Dose-response study of intrafollicular injection of insulin-like growth factor-I on follicular fluid factors and follicle dominance in mares

The effect of insulin-like growth factor-I (IGF-I) on the concentrations of follicular fluid factors during follicle deviation and the development of dominance was studied in mares in two experiments. Transvaginal ultrasound guidance was used for intrafollicular injection and subsequent sequential sampling of follicular fluid. Treatment involved a single injection of IGF-I into the second-largest follicle (F2) at the expected beginning of deviation (Hour 0) based on diameter (> or =20 mm) of the largest follicle (F1). Mares in IGF-I groups were given a dose of 500 microg (experiment 1) or 250, 25, or 2.5 microg (experiment 2). Ablation of F1 at Hour 24 was done in experiment 1, but not in experiment 2. The 500- and 250-microg doses stimulated growth, leading to ovulation of F2 in 10 of 10 and 4 of 5 mares in the two experiments, respectively, compared to 4 of 12 and 0 of 5 in saline-injected controls. These doses prevented (P < 0.05) the increase in IGF binding protein-2 and androstenedione that occurred in F2 of controls and increased (P < 0.05) the concentrations of activin-A, inhibin-A, and vascular endothelial growth factor (VEGF). The 500-microg dose stimulated higher (P < 0.05) concentrations of estradiol, but not until Hour 48, whereas the lower doses were ineffective. In experiment 2, free IGF-I concentrations in F2 at Hour 24 decreased progressively as the dose decreased so that concentrations for the 2.5-microg dose were higher (P < 0.05) than in F2 of controls and similar (not significantly different) to endogenous concentrations in F1. Correspondingly, concentrations of androstenedione in F2 at Hour 24 were lower (P < 0.05) and concentrations of activin-A, inhibin-A, and VEGF were higher (P < 0.05) after treatment of F2 with the 2.5-microg dose than in F2 of controls and were similar to concentrations in F1. Hence, a physiologic intrafollicular dose of IGF-I did not stimulate estradiol production but reduced the production of androstenedione and stimulated the production of activin-A, inhibin-A, and VEGF during follicle selection in mares.     

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.     

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.     

Critical role of insulin-like growth factor system in follicle selection and dominance in mares

The role of the insulin-like growth factor (IGF) system in the deviation in growth rates among follicles (follicle selection) was studied in mares using an IGF binding protein (BP) to reduce the follicular-fluid concentrations of IGFs. The future dominant follicle (F1) was treated by intrafollicular injection at the expected beginning of deviation (F1 > or = 20 mm; Day 0). The experimental groups were control (no injection, n = 8), vehicle (injection of vehicle; n = 6), and BP (injection of 250 microg of recombinant human IGFBP-3; n = 6). A sample of follicular fluid was taken from F1 on Day 1 in all groups. Compared with the control group, IGFBP-3 reduced (P < 0.05) the follicular-fluid concentration of free IGF-1 by 90%; lowered (P < 0.05) the concentrations of estradiol, activin-A, inhibin-A, and vascular endothelial growth factor; and increased (P < 0.05) the concentration of androstenedione. The diameter of F1 decreased and the diameter of F2 increased after Day 0 in the BP group, compared with the control and vehicle groups. A greater (P < 0.05) increase in circulating concentrations of FSH between Days 0 and 1 occurred in the BP group than in the other groups and accounted for the increased growth of F2. Dominance and ovulation from F1 occurred from fewer (P < 0.03) mares in the BP group (1 of 6) than from the control and vehicle groups combined (11 of 14); the remaining mares in the BP group ovulated from F2. Results indicated that the IGF system has a critical intrafollicular role in the differential changes in concentrations of follicular-fluid factors between the future dominant and subordinate follicles, leading to the development of follicle dominance (selection) and ovulation in mares.     

Follicle selection in cattle: dynamics of follicular fluid factors during development of follicle dominance.

