Proteolytic degradation of insulin-like growth factor binding proteins by ovarian follicles: a control mechanism for selection of dominant follicles

This review summarizes evidence for the role of proteolytic enzymes that degrade and inactivate insulin-like growth factor binding proteins (IGFBP) during follicular development in mammals. In some species (e.g., bovine), evidence indicates that decreases in IGFBP-4 and -5 levels in estrogen-dominant preovulatory follicles are likely due, in part, to increased protease activity, whereas lower levels of IGFBP-2 are not due to increased proteolysis. Increased IGFBP-4 and -5 protease along with lower amounts of IGFBP-4 binding activity and greater amounts of free IGF-I are some of the earliest developmental changes documented in bovine growing antral follicles. This protease activity has recently been ascribed to serine metalloprotease(s), including pregnancy-associated plasma protein-A (PAPP-A), which was first detected in human follicular fluid nearly 20 yr ago. Other recent studies verified the presence of PAPP-A mRNA in granulosa cells of humans, monkeys, cattle, mice, and pigs. Increases in the amount of PAPP-A mRNA in granulosa cells during follicular development occurs in some but not all species, indicating that other proteases or protease inhibitors may be involved in IGFBP degradation. Whether the hormonal control of PAPP-A production/activity by the ovary differs between monotocous and polytocous animals will require further study. These protease-induced decreases in IGFBP-4 and -5 likely cause increased levels of bioavailable (or free) IGFs that stimulate steroidogenesis and mitogenesis in developing dominant follicles, which ultimately prepare the follicle(s) and oocyte(s) for successful ovulation and fertilization.     

Concentrations of insulin-like growth factor-I, estradiol and progesterone in follicular fluid of ovarian follicles during early pregnancy in cattle

To determine if the presence of the developing conceptus is associated with changes in intrafollicular concentrations of insulin-like growth factor-I (IGF-I), estradiol (E2) and/or progesterone during early pregnancy in cattle, either pregnant (n=16) or nonpregnant (n=15) cows were slaughtered on Day 10, 15 or 18 postestrus. Ovaries and follicular fluid were collected. Follicles were grouped by diameter: 1.0 to 3.9 mm (small; n=63), 4.0 to 7.9 mm (medium; n=128), and >/= 8.0 mm (large; n=38). The average diameter of large follicles was greater (P<0.05) in pregnant than in nonpregnant cows on Day 10, but on Day 18 it was greater (P<0.05) in nonpregnant than in pregnant cows (11.3 vs 9.7 mm). Status (pregnant vs nonpregnant) did not affect (P>0.10) follicular fluid progesterone nor IGF-I concentrations. In contrast, the status and days postestrus affected (P<0.05) follicular fluid E2 concentrations. Follicular fluid E2 levels in the three follicle size-categories on Day 10 did not differ (P>0.10) between pregnant and nonpregnant cows. However, on Days 15 and 18 postestrus, follicular fluid E2 concentrations in pregnant cows was lower (P<0.05) in large follicles than in nonpregnant cows. We conclude that the presence of a developing conceptus early in pregnancy may alter follicular growth and inhibit follicular E2 production in cattle. These effects appear to be mediated by factors other than IGF-I.     

Relationships between concentrations of ovarian steroids,insulin-like growth factor-1 and IGF-binding proteins during follicular development in the ewe

The objective of the present study was to determine how insulin-like growth factor-1 (IGF-1) and IGF-binding proteins (IGFBPs) are related to in vivo follicular development in the sheep. Oestrus was synchronised in 20 cyclic ewes and the animals were slaughtered 44 h after the second injection, just before the start of preovulatory luteinising hormone (LH) surge. Normal growing follicles were dissected from the ovaries of each ewe and their diameters measured. The follicular fluid was aspirated and assayed for oestradiol, testosterone and total IGF-1 content. The follicles were classified as either non-oestrogenic or oestrogenic if the follicular fluid content of oestradiol was less than 60 ng ml⁻¹ or more than 60 ng ml⁻¹, respectively. The mean diameter of oestrogenic follicles was significantly (P < 0.001) higher than that of non-oestrogenic ones, but testosterone concentrations did not differ. IGF-1 concentrations in oestrogenic follicles were significantly (P < 0.01) lower than those in non-oestrogenic ones, with a significant (P < 0.01) negative correlation between follicular oestradiol content and IGF-1 concentration. IGFBPs were identified by Western ligand blot analysis using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions and band intensities on autoradiographs were quantified by scanning densitometry. The intensity of the doublet of IGFBP at 42–44 kDa was significantly (P < 0.02) higher in follicular fluid from oestrogenic follicles, whereas the intensity of the band at 35 kDa was significantly (P < 0.001) higher in follicular fluid from non-oestrogenic follicles. Some of the non-oestrogenic follicles also exhibited bands at 32.0-28.5 kDa with variable intensities, but such bands were totally absent in oestrogenic follicles. The results of this study suggest an involvement of both IGF-1 and IGFBPs in ovine follicular development.     

