Follicular fluid concentrations of free insulin-like growth factor (IGF)-I during follicular development in mares.

The objective of the present study was to evaluate changes in concentrations of free insulin-like growth factor (IGF)-I in follicular fluid (FFL) during follicle development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6-15 mm; n = 54), medium (16-25 mm; n = 23) or large (>25 mm; n = 15) and FFL was collected. Free IGF-I levels in FFL in large follicles of follicular phase mares were greater (P < 0.05) than in large follicles of luteal phase mares and small or medium follicles of luteal and follicular phase mares. Free IGF-I concentrations were greater (P < 0.05) in large follicles of luteal phase mares than small but not medium follicles of luteal phase mares. FFL ratio of estradiol:progesterone paralleled changes in free IGF-I. Free IGF-I concentrations were negatively correlated (P < 0.05) with insulin-like growth factor binding protein (IGFBP)-2, -4 and -5 but not IGFBP-3 levels. In addition, free IGF-I concentrations in FFL were positively correlated (P < 0.01) with FFL estradiol, progesterone, androstenedione, estradiol:progesterone ratio, total IGF-I and total IGF-II. We conclude that increases in intrafollicular levels of bioavailable (free) IGF-I are associated with increased steroidogenesis in developing mare follicles.

Insulin-like growth factor-I and its binding proteins in colostrum compared to measures in serum of Holstein neonates.

Colostral insulin-like growth factor-I (IGF-I) may be beneficial in the development of gastrointestinal tracts of bovine neonates. Thus, the purpose of this study was to examine relationships among concentrations of IGF-I and IGF-binding proteins (IGFBP) in colostrum used at two initial feedings and serum concentrations of IGF-I, IGFBP, total protein, gamma glutamyltransferase (GGT), and immunoglobulin G at 0 and 48 h after birth in Holstein neonates. Calves (n = 22) were separated from dams immediately after birth. Blood samples were taken before initial feeding and at 48 h after birth. Calves were fed 2 L of colostrum twice and milk replacer thereafter. Linear regression of serum IGF-I at 48 h and colostral IGF-I revealed a significant positive relationship (R2 = 0.204). Serum IGFBP-3 at 48 h and colostral IGFBP-3 also had a positive relationship (R2 = 0.143). However, linear regression of colostral IGF-I on the difference in serum IGF-I at 48 and 0 h was not significant. Calves were assigned to group 1 (0-h serum IGF-I < 10 ng/ml; n = 11) or group 2 (0-h serum IGF-I > or = 10 ng/ml; n = 11) for further analysis. There were no differences in serum IGF-I or IGFBP-2, -3, -4, and -5 concentrations at 48 h between groups 1 and 2. Correlation coefficients revealed negative relationships of serum IGF-I at 0 h to the difference between serum IGF-I at 48 and 0 h (r = -0.824), as well as birth weight of the calf to the amount of GGT at 48 h (r = -0.604). Females had lower birth weights than males, but sex of calf did not affect serum measures. At 0 h, but not 48 h, total serum protein was correlated to serum GGT concentrations (r = 0.573). From indirect evidence, absorption of colostral IGF-I and IGFBP-3 into systemic circulation may occur, but relative importance compared to endogenous sources is uncertain.

Propionibacteria fed to dairy cows: effects on energy balance, plasma metabolites and hormones, and reproduction.

To determine the effect of feeding Propionibacteria on energy balance, milk yield, and composition, metabolites and hormones of early-lactating dairy cows, multiparous Holstein cows were individually fed a total mixed ration from -2 to 12 wk postpartum with no addition (control, n = 10) or with an additional 17 g of Propionibacteria culture daily (Treated, n = 9). Daily feed intake and milk production were recorded. Plasma cholesterol, nonesterified fatty acids (NEFA), leptin, insulin, glucose, insulin-like growth factor-I (IGF-I), IGF-binding proteins (IGFBP), and progesterone concentrations were measured up to twice weekly. Cows fed supplemental Propionibacteria had improved energy balance at wk 1 of lactation and had lower DMI per kg of body weight than control cows on wk 3 to 7, 10, and 12. Cows fed Propionibacteria had a greater percentage of milk protein and solids-not-fat and plasma NEFA concentrations than did control cows only at wk 1 of lactation. Treatment did not affect milk production or percentage of milk fat and lactose. Leptin levels were greater in treated than control cows throughout the study. Plasma glucose, insulin, cholesterol, IGFBP-3, and IGF-I concentrations were not affected by feeding Propionibacteria, but those variables increased with week postpartum. Plasma IGFBP-2 and IGFBP-5 levels decreased with week postpartum. Measures of reproductive and ovarian function did not differ between Propionibacteria-treated and control cows. Feeding Propionibacteria culture to transition and early lactating dairy cows may hold potential for improved milk protein production and metabolic efficiency during early lactation, without affecting reproductive function.