Follicle diameter deviation during follicular waves in cattle begins with a reduction in growth rates of developing subordinate follicles, in contrast to the maintenance of a constant growth rate by a developing dominant follicle. In experiment 1, the temporal changes encompassing deviation in concentrations of follicular fluid factors relative to one another in the three largest follicles (F1, F2, and F3) were studied. Follicular fluid samples were collected when F1 reached diameter ranges of 7.0-7.9, 8.0-8.9, 9.0-9.9, and 10.0-10.9 mm (n = 12 per range). The first increase (P < 0.05) in the difference between F1 and F2 for estradiol occurred at the 8.0- to 8.9-mm range, which was one range earlier than for diameter (P < 0.05). Free insulin-like growth factor (IGF)-1 concentrations in F1 were similar among diameter ranges, but concentrations in F1 were higher (P < 0.05) than in F2 for each range except 7.0-7.9 mm. Concentrations of free IGF-1 in F2 decreased (P < 0.05). No significant differences were detected in concentrations of progesterone, androstenedione, total inhibin, and inhibin-A. Averaged over follicles, inhibin-B decreased (P < 0.05) between the 8.0- to 8.9- and 10.0- to 10.9-mm ranges, and activin-A increased (P < 0.05) between the 7.0- to 7.9- and 9.0- to 9.9-mm ranges. However, no differences were found among follicles. In experiment 2, changes associated with the development of dominance by F2 were studied using ablation of F1 at the beginning of expected deviation (F1, 8.5 mm; Hour 0) as the reference point. Follicular fluid factors were compared at Hour 12 between F2 of a control group (F1 intact; n = 10) and an ablated group (F1 ablated; n = 10). Diameter (P < 0.02), estradiol (P < 0.001), free IGF-1 (P < 0.002), and progesterone (P < 0.003) were greater and IGF-binding protein-2 was lower (P < 0.01) in F2 of the ablated group at Hour 12. No differences were detected in concentrations of androstenedione, total inhibin, and inhibin-A. The results of the two experiments indicated, on a temporal basis, that intrafollicular changes in estradiol and the IGF system, but not in the inhibin/activin system, could account for a reported greater FSH responsiveness by the future dominant follicle than by the future subordinate follicles by the beginning of diameter deviation in cattle.     

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)     

Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization.

This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte-corona-cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0.001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.     

Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary.

In the study reported here, we investigated the effect of androgens on recruitment of resting, primordial follicles into the actively growing pool. Healthy, random-cycling female rhesus monkeys were treated with testosterone, dihydrotestosterone (DHT), or vehicle for 3-10 days, after which ovaries were collected for histological analysis. The first stage of follicle growth is the formation of the primary follicle, consisting of an oocyte surrounded by a single layer of cuboidal granulosa cells. The number of primary follicles was significantly increased over time in testosterone-treated animals. In situ hybridization showed that androgen treatment resulted in an increase to 3-fold in insulin-like growth factor I (IGF-I) and to 5-fold in IGF-I receptor mRNA in primordial follicle oocytes. DHT effects were comparable to those of testosterone, showing that these are androgen receptor-mediated phenomena. These data show that androgens promote initiation of primordial follicle growth and implicate oocyte-derived IGF-I in this activation process.     

Post-thaw culture in presence of insulin-like growth factor I improves the quality of cattle cryopreserved embryos

The goal of this study was to examine the effect of insulin-like growth factor I (IGF-I; added during post-thaw culture (48 h)) on the preimplantation viability and quality of cryopreserved bovine in vivo recovered embryos. The morula stage embryos, non-surgically recovered from superovulated dairy cows of Czech Fleckvieh cattle breed, had previously been cryopreserved by a slow freezing technique and stored in liquid nitrogen since 1989-1990. Following thawing, the embryos were cultured for 48 h either alone (no IGF-I) or in the presence of IGF-I (10 or 100 ng/ml); non-cultured embryos served as a control. Thereafter, the embryos were analyzed for cleavage to the blastocyst stage, apoptosis (TUNEL), embryo cell number and quality of actin cytoskeleton. Following post-thaw culture 41% of embryos developed to advanced blastocysts. IGF-I increased this per cent and, at a higher dose, essentially reduced the per cent of degenerated embryos. In cultured embryos, IGF-I at both doses elevated the cell number compared with non-cultured embryos. However, in comparison with embryos cultured without IGF-I, only the higher IGF-I dose resulted in elevating the embryo cell number. The TUNEL index was significantly lowered by IGF-I treatment. Thawed embryos were mostly of the grade III actin type and fewer (12%) had grade II actin, whilst no grade I actin embryos were noted. The addition of IGF-I resulted in the appearance of grade I actin embryos (8.33 and 6.9% for 10 and 100 ng/ml, respectively). These observations indicate that the addition of IGF-I during post-thaw culture can improve the quality of bovine cryopreserved embryos.     

