Flow cytofluorometric analysis of granulosa cell proliferation in rats

Abstract

Immature rats were given PMSG followed by an ovulatory dose of hCG. Mature cyclic rats were given an ovulatory dose of hCG on the day of dioestrus, 24 h before the endogenous LH surge was expected to occur. Granulosa cells from individual follicles were subjected to flow cytometry at various times after hormone administration. Administration of PMSG to immature rats caused a marked, transient increase in 'cycling' granulosa cells (cells in S, G2, or M phases of the cell cycle). Administration of hCG to adult cyclic rats caused a slight, but significant, increase in 'cycling' granulosa cells. The results could be taken to indicate that hCG-induced luteinization is not, in fact, accompanied by cessation of cellular proliferation. However, the data lend themselves to alternative explanations. The observed phase shifts may be due to the lengthening of the duration of S and G2 phases, indicating an actual decrease in the rate of cell proliferation. Flow cytometry was found to be a rapid, inexpensive tool for the study of follicular growth in the rat, but, due to variation between follicles and the need for a minimal sample size of 2000 cells, its application is limited to large follicles.

Attenuation of cell cycle progression by 2,3,7,8-tetrachlorodibenzo-p-dioxin eliciting ovulatory blockade in gonadotropin-primed immature rats

“…The current data showing the simultaneous increase in the number of S-phase granulosa cells and decrease in G0/G1-phase cells after 24 h of PMSG stimulation is in agreement with reports on gonadotropin-primed immature rats and adult cycling rats [28,29]. Interestingly, TCDD exerted its action 24 h after PMSG treatment, decreasing the number of S-phase cells, increasing the number of G2/M-phase cells, and not affecting G0/G1-phase cells.…”

Association of luteinizing hormone receptor gene expression with cell cycle progression in granulosa cells

“…For example, direct injection of the cell cycle inhibitor olomoucine under the bursa of exteriorized ovaries results in abnormal follicular morphology within 24 h (data not presented). Granulosa cells in the latter stages of follicle growth (36 -48 h post-PMSG) are largely arrested in G1 phase of the cell cycle (3,13). By administering a second injection of PMSG at 24 h, the proportion of cells in S phase could be maintained or augmented.…”

“…Granulosa cell proliferation during antral follicle growth in immature rats peaks 24 -42 h after stimulation with pregnant mare's serum gonadotropin (PMSG) and returns to baseline (pre-PMSG) levels at 48 h [prior to the administration of an ovulatory human chorionic gonadotropin (hCG) bolus] (3,13,20). During this time, the expression of LHCGR mRNA increases in a time-dependent manner until 42 h, after which expression levels plateau between 42-48 h post-PMSG, suggesting that cell cycle progression and LHCGR expression may be mechanistically linked.…”

Multiparametric study of atresia in ewe antral follicles: Histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

“…After flushing, the follicles were cut open with small iris scissors and gently scraped with a blunt probe into the same flushing fluid to insure the adequate recovery of granulosa cells (Hirshfield et al, 1988;Ballesteros et al, 1992;Guthrie et al, 1994). Care was taken to avoid contaminating the follicular fluid with blood.…”