Characterization of insulin-like growth factor-binding proteins of porcine ovarian follicular fluid.

Using competitive ligand-binding studies, ligand blotting, and immunoprecipitation, we have characterized the insulin-like growth factor (IGF)-binding proteins (BPs) of porcine follicular fluid. Competitive ligand-binding studies revealed a preference of ovarian IGFBPs for IGF-II over IGF-I. Follicular fluid from small, 1-3-mm follicles had nearly twice the binding capacity for IGFs as that from large, 6-10-mm follicles. Ligand blots of porcine follicular fluid resolved 5 major bands of IGF-binding activity having apparent molecular sizes of 44, 40, 34, 29, and 22 kDa. The 40-44-kDa bands were immunoprecipitated by an antibody to porcine IGFBP-3, the acid-stable subunit of the 150-kDa growth hormone-dependent IGF-binding protein complex of porcine serum. The 34-kDa band was immunoprecipitated by an antibody to rat IGFBP-2, the major IGF-binding protein found in fetal rat serum. To date we have been unable to immunoprecipitate the 29- and 22-kDa bands with any of the antibodies tested, including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGF-binding protein. The 40-44-kDa species (IGFBP-3) was the predominant form and was equally abundant in fluid from large and small follicles. In contrast, the smaller forms, including IGFBP-2 and the 29- and 22-kDa forms were significantly more prominent in fluid from small follicles. In view of other studies indicating a significant effect of IGFBPs on ovarian cell function, follicular IGFBPs may play an important role in the IGF autocrine/paracrine regulatory system of the ovary.     

Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins

Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2 alpha (PGF2 alpha). Blood and follicular fluid were collected 48-50 h post-administration of PGF2 alpha (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RIA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p less than 0.05) number of large (greater than or equal to 4 mm) follicles and 47% greater (p less than 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p less than 0.05) concentrations of IGF-I (+/- SE) in the two largest follicles than control (unselected) cows (327 +/- 28 vs. 243 +/- 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p less than 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (p less than 0.05) in small follicles than in large follicles, and was greater (p less than 0.05) in large follicles of control (93.2 +/- 5.3%) than twin-producing (76.2 +/- 4.4%) cattle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of days after calving on insulin-like growth factor-I, insulin-like growth factor binding proteins, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant follicles of postpartum beef cows

The effect of days after calving on IGF-I, IGFBP, progesterone, androstenedione, estradiol, and aromatase mRNA in dominant ovarian follicles (DF) was evaluated in Angus × Hereford cows. Growth of DF (>9 mm) was monitored daily by ultrasonography and fluid from DF was collected in vivo at either 30 ± 2 d or 47 ± 2 d postpartum. Follicular fluid (FF) was also aspirated from DF of contemporary ovulatory cows at proestrus. Estrous behavior was monitored continuously using the HeatWatch system, and progesterone in plasma collected twice weekly was used to assess luteal activity. Anovulatory DF aspirated 30 and 47 d postpartum had similar concentrations of IGF-I, IGFBP, progesterone, estradiol and androstenedione in FF and IGF-I and IGFBP in plasma. The intervals from aspiration to estrus were similar for cows aspirated 30 and 47 d postpartum. Proestrous follicles had greater (P < 0.01) estradiol (435 ± 79 ng/mL) than DF at 30 d (107 ± 63 ng/mL) or 47 d (68 ± 53 ng/mL) after calving. Concentrations of androstenedione in FF were also greater (P < 0.01) in proestrous follicles than in DF aspirated at 30 or 47 d after calving. Concentrations of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding proteins (IGFBP) in FF and plasma, and aromatase mRNA in granulosa cells were similar for anovulatory and proestrous cows. In conclusion, estradiol production by DF of postpartum anovulatory cows may be limited by inadequate production of androstenedione during the postpartum anovulatory interval and this may influence follicular maturation. Concentrations of IGF-I and IGFBP were similar in anovulatory and proestrous cows, an indication that alterations in the IGF-I system in the DF at 30–47 d after calving are not associated with delayed follicular development in postpartum beef cows.     