Changes in follicular fluid steroids, insulin-like growth factors (IGF) and IGF-binding protein concentration, and proteolytic activity during equine follicular development.

The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), and IGFBP-3 concentrations tended to be greater (P < 0.10) in large than in small or medium follicles, whereas IGFBP-2, -4, and -5 levels were less (P < 0.05) in large than in small or medium follicles. Estradiol and androstenedione concentrations were negatively correlated (P < 0.01) with IGFBP-2, -4, and -5 but not IGFBP-3 concentrations. To evaluate proteolysis of IGFBP, follicular fluid was incubated with human 125I-labeled IGFBP-2, -3, and -5 and protein separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of 125I-lableled IGFBP-2 or -3, and the small amount of proteolysis of IGFBP-2 and -3 did not differ (P > 0.10) among follicle classes. However, more 125I-labeled IGFBP-5 was cleaved (P < 0.05) by follicular fluid from large follicles collected during the follicular phase than large follicles during the luteal phase, and small or medium follicles from follicular and luteal phase mares indicating that a protease to IGFBP-5 exists in estrogen-dominant equine follicles. This IGFBP-5 protease was inhibited by kallikrein/serine protease and metalloprotease inhibitors. We conclude that the tendency of estrogen-dominant follicles of mares to have greater levels of IGFBP-3 and lesser levels of IGFBP-2 does not appear to be due to differences in proteolysis, whereas changes in IGFBP-5 levels are likely due to changes in activity of a serine protease or metalloprotease. Changes in IGFBP may alter levels of bioavailable IGF that stimulate steroidogenesis and mitogenesis in developing mare follicles.

Dexamethasone influences endocrine and ovarian function in dairy cattle.

Multiparous nonlactating Holstein cows were used to determine the effect of dexamethasone on ovarian follicular development and plasma hormone concentrations. Animals were randomly divided into two groups, control (C; n = 5) and treatment (T; n = 6), but managed as one group. Both groups were synchronized with two injections of PGF2alpha (25 mg i.m.) 11 d apart. One day after ovulation (d 0) the T group received a daily injection of dexamethasone (44 microg/kg of body weight; i.m.) until the first dominant follicle stopped growing or up to d 12 postovulation. The C group received vehicle injections. Blood samples were collected daily from all cows. Concentrations of LH and FSH did not differ between the C and T cows, whereas progesterone concentrations were lower in T than in C cows from d 4 onward. Treatment x day interaction influenced plasma insulin concentrations such that T cows had insulin concentrations 2.9- to 6.0-fold those of C cows between d 2 and 9. Dexamethasone decreased IGF-I and -II concentrations from d 5 onward. Concentrations of plasma leptin and the various IGF binding proteins were not affected by dexamethasone. Total number of follicles (> or = 5 mm) and plasma estradiol concentrations were less in T than in C cows on d 0, 1, and 4. The growth rate of the dominant follicles and maximum diameter of the dominant and subordinate follicles were not affected by dexamethasone. The diameter of the CL was 21 to 39% larger in T than in C cows between d 6 and 10. Treatment x day interaction influenced plasma cholesterol concentrations such that cholesterol levels decreased 46.8% in T cows and 19.5% in C cows between d 0 and 10. Plasma glucose concentrations were greater in T than in C cows between d 1 and 10. In summary, dexamethasone had significant effects on metabolism without a major impact on growth of the first-wave dominant follicle. Dexamethasone-induced suppression of luteal function was associated with decreased plasma IGF-I and -II concentrations.

Hormonal control of ovarian cell production of insulin-like growth factor binding proteins.

