| Object to set up a system of spermatogenesis in vitro for studying spermatogenesis of boars further, providing the model for assistant reproduction, illuminateing the mechanism of infertility caused by environmental pollution, offering new ideas for transgene and saving threatened species. We compared two different culture methods which were used often, observed morphological characteristics of germ cells during culture, and also detected the proliferation and differentiation of germ cells after culture.Germ cells mixed culture and tissue culture were used to study germ cells'surviving rate, surviving time and differentiation degree of germ cells. Results show that during germ cells mixed culture, germ cells surviving rate had the tendency of "low-high-low", the surving rates were (91.66±0.46)%, (95.41±1.11)%, (94.15±0.32)% on first, second, third detecting day respectively. However, during tissue culture, the rates were (96±0.58)%, (98±0)% and (98±0)% on first, second, third detecting day respectively. Besides, the differentiation degree of germ cells in testis tissue culture was much higher than in mixed culture. The highest differentiation degree of germ cell type were secondary spermatocytes and spermatids during mixed culture , tissue culture respectively.To understand the proliferation and differentiation of germ cells further, we used the culture method that showed higher germ cells' surving rate and differentiation degree in former experiment, basing on this culture condition: 1mL DMEM/F12 contained 100IU mycillin, 10-4Vc, 10μg/mL VE, 10-4mg/mL FSH, 10μg/mL insulin-transfer protein, 34℃, 5%CO2, 95%air. Brdu(5' -Bromo-2-deoxyuridine, BrdU) was labelled with S phrase germ cells to study the proliferation of germ cells, while HE staining and transmission electron microscope technique were also used to study the differentiation of germ cells. Results show that: (1) Those cells labeled with Brdu could proliferate in vitro. Colonies were obviously oberseved through fluorescence inverted microscope. The colonies consisted of 2-cell, 4-cell, 6-cell even number germ cells. Otherwise, we also observed colonies consisted odd number germ cells such as 3-cell, 5-cell, 9-cell. In conclusion most of the colonies consisted less than 10 germ cells. Besides, there were also many single cells present. (2) After HE staining, spermatonium cells could be seen jointed with intercullar bridges adhered to sertoli cells. Occasionally, we observed secondary spermatocytes differentiating into spermatids. After 20d culture, spermatids could be seen with amethyst nucleus, the nucleus tend to deviate from cytoplasm. (3)Ultrastructural pictures showed cytoplasm of sertoli cells were not abundant after culture than before; the number of mitochondria decreased, and engorgement of mitochondria could also be clearly seen. We were glad to observed two spermatids. Those two spermatids all had acrosomal vesicles on one side of nucleus showing high electron density, having the tendency to expand along the nucleus. By the side of nucleus, We also observed the Golgi complex, which was the typical characteristic of spermatid phrase.Conclusion: (1) germ cells during testis tissue culture show higher surving rate and differentiation degree. (2) Germ cells from testis tissue could better proliferate in vitro to form colonies. The colonies jointed with intercellular bridges were mostly consisted of less than 10 cells.Both even number and odd number germ cells' colonies could be seen during culture. (3) HE staing and Ultrastructure pictures showed that germ cells could differentiate into spermatids.
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