Effect of steroid- and inhibin-free ovine follicular fluid on ovarian follicles and ovarian hormone secretion

Treatment of ewes with steroid-free ovine follicular fluid (oFF) during the follicular phase of the oestrous cycle results in the immediate inhibition of the ovarian secretion of oestradiol, inhibin and androgens. An experiment was conducted to determine whether this effect of oFF was due to inhibin, or to direct inhibition of ovarian function by other factors in oFF. Eight ewes in which the left ovary and vascular pedicle had been autotransplanted to a site in the neck were studied during the breeding season. Luteal regression was induced in all animals by injection of cloprostenol (100 micrograms i.m.; PG) on Day 10 of the luteal phase. The animals were divided into two groups (n = 4) and treated with either steroid-free oFF (oFF; 3 ml s.c.; 3.2 microgram p1-26 alpha inhibin/ml) or steroid-free oFF in which the inhibin content had been reduced by greater than 90% (IFoFF; 3 ml s.c.; 0.3 microgram p1-26 alpha inhibin/ml) by affinity chromatography, 24 and 36 h after PG. Samples of ovarian and jugular venous blood were collected at (i) intervals of 4 h from 16 h before until 120 h after PG and (ii) intervals of 10 min from 48 to 52 h after injection of PG to investigate the pattern of pulsatile secretion of ovarian hormones. All ewes had previously been monitored during a normal PG-induced follicular phase. Injection of oFF resulted in an increase (P less than 0.05) in the concentration of inhibin in jugular venous plasma and a profound (P less than 0.001) and prolonged decrease in the peripheral concentration of follicle-stimulating hormone (FSH). Injection of IFoFF had no significant effect on peripheral concentrations of inhibin or FSH in the first 24 h after treatment; thereafter inhibin concentrations fell (P less than 0.01) progressively until 40 h and then increased (P less than 0.01) until 72 h after treatment. In both treatment groups, however, within 24-36 h of treatment the concentration of FSH increased 5-10-fold (P less than 0.001) to a peak that occurred within 48-60 h and then declined to basal concentrations within 72-84 h of treatment. The concentration of luteinizing hormone (LH) in jugular venous plasma increased in both groups after treatment (P less than 0.01), although the rise after injection of oFF only started after 24 h. Thereafter, there was a progressive increase in the concentration of LH, peaks occurring 48-60 h after treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

A potential role for insulin-like growth factor binding protein-4 proteolysis in the establishment of ovarian follicular dominance in cattle.

A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and growth of dominant ovarian follicles. We have investigated whether a component of the insulin-like growth factor (IGF) system, namely IGFBP-4 protease, is associated with the establishment of follicular dominance in cattle. IGFBP proteases degrade IGFBPs, freeing IGFs to interact with their receptors. In experiment 1, follicular fluid from preovulatory follicles (n = 4) degraded about 80% of the added recombinant human (rh) IGFBP-4 within 18 h of incubation. The IGFBP-4 protease exhibited optimal activity at neutral/basic pH and its sensitivity to various protease inhibitors suggested a metalloprotease. The decline in the intensity of the band corresponding to intact rhIGFBP-4 was accompanied by the appearance of immunoreactive fragments of molecular weights approximately 18 and 14 kDa, which were not detectable by ligand blot analysis. In experiment 2, follicular fluid samples were collected from dominant and subordinate follicles on Day 2 or 3 of the first follicular wave, after ovariectomy (experiment 2a, n = 3/day) or by ultrasound-guided follicular aspiration (experiment 2b, n = 4-5/day). Estradiol concentrations in follicular fluid from dominant vs. subordinate follicles confirmed their identities and indicated that the dominant follicle had been selected by Day 2 of the follicular wave. In both experiments 2a and 2b, IGFBP-4 proteolytic activity was 2- to 3.5-fold (P < 0.05) and 5-fold (P < 0.01) higher in follicular fluid from dominant than subordinate follicles on Days 2 and 3 of the follicular wave, respectively. The finding that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave, strongly suggests a role for IGFBP-4 protease in the establishment of ovarian follicular dominance.     

