Partial purification from bovine follicular fluid of a factor of low molecular mass with inhibitory effects on the proliferation of bovine granulosa cells in vitro and on rat follicular development in vivo

Abstract

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.

Inhibin A Increases Apoptosis in Early Ovarian Antral Follicles of Diethylstilbestrol-Treated Rats1

“…In this context, the relevance of interfollicular regulation in this process is well recognized. Factors from granulosa cells or in follicular fluid, presumably produced by the dominant follicle, that appear to inhibit the development of its subordinate neighbors have been described in various species [23,24,30,[47][48][49]. Our results suggest that inhibin produced by the dominant follicle may be an additional factor participating in this process.…”

Mouse Ovarian Follicles Secrete Factors Affecting the Growth and Development of Like-Sized Ovarian Follicles In Vitro1

“…Subsequently, steroid-free ovine follicular fluid was shown to lower steroid secretion from the ovary, with an associated decrease in P450 arom and inhibin-activin ␤ b subunit [12,24]. Granulosa cell-inhibitory factor (GCIF), a factor obtained from steroid-free bovine follicular fluid, has been shown to inhibit rat and bovine follicle growth and development [13,14], whereas immunization of sheep against GCIF increased the ovulation rate [25]. Similar factors have been reported both in the pig [26] and the rat [27].…”

“…Mizunuma et al [15] have also documented that the development of primary mouse follicles in vitro is inhibited by activin secreted by larger secondary preantral follicles in vitro. Production of stimulatory factors has also been reported (e.g., [13,16,27]), although they have been investigated less than the inhibitory ones.…”

“…Hypotheses regarding direct follicular interactions have been suggested now for many years, but have proven difficult to investigate in vivo [1]. Despite such difficulties, several extracts of follicular fluid have been proposed to play a role in folliclefollicle interactions [9][10][11][12][13][14], but their in vivo roles have yet to be established.…”

Evidence of intraovarian follicular dominance effects during controlled ovarian stimulation in a sheep model

“…Studies published elsewhere evidenced the production of follicle growth-inhibitory factors (34); specifically, by the characterization of granulosa cell inhibitory factors, which negatively affect the proliferation of small and medium follicles in the bovine (35,36). Follicle growth inhibitory factors have been hypothesized to act either by impairing the production and/or activity of local growth factors via subordinate follicles and, thus, the response of granulosa cells to FSH or by interfering with the appropriate vascularization of subordinate follicles, a key step in follicular development (37).…”