To determine if the hormonal effects on insulin-like growth factor binding protein (IGFBP) production differed between granulosa and thecal cells, both cell types were collected and cultured in serum-free medium with various hormone treatments, arranged in three experiments. Following treatment, cells were enumerated and media were collected, concentrated 10-fold and subjected to ligand blotting. Experiment 1 revealed that > or =1.5 x 10(5) viable cells at plating were needed for maximal IGFBP production by granulosa and thecal cells. The major forms of IGFBPs produced were a 27-34-kDa IGFBP (IGFBP-2 and -5), and a 20-22-kDa IGFBP (IGFBP-4) by the granulosa cells and a 40-44-kDa IGFBP (IGFBP-3), 34-kDa IGFBP (IGFBP-2), 27-29-kDa IGFBP (IGFBP-5) and a 20-22-kDa IGFBP (IGFBP-4) by the thecal cells. In Experiment 2A, insulin stimulated production of IGFBP-5 by thecal cells, and basic fibroblast growth factor (bFGF) inhibited the insulin-induced increase in IGFBP-5 production; epidermal growth factor (EGF) and luteinizing hormone were without effect. The small amounts of IGFBP-2 and -3 produced by thecal cells of Experiment 2A were not affected by treatment. Production of IGFBP-2/-5 by granulosa cells in Experiment 2B was inhibited by insulin, with EGF and bFGF further enhancing insulin's inhibitory effect; follicle-stimulating hormone was without effect. In Experiment 3A, insulin enhanced production of IGFBP-5 by thecal cells whereas glucagon blocked insulin's stimulatory effect. In contrast, insulin or glucagon alone had no effect on production of the IGFBP-4 by thecal cells but when combined inhibited IGFBP-4 production. The small amounts of IGFBP-2 and -3 produced by thecal cells of Experiment 3A were not affected by treatment. In Experiment 3B, production of IGFBP-2/-5 by granulosa cells was attenuated in the presence of cortisol with or without insulin and insulin plus glucagon; glucagon and cortisol decreased production of IGFBP-4 by granulosa cells. These results suggest that production of IGFBP-2, -4, and -5 by granulosa and thecal cells are differentially affected by hormonal stimuli, and that IGFBP-3 is more consistently produced by thecal cells than granulosa cells of cattle although its production was not hormonally regulated.

Proteolysis of insulin-like growth factor binding proteins during preovulatory follicular development in cattle.

The present study was conducted to evaluate changes in follicular fluid (FF) insulin-like growth factor binding protein (IGFBP) proteolytic activity and levels of steroids and IGFBP during follicular development in cattle. Estrous cycles of cows were synchronized with two injections of prostaglandin F2alpha (PGF) 11 d apart and follicular growth monitored via daily rectal ultrasonography in order to identify the dominant follicle. All cows were ovariectomized 48 hr after the second injection of PGF. Follicular fluid was collected individually for all follicles > 5 mm and pooled for small (1 to 5 mm) follicles. Follicular fluid estradiol and androstenedione levels were greater (P < 0.05) and progesterone and IGFBP-3 levels not different (P > 0.10) in large dominant than in small (1 to 5 mm) or large (>5 mm) subordinate follicles, whereas IGFBP-2, -4 and -5 levels were less (P < 0.05) in large dominant than in small or large subordinate follicles. To evaluate proteolysis of IGFBPs, FF was incubated with recombinant human (125) I-labeled IGFBP-2, -3, -4, and -5 and proteins separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of (125)I-lableled IGFBP-2 or -3. However, cleavage of (125)I-labeled IGFBP-4 and -5 by FF from large dominant follicles was greater (P < 0.05) than by FF from small or large subordinate follicles indicating that a protease to IGFBP-4 and -5 exists in estrogen dominant follicles. We conclude that lower levels of IGFBP-2 in estrogen dominant follicles of cattle are not due to increased proteolysis, whereas decreases in IGFBP-4 and -5 levels are likely due, in part, to increased protease activity. Changes in IGFBP may alter levels of bioavailable IGFs that stimulate steroidogenesis and mitogenesis in developing bovine follicles.

Possible role of kallikrein in proteolysis of insulin-like growth factor binding proteins during the oestrous cycle and early pregnancy in pigs.