Molecular evidence that growth of dominant follicles involves a reduction in follicle-stimulating hormone dependence and an increase in luteinizing hormone dependence in cattle

The bovine dominant follicle (DF) model was used to identify molecular mechanisms potentially involved in initial growth of DF during the low FSH milieu of ovarian follicular waves. Follicular fluid and RNA from granulosa and theca cells were harvested from 10 individual DF obtained between 2 and 5.5 days after emergence of the first follicular wave of the estrous cycle. Follicular fluid was subjected to RIA to determine estradiol (E) and progesterone (P) concentrations and RNA to cDNA microarray analysis and (or) quantitative real-time PCR. Results showed that DF growth was associated with a decrease in intrafollicular E:P ratio and in mRNA for the FSH receptor, estrogen receptor 2 (ER beta), inhibin alpha, activin A receptor type I, and a proliferation (cyclin D2) and two proapoptotic factors (apoptosis regulatory protein Siva, Fas [TNFRSF6]-associated via death domain) in granulosa cells. In contrast, mRNAs for the LH receptor in granulosa cells and for two antiapoptotic factors (TGFB1-induced antiapoptotic factor 1, LAG1 longevity assurance homolog 4 [Saccharomyces cerevisiae]) and one proapoptotic factor (tumor necrosis factor [ligand] superfamily, member 8) were increased in theca cells. We conclude that the bovine DF provides a unique model to identify novel genes potentially involved in survival and apoptosis of follicular cells and, importantly, to determine the FSH-, estradiol-, and LH-target genes regulating its growth and function. Results provide new molecular evidence for the hypothesis that DF experience a reduction in FSH dependence but acquire increased LH dependence as they grow during the low FSH milieu of follicular waves.     

Influence of exercise duration on serum insulin-like growth factor and its binding proteins in athletes

The changes in circulating concentrations of insulin-like growth factors during exercise have to date remained incomplete in their documentation. Therefore, we examined in 25 healthy athletes the effects of three different durations of three types of exercise – incremental ergometer cycling exercise (ICE), long-distance Nordic ski race (NSR) and a treadmill-simulated soccer game (TSG) lasting 20 min, 3 h, and 2 × 45 min separated by a 15-min half-time rest respectively, on plasma concentrations of growth hormone ([GH]), insulin-like growth factor-1 ([IGF-I]) and its binding proteins 1 and 3 ([IGFBP-1], [IGFBP-3]). Compared to baseline, serum [GH] increased by 15.2-fold after ICE (P < 0.001), 2.9-fold after NSR (P < 0.01) and 4.6-fold after TSG. Serum [IGF-I] rose by 11.9% after ICE (P < 0.001), while it decreased by −14.6% after NSR (P < 0.001) and was unchanged after TSG. Serum [IGFBP-1] was slightly increased (1.7-fold) after ICE (P < 0.01), but increased markedly (11.8-fold) after NSR (P < 0.001) and by 6.3-fold after the second session of TSG (P < 0.01) (it remained unchanged at the end of the first period of TSG, i.e. after 45-min exercise). The [IGFBP-3] increased by 14.7% after ICE (P < 0.001) and by 6% after TSG (P < 0.05) while it did not change after NSR. From our results it would appear that [IGFBP-1] increase to bind free IGF and hinder their insulin-like action during long-term exercise (lasting beyond 45 min). It is suggested that IGFBP-1 might thus contribute both to preventing hypoglycaemic action of IGF and to facilitating glucose uptake by muscle cells when muscle glycogen stores become deplete. Accepted: 27 May 1998     

Activities of glucose metabolic enzymes in human preantral follicles: in vitro modulation by follicle-stimulating hormone, luteinizing hormone, epidermal growth factor, insulin-like growth factor I, and transforming growth factor beta1

