This study was evaluated the effect of different synchronization protocols such as serum starvation for 1–3 days, confluency and chemical inhibitors on synchronization accuracy at G0/G1, apoptosis, and DNA synthesis in sheep granulosa cells. The cells were obtained from ovarian antral follicles of slaughtered sheep and used at first and fifth passages. Flow cytometry analysis showed that confluent cells, serum starvation for 24, 48, and 72 hours, and mimosine treatment significantly increased G0/G1 phase cells when compared to normally growing cells ( ). Nocodazole treatment increased the cell population in the G0/G1 stage when compared with the control group but did not change the G2/M stage population. Treatment of cells with mimosine, nocodazole, and serum starvation in three groups resulted in proliferation arrest ( ). Serum starvation for 72 hours significantly promoted apoptosis in granulosa cells ( ). The results of the primary culture and 5th passage were the same. The use of 48-hour serum starvation and mimosine treatments has been recommended because cell death in these groups was very similar to the control group. 1. Introduction Recently, animal cloning by nuclear transfer (NT) has undergone rapid development. Somatic cell cloning has been successful in sheep [1], cattle [2], mice [3], goats [4], pigs [5], cats [6], rabbits [7], and horses [1]. Production of offspring by NT using somatic cells is possible, not only for the production of domestic animals [8, 9] but also for producing transgenic [10] or gene-targeted [11] offspring. However, somatic cell NT has a very low success rate, generally not more than 3%, with a high frequency of fetal loss and an increase in prenatal mortality [12, 13]. Many factors affect the efficiency of NT. One important factor is the cell cycle phase of the donor cells at NT [14]. Synchronization of the cell cycle stage of the recipient cytoplasm and the donor nucleus is essential for maintaining correct ploidy and for preventing DNA damage in NT reconstructed embryos. Previous studies have indicated that S phase nuclei transferred to MII oocytes undergo premature chromosome condensation (PCC) resulting in major chromatin aberrations and a reduction in development of NT embryos [15, 16]. The cell cycle stage of the donor nucleus must be compatible with the high levels of maturation promoting factor (MPF) activity present in the metaphase II oocyte [17]. For this reason, only nuclei in the G1 [2, 18–20] or G0 phases [3, 21, 22] of the cell cycle can be used for nuclear transplantation to enucleated MII oocytes.
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