| Although our knowledge of spermatogenesis has advanced considerably in recent years, numerous questions remain unanswered due to the lack of proper in vivo or in vitro models.Mammalian spermatogenesis is a complex cyclic process of proliferation and differentiation of male germ cells that results in production of spermatozoa capable of fertilizing the oocyte. The study of testicular development, maturation, and spermatogenesis has largely been limited to animal experimentation and frequently focused on laboratory rodents as models for humans. Information on testis function in livestock species is relatively limited. The development of in vitro models for testicular reproductive system may provide important tools for investigating specific mechanisms of spermatogenesis and toxicity in the testis. Although various systems have been reported, their application in reproductive studies has been limited by the poor ability to replicate the complex biochemical,molecular, and functional interactions observed in the testis.Spermatogenesis is the process to produce fertile spermatozoa. Spermatogenesis occurs in the seminiferous epithelium and consists of three phases. In the proliferative phase, a SSC starts to divide while others remain inactive as reserve. After more mitotic divisions, the daughters become A-spermatogonia which differentiate into A1-spermatogonia. Several divisions occur, dependent on the species, occur to produce more A1-spermatogonia, followed by intermediate (Ⅰ)-spermatogonia and ending with in B-spermatogonia. B-spermatogonia then divide to form preleptotene spermatocytes. The diploid primary spermatocytes pass through the Sertoli cells’ tight junctions to develop in the adluminal compartment and away from the body’s immune system to undergo meiosis.Primary spermatocytes develop into haploid spermatids through meiosis. Preleptotene spermatocytes become leptotene, zygotene and pachytene primary spermatocytes after DNA synthesis. The first meiotic division results in the formation of2secondary spermatocytes which undergo the second meiotic division fairly quickly to form4haploid round spermatids. The next step is spermiogenesis when the round spermatids give rise to elongated spermatids. There are several stages in spermiogenesis including acrosome appearance, nucleus condensation, cell elongation, flagellum formation and finally cytoplasmic resorption and spermiation.The study of SSC biology and the factors regulating their self-renewal or differentiation is difficult because of their low number and difficulties in isolating and identifying them. Although the morphological characteristics and location within the tubule may help distinguish SSCs in situ, morphology cannot be used for identification of SSCs after single cell preparation from the testis or cell culture.1. Long-term culture and spermatogenesis analysis of sertoli cell/germ cell co-culture system from seminiferous tubules of rat testisIt was the first time to establish a sertoli cell/germ cell co-culture system for rat germ cells in this study.The rat testicular pieces from15days old was cultured in DMEM/F12with10%FBS for1to2weeks and the sertoli cells were observed attaching to the surface of the culture dish from seminiferous tubules. The germ cells and Sertoli cells came from pieces of the testis seminiferous tubules of the rat. The co-cultured cells were survival in vitro for over two months without extra growthfactor.The spermatocytes and spermatids were able to be observed and, it suggested that the spermatogonial stem cells kept the potency of differentiation into the spermatocytes and spermitids.2. Identifiction of Long-term culture a of Sertoli cell/germ cell co-culture system from seminiferous tubules of rat testisThe development of in vitro models for testicular reproductive system may provide important tools for investigating specific mechanisms of spermatogenesis and toxicity in the testis. Although various systems have been reported, their application in reproductive studies has been limited by the poor ability to replicate the complex biochemical,molecular, and functional interactions observed in the testis. In the present study, we evaluated a significantly improved Sertoli cell/germ cell co-culture (SGC) system that the proliferation and differentiation of testicle germ cells of the rat could be maintained for long-term in vitro, and spermatids produced ceaselessly. The germ cells and Sertoli cells came from pieces of the testis seminiferous tubules of the rat. The co-cultured cells were survival in vitro for over two months without extra growthfactor. Furthermore,RT-PCR analysis cells stabilization of cdh1、scp3、tnp2expression confirmed the integrity of this co-culture system. Immunocytochemistry display the CDHI、PLZF、SCP3、SOX9positive cell was existed in system. All the results were proved that the SGC system included the spermatogonial stem cell、 spermatocytes and round spermatid. These events can be considered the complementary evidences for the morphological observation of germ cell cultivation in our laboratory.We conclude that this modified this system will provide investigators with a simple, efficient, and highly reproducible alternative in the screen for the assessment of molecular mechanisms associated with both normal development and reproductive toxicity induced by environmental toxicants.3.Long-term culture, isolation and purification of porcine SSCsIn this study,10-week-old porcine were used. Testicular cell suspension was obtained by two-step enzymatic digestion. Then, according to the principle that testicular somatic cells bind tightly to plastic and collagen matrics when cultures in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen matrics when culture in serum-containing medium. Porcine SSCs were then easily isolated from the purified spermatogenic population during a short incubation step on culture on laminin matrix. After purification,2×105SSCs were obtained from2g porcine testis tissus. So, it is a highly efficient separation and purification methods.We established porcine SSCs long-term culture system. In this system, the SSCs were cultured on STO feeder layers, using a defined serum-free mediumt that the presence of GDNF, bFGF,GFRal and B-27growth factors. Clump-forming germ cells kept on expanding for more than20passages and up to6months. The in vitro cultured SSCs can be cryopreserved at any time of the culture period, and resume growth after thawing rapidly.We used frozen sections of testis immunofluorescence detect the topographic arrangement and cell morphology of procine SSCs in the seminiferous cutubles, and testified that the SSC specific marker genes DBA,PGP9.5,OCT4and PLZF are reliable. Immunocytochemistry showed that the SSCs clones were also positive for DBA,PGP9.5,OCT4and PLZF. So, it will be laid the foundation for the identification of SSCs of other large animals. |
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