Modulation of glucose metabolic capacity of human preantral follicles in vitro by gonadotropins and intraovarian growth factors was evaluated by monitoring the activities of phosphofructokinase (PFK) and pyruvate kinase (PK), two regulatory enzymes of the glycolytic pathway, and malate dehydrogenase (MDH), a key mitochondrial enzyme of the Krebs cycle. Preantral follicles in classes 1 and 2 from premenopausal women were cultured separately in vitro in the absence or presence of FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF-I), or transforming growth factor beta1 (TGFbeta1) for 24 h. Mitochondrial fraction was separated from the cytosolic fraction, and both fractions were used for enzyme assays. FSH and LH significantly stimulated PFK and PK activities in class 1 and 2 follicles; however, a 170-fold increase in MDH activity was noted for class 2 follicles that were exposed to FSH. Although both EGF and TGFbeta1 stimulated glycolytic and Krebs cycle enzymes for class 1 preantral follicles, TGFbeta1 consistently stimulated the activities of both glycolytic enzymes more than that of EGF. IGF-I induced PK and MDH activities in class 1 follicles but negatively influenced PFK activity for class 1 follicles. In general, only gonadotropins consistently stimulated both glycolytic and Krebs cycle enzyme activities several-fold in class 2 follicles. These results suggest that gonadotropins and ovarian growth factors differentially influence follicular energy-producing capacity from glucose. Moreover, gonadotropins may either directly influence glucose metabolism in class 2 preantral follicles or do so indirectly through factors other than the well-known intraovarian growth factors. Because growth factors modulate granulosa cell mitosis and functionality, their role on energy production may be related to specific cellular activities.     

Relationship of macroscopic appearance of the surface of bovine ovarian follicles concentrations of steroids in follicular fluid, and maturation of oocytes in vitro

The relationship among opaqueness of the surface of bovine ovarian follicles, concentrations of follicular steroids, and capacity of oocytes to achieve nuclear maturation in vitro was examined in this study. Follicles greater than or equal to 5 mm in diameter were classified as clear (n=68) or opaque (n=72) based on their surface appearance. An oocyte and follicular fluid (FF) were removed from each follicle. Each oocyte was cultured, and the concentration of estradiol (E), progesterone (P), and testosterone (T) was determined for each sample of FF. Oocytes that extruded the first polar body by 30 h in culture were considered mature. All other oocytes were immature. More (p less than 0.05) mature oocytes came from clear (56%) than opaque follicles (29%). Clear follicles had lower concentrations of E (p less than 0.05) and P (p less than 0.10) in FF than opaque follicles. Follicles with mature oocytes had greater (p less than 0.05) concentrations of P than follicles with immature oocytes. Follicles were separated into three categories based on ratio of P:E in FF: high = P:E greater than or equal to 10, medium = P:E greater than or equal to 1 less than 10, and low = P:E less than 1. The percentage of mature oocytes from clear follicles was similar for high (64%), medium (48%), and low (57%) P:E groups; however, the percentage of mature oocytes from opaque follicles was greater (p less than 0.05) for the high (59%) than for the medium (21%) or low (19%) P:E groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive hormones and follicular growth during development of one or multiple dominant follicles in cattle

The mechanisms regulating ovulation rate under natural conditions are not yet defined, particularly for monovular species. In the present study, we evaluated ovarian structures (every 12 h by ultrasonography) and circulating hormones (every 6 h) to determine the differences between cows that developed one (single dominant; n = 16), two (double dominant; n = 8), or three (triple dominant; n = 3) dominant follicles. The four largest follicles were tracked retrospectively, and the data were normalized to the time of expected follicular deviation (F1 >/= 8.5 mm; hour 0). Follicular dynamics from emergence to deviation were similar, whereas after deviation, expected subordinate follicles continued to grow at a rate similar to the dominant follicle. Triple dominants had greater FSH than double dominants (hour -24 to hour -12) and single dominants (hour -42 to hour -6), and double dominants had greater FSH than single dominants (hour -24 to hour -12). Increased circulating estradiol but lower inhibin were observed in cows that developed multiple follicles. In addition, double dominants had greater LH than single dominants (hour -42 to hour -24 and hour -6 to hour 0) and lower progesterone than single dominants (hour -12 and hour -6). Luteal volume was similar between groups, but milk production was greater for codominant than for single-dominant cows. Thus, selection of multiple dominant follicles during high milk production is related to a transient increase in circulating FSH and LH during the 24 h before follicular selection, producing continued postdeviation growth of follicles that ordinarily would have regressed. Increased FSH and LH probably result from decreased circulating inhibin and progesterone in cows that develop codominant follicles.     

