Purpose To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat. Methods The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR). Results The viability of isolated testicular cells was>90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers. Conclusion Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats.Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.

We reported previously testis-mediated germline chimera production and characterization of germline stem cell-like cells from chicken testes. The present study aimed to establish an in vitro system for culture of quail spermatogonial stem cells (SSCs) for practical applications in germline preservation and transgenesis. Testicular cells (TCs) from juvenile (4 weeks old) or adult (8 weeks old) quail testis were isolated using sequential enzymatic digestion. The percentages of viability of isolated TCs were 91.00% ± 2.12% and 88.00% ± 1.87% in juvenile and adult testes, respectively, and immunohistochemical evaluation indicated the expression of integrin alpha-6 (ITGA6), GDNF family receptor alpha-1 (GFRA1), and Deleted in azoospermia-like (DAZL) in specific TCs. SSCs were purified by differential plating of TCs and then subjected to quantitative reverse transcription-polymerase chain reaction, which showed differential expression of SSC-specific, and germness and stemness-related genes. Coculture of quail SSCs with mouse embryonic fibroblasts and Sertoli cells as a feeder layer resulted in the generation of stable SSC colonies and short-term cultivation, and the expression of SSC and germ cell markers was maintained during several passages of culture. Collectively, these results demonstrate the efficient isolation and characterization of quail SSCs and the suitability of Sertoli cells as a feeder layer for in vitro culture of quail SSCs. Quail SSCs will facilitate the production of germline chimeras and transgenesis.

The production of transgenic livestock is constrained due to the limited success of currently available methods for transgenesis. Testis mediated gene transfer (TMGT) is an emerging method that shows a high success rate in generating transgenic mice. In this study, we report a newly developed protocol for electroporation-aided TMGT to produce a transgenic goat. The injectable volume and concentration of the transgene were first standardized, and then electroporation conditions were optimized in vitro. In vivo experiments were performed by injecting a transgenic construct (pIRES2-EGFP; enhanced green fluorescent protein) into the testicular interstitium followed by electroporation. Immunohistochemistry, quantitative real-time PCR (qPCR) and western blotting analyses confirmed the successful transfer of the transgene into seminiferous tubules and testicular cells. Furthermore, chromosomal integration of the transgene and its expression in sperm were evaluated d60 and d120 post-electroporation. Our protocol neither altered the seminal characteristics nor the fertilization capacity of the sperm cells. In vitro fertilization using transgenic sperm generated fluorescent embryos. Finally, natural mating of a pre-founder buck produced a transgenic baby goat. The present study demonstrates the first successful report of an electroporation-aided TMGT method for gene transfer in goats.

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The survivability and opportunity of successful development of an embryo are influenced directly or indirectly by factors controlling uterine microenvironment. Out of all factors, hormones such as prostaglandins (PGs) released during the preimplantation period influence molecular interactions involved in maintenance of pregnancy through reciprocal interactions between the conceptus and endometrium. PGs are important regulators of female reproductive functions, namely, ovulation, uterine receptivity, implantation, and parturition. Among different classes of PGs, prostaglandin F2 (PGF2 ) and prostaglandin E2 (PGE2) are main prostanoids produced by human and bovine endometrium for successful growth and development of the posthatching blastocyst. In ruminants, PGF2 produced by endometrium is the major luteolytic agent, whereas PGE2 has luteoprotective and antiluteolytic properties. Therefore, the development and maintenance of the corpus luteum (CL), as well as establishment of pregnancy, depend on the balance of luteolytic PGF2 and luteotropic PGE2. In this review, we discussed the expression and function of genes which predominantly regulate the synthesis and their secretion of PGF2 and PGES, namely, PGFS (AKR1B5/AKR1C3), PGES, PGFR, and COX-2.

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