During early pregnancy, pig conceptuses initiate the synthesis of oestrogens and on day 12 their trophoblastic membranes undergo a rapid expansion throughout the uterine horns. The insulin-like growth factor (IGF) system may be involved with conceptus development and steroidogenesis in pigs. Changes in uterine luminal IGF, insulin-like growth factor binding proteins (IGFBPs) and enzymatic activity for cleavage of IGFBPs during the oestrous cycle and early pregnancy were investigated. Uterine luminal content of IGF-I and IGF-II in uterine flushings from pigs on day 12 of pregnancy were two and three times greater, respectively, compared with uterine flushings collected from gilts during the oestrous cycle. Both IGF-I and -II content decreased on day 15 of gestation but content of IGF-II in uterine flushings remained three times greater than that of cyclic gilts. IGFBP-2 and -3 were the predominant binding proteins present in uterine flushings during days 0-10 of the oestrous cycle or day 10 of pregnancy. No IGFBPs were detected in the uterine flushings of either cyclic or pregnant pigs after day 10 by ligand blotting. Incubation of [125I]-labelled IGFBPs with various protease inhibitors indicated that cleavage of [125I]-labelled IGFBP-2 and -3 in uterine flushings involved serine proteases such as tissue kallikrein and matrix metalloproteinases. The results of the present study indicate that an increase in tissue kallikrein activity on day 12 of the oestrous cycle and pregnancy in pigs can directly, or indirectly through activation of matrix metalloproteinases, cleave IGFBP-2 and -3, thus allowing uterine release of IGF-I and -II in the uterine lumen to stimulate conceptus development.

Effects of thyroid hormones on bovine granulosa and thecal cell function in vitro: dependence on insulin and gonadotropins.

The objective of this study was to evaluate the influence of thyroxine (T4) and triiodothryonine (T3) on steroid production by bovine granulosa and thecal cells. Granulosa and thecal cells were obtained from small (1 to 5 mm) and large (> or = 8 mm) follicles of cattle, respectively, and cultured for 4 d. We conducted six experiments to evaluate the effect of 2 d of exposure to various doses of T3 or T4. In insulin- or insulin plus FSH-treated granulosa cells of experiment 1, 30 and 100 ng/ml of T4 had no effect on aromatase activity or progesterone production. In experiment 2, in the presence of insulin and FSH, 1 and 3 ng/ml of T3 weakly (<1.4-fold) increased aromatase activity of granulosa cells but had no effect on progesterone production. Low doses of T4 (3 to 30 ng/ml) tested in experiment 3 had no effect on aromatase activity but increased (to as much as 1.4-fold) progesterone production by granulosa cells. In experiment 4, T4 (30 ng/ml) increased (to 1.2-fold) progesterone production by granulosa cells only in the presence of FSH and had no effect on aromatase activity. In thecal cells of experiment 5, in the presence of insulin and LH, 30 and 100 mg/ml of T4 increased androstenedione production to 2.3- and 2.8-fold, respectively; only 100 ng/ml of T4 was effective at stimulating progesterone production by thecal cells. In experiment 6, 1 ng/ml of T3 increased thecal cell androstenedione production to 3.9-fold, whereas 3 ng/ml of T3 was without effect; progesterone production was not affected by T3. These results support the hypothesis that thyroid hormones may have direct stimulatory effects on ovarian function in cattle, acting at the level of granulosa and thecal cells.

Production of insulin-like growth factor-I by granulosa cells but not thecal cells is hormonally responsive in cattle.

