Study On Preparation And Culture Of Bull Spermatogonial Stem Cells

In order to observe morphology and histologic structure , spermatogonial cells of newly born bull were cultured in vitro by three methods.①10-20mg testis tissue was cultured in DMEM supplemented with 10% fetal bovine serum(FBS)for one and two weeks in vitro. Fresh testis tissue was control.The structure of tissues and cells were studied by light microscope and electron microscope.②The snipped tissues were blowed repeatedly to make seminiferous tubules broken into pieces,and then cultured them in DMEM supplemented with 10% FBS,so that growth behaviors of somatic cells and spermatogonial cells could be studied.After culture the spreaded cells were fixed to observe the changes of cellular structure by electron microscope.③In order,to contrast with pieces-culture method and study the effect of long-term culture on spermatogonial cells,cells were cultured were cultured in DMEM supplemented with 2.5% FBS. In addition to investigated the effect of serum concentration on growth of somatic cells and spermatogonial cells,the isolated spermatogonial cells were cultured in the DMEM supplemented with 0%, 2.5%, 5%, or 10% FBS.The results were showed as followed: At 1 wk after culture, lumina diameter of testis tissue of newly born bull increased obviously, number of vessel wall cells increased, lumina of seminiferous tubules became hollow from solid center.At 2 wk, lumina diameter kept on increasing, number of vessel wall cells had no obvious change. Following the raise of FBS concentration, proliferation rate of somatic cells speeded up. The rate in 0% FBS was descent. Among 0%,2.5%,5% and 10% groups,2.5% FBS had profit on spermatogonial cells to grow and form more cell colonies, and profited to keep their undifferentiation state. The pieces-culture,cell suspension culture and different FBS concentration culture all obtained typical spermatogonial colonies,the pieces-culture obtained more and great colonies. Histological observation showed that there were two types different size spermatogonial cells with different size in newly born bull testis. At 1 wk after culture, some spermatogonia have migrated to basement membrane from lumina of seminiferous tubules. Seminiferous tubules basement membrane of testis tissue was multi-lamellar structure, thickness was 1.27±0.11μm,this structure did not changed after culture.