cells were used. The optimal feeder culture system for growth and proliferation of chicken iPSC consisted of MEF feeder cells derived from ICR mice that were inactivated by mitomycin C combined with embryonic germ cell culture medium.

Current status of assisted reproductive technologies in buffaloes.

Buffaloes are raised by small farm holders primarily as source of draft power owing to its resistance to hot climate, disease, and stress conditions. Over the years, transformation of these animals from draft to dairy was deliberately carried out through genetic improvement program leading to the development of buffalo-based enterprises. Buffalo production is now getting more attention and interest from buffalo raisers due to its socioeconomic impact as well as its contribution to propelling the livestock industry in many developing countries. Reproduction of buffaloes, however, is confronted with huge challenge and concern as being generally less efficient to reproduce compared with cattle due to both intrinsic and extrinsic factors such as poor estrus manifestation, silent heat, marked seasonal infertility, postpartum anestrus, long calving interval, delayed puberty, inherently low number of primordial follicles in their ovaries, high incidence of atresia, and apoptosis. Assisted reproductive technologies (ARTs) are major interventions for the efficient utilization of follicle reserve in buffaloes. The present review focuses on estrus and ovulation synchronization for fixed time artificial insemination, in vitro embryo production, intracytoplasmic sperm injection, cryopreservation of oocytes and embryos, somatic cell nuclear transfer, the factors affecting utilization in various ARTs, and future perspectives in buffaloes.

Constant light exposure causes oocyte meiotic defects and quality deterioration in mice.

Artificial light at night (ALAN) exposes us to prolonged illumination, that adversely affects female reproduction. However, it remains to be clarified how prolonged light exposure affects oocyte meiotic maturation and quality. To this end, we exposed female mice to a constant light (CL) of 250 lux for different durations. Our findings showed that CL exposure for 7 weeks reduced the oocyte maturation rate. Meanwhile, CL exposure caused greater abnormalities in spindle assembly and chromosome alignment and a higher rate of oocyte aneuploidy than the regular light dark cycle. CL exposure also induced oxidative stress and caused mitochondrial dysfunction, which resulted in oocyte apoptosis and autophagy. Notably, our results showed that CL exposure reduced the levels of α-tubulin acetylation, DNA methylation at 5 mC, RNA methylation at m6A and histone methylation at H3K4me2 but increased the levels of histone methylation at H3K27me2 in oocytes. In summary, our findings demonstrate that constant bright light exposure causes oocyte meiotic defects and reduces cytoplasmic quality. These results extend the current understanding of ALAN-mediated defects in female reproduction.

Developmental potential of buffalo embryos cultured in serum free culture system.

The presence of serum in embryo culture medium has been implicated for increased embryo's sensitivity to cryopreservation, compromised viability, abnormal embryo and fetal development. Hence, designing a serum free culture system is indispensable. The present study aims to compare the efficiency of the serum and granulosa cells monolayer free commercial culture system (SFCS) with the conventional serum supplemented co-culture system (SSCS) and optimized culture system (OCS). Generally, SFCS is designed explicitly for bovine oocyte maturation and embryo culture (SF-IVM and SF-IVC), and SSCS (based on M199, SS-IVM, and SS-IVC) is utilized for buffalo in vitro embryo production. However, OCS is a newly designed culture system in which oocyte maturation is performed in serum supplemented maturation medium, and the subsequent embryos are co-cultured with granulosa cells in serum free culture medium. To evaluate the effect of serum on buffalo embryo production, buffalo oocytes, and their subsequent embryos were cultured in SSCS, SFCS, and OCS, simultaneously. The percentage of cleaved embryos cultured in SSCS and OCS was approximately 4% higher as compared to SFCS. However, OCS significantly showed the maximum proportion of embryos that developed to the blastocyst stage (7d) and hatched (6d) as compared to the SFCS and SSCS. Additionally, OCS promoted the expression of developmentally important genes (BCL2-L1 and VEGF-A), cell number, and cryo-survival ability of blastocysts in comparison with SSCS. Taken together, OCS is more suitable for the oocyte maturation and culture of buffalo embryos. However, to design the serum free culture system, it is recommended to find suitable serum alternatives for in vitro oocyte maturation.

Mogroside V improves porcine oocyte in vitro maturation and subsequent embryonic development.

Oocyte in vitro maturation (IVM) plays a pivotal role in in vitro embryo production. However, the efficiency of IVM is still low and needs to be further improved. In the present study, we evaluated the beneficial effects of mogroside V, an extract derived from Siraitia grosvenorii, on oocyte IVM. Porcine cumulus-oocyte complexes were cultured in IVM medium supplemented or not supplemented with mogroside V for 40 h. We found that mogroside V supplementation increased the percentage of oocyte first polar body extrusion and improved subsequent blastocyst formation after parthenogenetic activation. Furthermore, mogroside V reduced the levels of reactive oxygen species (ROS) and increased the mRNA expression of oxidative stress-related genes (SOD, CAT and SIRT1). Moreover, mogroside V supplementation enhanced the mitochondrial content, mtDNA copy number, mitochondrial membrane potential (ΔΨm), ATP generation, and the relative mRNA expression of mitochondria-related genes (PGC-1α and TFAM). In summary, our findings demonstrate that mogroside V supplementation reduces intracellular ROS levels and enhances mitochondrial function to promote porcine oocyte IVM.