Effect of follicular fluid treatment on follicle-stimulating hormone, luteinizing hormone and compensatory ovarian hypertrophy in prepuberal gilts

Prepuberal 130-day-old gilts were treated with 10 ml of charcoal-stripped porcine serum (PS), whole porcine follicular fluid (WpFF) or charcoal-stripped pFF (CpFF) twice daily beginning the day before and continuing 8 days after unilateral ovariectomy (ULO). Follicle-stimulating hormone (FSH) declined for the first 14 h after ULO in WpFF and CpFF gilts and then by 24 h returned to values observed at or before ULO, whereas FSH was increased nearly twofold at 14 h in PS gilts. At 8 days after ULO the remaining ovaries from PS-treated gilts were heavier than ovaries from follicular fluid-treated gilts. In a second experiment, ovariectomized 130-day-old gilts were assigned to either a group infused with PS, a group infused with 5 ml CpFF, or a group infused with 10 ml Cpff at 18 and 2 h before a gonadotropin-releasing hormone (GnRH) challenge. Porcine follicular fluid had no effect on luteinizing hormone (LH) response to GnRH, depressed the FSH response to a 10-micrograms challenge of GnRH, but had no effect on FSH response to a 50-micrograms challenge of GnRH. In a third study, gilts were subjected to sham ovariectomy (Sham) or ULO at 130 days of age. GnRH (10 micrograms) was given on Days 1, 2 or 8 after surgery. The response to GnRH in ULO versus Sham gilts did not differ for FSH or LH on any day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Suppression of dominant and subordinate ovarian follicles by a proteinaceous fraction of follicular fluid in heifers

Nulliparous Holstein heifers were examined ultrasonically once daily during an interovulatory interval (ovulation = Day 0). Follicles with a diameter >/=4 mm were sequentially identified. Heifers were randomized into four groups (n = 4 heifers per group): untreated control heifers and those treated on Days 0 to 3, Days 3 to 6, or Days 6 to 11. Heifers designated for treatment were given an intravenous injection, twice daily, of a proteinaceous fraction of follicular fluid (PFFF; 16 ml) prepared by extracting bovine follicular fluid with activated charcoal. Mean cessation of growth of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 5.5) than in heifers treated on Days 0 to 3 (Day 1.5) or Days 3 to 6 (Day 3.5). Mean onset of regression of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 12.0) than in heifers treated on Days 0 to 3 (Day 5.0) or Days 3 to 6 (Day 7.5). Mean cessation of growth of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 3.0) than in heifers treated on Days 0 to 3 (Day 1.2). Mean onset of regression of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 7.0) than in heifers treated on Days 0 to 3 (Day 4.8). In heifers treated on Days 6 to 11, cessation of growth and onset of regression of the dominant follicle (means, Days 5.2 and 12.0, respectively) were not significantly different from those of the controls. The hypothesis that PFFF treatment on Days 0 to 3 would cause suppression of all follicles of Wave 1 was supported. The hypothesis that PFFF treatment on Days 3 to 6 would not alter growth of the dominant follicle of Wave 1 was not supported. The mean day of detection of the dominant follicle of Wave 2 was different (P<0.005) in control heifers (Day 8.5) than in heifers treated on Day 0 to 3 (Day 5.5) or Days 6 to 11 (Day 14.2). The mean length of the interovulatory interval was shorter (P<0.05) in control heifers (20.5 d) than in heifers treated on Days 6 to 11 (23.2 d). The hypothesis that PFFF treatment on Days 6 to 11 would delay the emergence of Wave 2 was supported. The proportion of heifers with 2-wave interovulatory intervals was 3 4 for control heifers and 0 4 , 1 4 , and 4 4 for heifers treated on Days 0 to 3, Days 3 to 6, and Days 6 to 11, respectively (3 4 vs 0 4 , P<0.05); the remaining heifers had 3-wave interovulatory intervals. On average, in PFFF-treated heifers, follicles stopped growing 1 d after treatment was started, and Wave 2 was detected 3 d after treatment was stopped.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone and insulin-like growth factor I concentrations in bulls of various growth hormone genotypes

A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.