22:T555,Bovine follicular fluid was aspirated from follicles of 2-20 mm in diameter, charcoal-treated to remove steroids and then separated into low and high molecular mass fractions. The low molecular mass (< 10 kDa) fraction was purified on a Sephadex G-25 chromatography column with formic acid as the eluent. Seven peaks were isolated and assayed for biological activity in cultures of bovine granulosa cells at concentrations of 10, 100 and 1000 ng ml-1. One peak (peak 4) inhibited (P < 0.001) the proliferation of granulosa cells when measured by cell counting and by [3H]thymidine incorporation (33-37% inhibition). This peak inhibited proliferation of granulosa cells from both small (< 2 mm) and medium (2-10 mm) follicles, but not large (> 10 mm) follicles. The inhibitory effect of peak 4 was not due to a toxic effect on cells. Administration of peak 4 to rats did not affect liver or kidney masses but did decrease uterine (25%, P < 0.01) and ovarian (35%, P < 0.01) masses. Peak 4 also caused a reduction in the number of large follicles (65%, P < 0.01) but increased the number of small follicles (55%, P < 0.01). We have named the inhibitory factor associated with peak 4, granulosa cell-inhibitory factor (GCIF). The results presented suggest that GCIF may be a factor secreted by dominant follicles that inhibits the development of subordinate follicles.23:T516,In the dairy cow, low systemic concentrations of progesterone are known to be a major factor associated with early embryo loss. Endometrial expression of the gene encoding retinol-binding protein (RBP) is sensitive to small changes in progesterone on day 7 of the oestrous cycle. The objectives of the present study were to measure RBP concentrations in bovine uterine flushings and plasma across different days of the oestrous cycle and to examine the relationship between uterine RBP and systemic concentrations of progesterone. Uterine flushings and plasma were collected from cows on days 3, 7, 11 and 15 of the oestrous cycle. Uterine RBP concentrations were five- to 15-fold higher (P < 0.001) on day 15 compared with the other days and twofold higher (P < 0.001) in the uterine horn ipsilateral to the corpus luteum on day 15. RBP concentrations were similar in flushings and plasma across days 3-11; however, day 15 RBP concentrations were six- to 15-fold higher (P < 0.001) in uterine flushings. No significant relationship was found between the concentration of systemic progesterone and RBP concentrations on day 7. Overall, the results of the present study indicate a local controlling mechanism operating at the level of the endometrium to regulate RBP secretion, most likely progesterone.24:T50e,A granulosa cell-inhibitory factor (GCIF) of low molecular mass from bovine follicular fluid inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. The present study examined the ability of GCIF to modulate the effects of FSH, epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) and androstenedione on the proliferation of bovine granulosa cells and on aromatase activity in vitro. Granulosa cell proliferation was assayed by counting haemocytometric cells and by measuring the incorporation of [3H]thymidine into acid-precipitable material. Assay of aromatase activity was based on the conversion of [3H]androstenedione to [3H]H2O. FSH, androstenedione, EGF and IGF-I all stimulated (P < 0.01) granulosa cell proliferation; however, the addition of GCIF reduced (P < 0.01) cell proliferation in their presence. In the case of EGF, the addition of GCIF almost abolished the stimulatory response. FSH and IGF-I, but not EGF, stimulated (P < 0.01) aromatase activity of granulosa cells. The stimulatory effect of IGF-I was decreased by GCIF. The inhibitory effects of GCIF indicate that it may play a significant role in regulating the effects of intraovarian growth factors on granulosa cells and the growth of follicles.

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