To determine whether the hormonal regulation of IGF-I production differs between granulosa and thecal cells in cattle, granulosa and thecal cells from bovine follicles were collected, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 24 h in serum-free medium with various hormones. In Exp. 1, granulosa cells were treated with 0 or 100 ng/mL of insulin and(or) 50 ng/mL of follicle-stimulating hormone (FSH), insulin plus 10 ng/mL of epidermal growth factor, or insulin plus 10 ng/mL of basic fibroblast growth factor. In Exp. 2, thecal cells were treated as described in Exp. 1 except that 100 ng/mL of luteinizing hormone (LH) was used instead of 50 ng/mL of FSH. In Exp. 3, granulosa and thecal cells were treated with 0 or 30 ng/mL of cortisol with or without 100 ng/mL of insulin, 300 pg/mL of glucagon, or glucagon plus insulin. In Exp. 4, granulosa and thecal cells were treated with 0 or 300 ng/mL of estradiol with or without 100 ng/mL of insulin and(or) 100 ng/mL of LH. At the end of treatment, medium was collected, concentrated with Centricon-3 concentrators, and assayed for IGF-I by radioimmunoassay. Cell numbers were determined by Coulter counting at the end of culture. Thecal cells produced low amounts of IGFI (0.48 +/- 0.04, 0.63 +/- 0.03, and 0.82 +/- 0.03 ng per 100,000 cells per 24 h in Exp. 2, 3, and 4, respectively), and this production was not influenced (P > 0.05) by the various treatments. In contrast, IGF-I production by granulosa cells (2.0 to 6.2 ng per 100,000 cells per 24 h) was influenced by treatment in Exp. 1, 3, and 4 and was greater than IGF-I production by thecal cells (Exp. 2, 3, and 4). Alone, insulin, FSH, LH, and cortisol (but not estradiol) each decreased (P < 0.05) granulosa-cell IGF-I production by 20 to 57%; combined treatments of insulin plus FSH or insulin plus cortisol decreased IGF-I production to levels seen with insulin alone. Glucagon had no effect (P > 0.10) on IGF-I production in the absence or presence of insulin. In the presence of insulin, epidermal growth factor, basic fibroblast growth factor, and estradiol decreased (P < 0.05) IGF-I production below that observed for insulin alone. These results indicate that, during follicular development in cattle, changes in intrafollicular levels of IGF-I may be due to hormonally-induced changes in granulosa-cell, but not thecal-cell, IGF-I production.

Ovarian action of leptin: effects on insulin-like growth factor-I-stimulated function of granulosa and thecal cells.

To test the hypothesis that leptin signals metabolic information to the reproductive system in cattle by directly affecting IGF-I-induced ovarian cell function, granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in serum-free medium with added hormones. Recombinant human leptin at 30 and 300 ng/mL had no effect on basal thecal cell steroidogenesis or thecal cell numbers. However, 300 but not 30 ng/mL of leptin attenuated (p < 0.05) luteinizing hormone-induced androstenedione production by 24% in the absence of IGF-I and by 16% in the presence of IGF-I. Leptin had no effect on IGF-I-induced estradiol production in the presence of follicle-stimulating hormone (FSH), but at 100 ng/mL, leptin inhibited (p < 0.05) FSH plus IGF-I-induced progesterone production and granulosa cell proliferation by 29 and 31%, respectively. Leptin did not compete for 125I-IGF-I binding to granulosa or thecal cells, whereas unlabeled IGF-I did. In conclusion, leptin has weak inhibitory effects on gonadotropin- and/or IGF-I-induced steroidogenesis of thecal and granulosa cells.

Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells.

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.

Influence of cortisol on insulin- and insulin-like growth factor 1 (IGF-1)-induced steroid production and on IGF-1 receptors in cultured bovine granulosa cells and thecal cells.

During stress, hyperactivity of the adrenal gland can directly and indirectly inhibit ovarian function. However, little evidence existed to support the notion that glucocorticoids could influence insulin-like growth factor 1 (IGF-1) action within the ovary. Therefore, the effect of cortisol on IGF-1-induced granulosa and thecal cell function was evaluated. Granulosa and thecal cells from bovine ovarian follicles were cultured for 2 d in the presence of 10% fetal calf serum and then cultured for an additional 2 d in serum-free medium with added hormones. Cortisol had little or no effect (p > 0.05) on IGF-1-induced progesterone production by granulosa cells from both small (1-5 mm) or large (> or =8 mm) follicles. Also, cortisol had little or no effect (p > 0.05) on basal, insulin-, or IGF-1-induced estradiol production by granulosa cells from small or large follicles, or on the number of IGF-1 receptors in granulosa cells from small follicles. Cortisol had no effect (p > 0.10) on insulin-induced granulosa cell numbers, but increased IGF-1-induced granulosa cell numbers. In thecal cells, doses of 1-100 ng/mL of cortisol increased (p< 0.05) insulin- and IGF-1-induced thecal cell numbers by 10-20%, progesterone production by 18-36%, and androstenedione production by two- to fourfold. The estimated dose of cortisol necessary to stimulate 50% of the maximum androstenedione production in the presence of IGF-1 was 7 ng/mL. In contrast, cortisol decreased (p < 0.05) the number of IGF-1 receptors in thecal cells by 45%. In conclusion, cortisol at physiological levels can directly influence ovarian follicular function in cattle, especially thecal androstenedione production.