Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers.

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.

Canty MJ ，Boland MP ，Evans AC ，Crowe MA ... -  《ANIMAL REPRODUCTION SCIENCE》

Pregnancy-associated plasma protein-A and insulin-like growth factor binding protein mRNAs in granulosa cells of dominant and subordinate follicles of preovulatory cattle.

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.

Santiago CA ，Voge JL ，Aad PY ，Allen DT ，Stein DR ，Malayer JR ，Spicer LJ ... -  《DOMESTIC ANIMAL ENDOCRINOLOGY》

Gonadotrophin responsiveness, aromatase activity and insulin-like growth factor binding protein content of bovine ovarian follicles during the first follicular wave.

The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.

Rhodes FM ，Peterson AJ ，Jolly PD 《REPRODUCTION》

Modulating peripheral gonadotrophin levels affects follicular expression of mRNAs encoding insulin-like growth factor binding proteins in sheep.

Evidence suggests that the insulin-like growth factor binding proteins (IGFBPs) are involved in modulating the role that IGF-I and -II play in regulating follicular growth and development in sheep. However, little information exists as to the role that key peripheral factors play in regulating the expression of IGFBP components within the follicle. The present study investigated the regulatory effects of FSH and LH on gene expression for IGFBP-2 to -6 in ovine follicles, using bovine follicular fluid (bFF) and gonadotrophin-releasing hormone antagonist (GnRHa) model systems to perturb endogenous gonadotrophin secretion. Gene expression studies were carried out using in situ hybridisation with sheep-specific ribonucleotide probes. Treatment of ewes with bFF had few consistent effects on IGFBP-3 and -4 mRNA expression in follicles, whereas bFF treatment resulted in significant decreases in IGFBP-2 and IGFBP-6 mRNA expression in only medium follicles 60 h after onset of bFF, and in follicles 12h after the onset of bFF, respectively. Treatment of ewes with GnRHa resulted in transient significantly increased IGFBP-2 mRNA in healthy follicles in comparison to early atretic or atretic follicles 12h post-GnRHa (P<0.05), in IGFBP-3 mRNA in early atretic and atretic follicles 36 h post-GnRHa (P<0.001), and IGFBP-5 mRNA in follicles 12h post-GnRHa (P<0.001). In contrast, GnRHa treatment resulted in significant decreases in IGFBP-4 (P<0.001) and IGFBP-6 (P<0.01) mRNA expression in large follicles 36 h post-GnRHa, and atretic follicles by 60 h post-GnRHa, respectively. These data highlight that FSH and LH are involved, at least in part, in mediating the proliferative and differentiative changes in intrafollicular IGFBP levels that are observed during follicular growth and atresia in the sheep.

Hastie PM ，Haresign W 《-》

Levels of insulin-like growth factor (IGF) binding proteins, luteinizing hormone and IGF-I receptors, and steroids in dominant follicles during the first follicular wave in cattle exhibiting regular estrous cycles.

Objectives of this study were to determine if concentrations of steroids, insulin-like growth factor -I (IGF-I), and IGF binding proteins (IGFBP) in follicular fluid and numbers of LH and IGF-I receptors change during growth of the dominant follicle. Ovarian follicular development was monitored daily via ultrasound in lactating Holstein cows. Animals underwent bilateral ovariectomy when the dominant follicle was first identified (days 4-6; estrus = day 0; early; n = 5) or when it stopped growing (days 8-12; late; n = 8). All follicles were classified as dominant (DF), large (LG; > = 6 mm in diameter, excluding DF) or small (SM; < 6 mm), follicular fluid was aspirated, and theca and granulosa cells were collected. Levels of IGFBP-2, assessed via ligand blotting, were greater (P < 0.05) in LG and SM follicles compared with DF in early cows. Levels of IGFBP-3 in follicular fluid were unaffected by follicle class. Numbers of specific 125I-hCG/LH binding sites in thecal cells were greater (P < 0.01) in DF compared with LG and SM follicles of both early and late cows. Numbers of specific 125I-hCG/LH binding sites in granulosa cells were similar for follicle sizes in early cows, but, in late cows, were greater (P < 0.01) in DF compared with SM follicles and were severalfold greater (P < 0.01) in late DF compared with early DF. Numbers of receptors for IGF-I in thecal cells were 2-fold greater (P < 0.05) in DP and LG compared with SM in late cows. Numbers of IGF-I receptors in granulosa cells were unaffected by size or growth of follicles, but were severalfold greater than in theca cells. Concentrations of estradiol were severalfold greater (P < 0.01) in DF compared with LG and SM in both early and late cows. Concentrations of androstenedione in early cows were greater (P < 0.05) in DF and SM compared with LG follicles. Concentrations of progesterone and IGF-I did not differ (P > 0.10) among follicle classes, but both were greater (P < 0.10) in late LG compared with early LG follicles. Concentrations of IGF-II in follicular fluid did not differ (P > 0.10) between early and late cows but were greater (P < 0.10) in SM than DF or LG follicles. We conclude that low amounts of IGFBP-2 and increased thecal binding sites for hCG/LH appear to be related to establishment of the dominant follicle during the first follicular wave in cattle exhibiting regular estrous cycles during late lactation.

Stewart RE ，Spicer LJ ，Hamilton TD ，Keefer BE ，Dawson LJ ，Morgan GL ，Echternkamp SE ... -  《ENDOCRINOLOGY》