Frequency of luteinizing hormone pulses in cattle influences duration of persistence of dominant ovarian follicles,follicular fluid concentrations of steroids,and activity of insulin-like growth factor binding proteins

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.     

Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon.

Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after 16 days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.     

Growth hormone,insulin-like growth factor I,and motoneuron size

In this study we asked whether growth hormone (GH) and one of its key mediators, insulin-like growth factor I (IGF-I), influence spinal motoneuron size in conjunction with whole body size. We present evidence that GH has such a role, possibly without the mediation of IGF-I. Both lumbar motoneuron and body size were found to be increased relative to littermate controls in transgenic mice overexpressing GH, while body size, but not motoneuron size, was increased in mice overexpressing IGF-I. GH overexpression coordinately increased nucleolar, nuclear, and cell body size in lumbar spinal motoneurons, so that their normal size relationships were preserved in the transgenic mice. In addition, spinal cord and brain weights were significantly increased in both types of transgenic animal. We conclude that GH can regulate motoneuron, central nervous system, and body size in the same animal, and that IGF-I can mimic the effects of GH on at least two of these three parameters. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 202–212, 1997.     

Selection of the dominant follicle in cattle: role of estradiol

Involvement of estradiol in the deviation in growth rates between the two largest follicles of a wave was studied in 39 heifers. In experiment 1, the largest follicle remained intact in a control group and was ablated in five estradiol-treated groups when the largest follicle reached 8.5 mm or larger (expected beginning of deviation; Hour 0). The ablation groups were given a single injection of 0, 0.004, 0.02, 0.1, or 0.5 mg of estradiol. Blood samples were taken from a jugular vein every hour at Hours 0 to 16. By Hour 8, FSH concentrations were greater (P < 0.05) in the ablation group that received 0 mg of estradiol than in the controls. Among the estradiol groups, that receiving 0.02 mg had the lowest detectable increase in estradiol. In this group, FSH concentrations were not suppressed below the control concentrations, but the increase in FSH concentrations following ablation of the largest follicle was delayed for 2 or 3 h. This delay in the increase of FSH concentrations corresponded to the hours that estradiol was maximal. In experiment 2, blood samples were taken every 4 h from the caudal vena cava cranial to the junction with the ovarian veins in heifers with the largest follicle intact (controls) or ablated at 8.5 mm or larger (Hour 0). Averaged over Hours 4 to 48, estradiol concentrations were higher (P < 0.04) in the controls than in the ablation group. During Hours 0 to 12, estradiol concentrations increased (P < 0.05) in the controls, whereas FSH concentrations decreased (P < 0.05). In the ablation group, estradiol concentrations were lower than in the controls by Hour 4, and FSH concentrations increased (P < 0.05) between Hours 4 and 12. These results support the hypothesis that the largest follicle releases increased estradiol into the blood at the beginning of follicular deviation, and that the released estradiol is involved in the continuing depression of FSH concentrations to below the requirement of the smaller follicles.     

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.     

Affinity chromatography with amniotic fluid somatomedin binding protein in the purification of insulin-like growth factor I

A simplified procedure has been developed for the isolation of insulin-like growth factor I from human plasma by use of affinity chromatography with the somatomedin binding protein. After acidification of human plasma and separation of insulin-like growth factor I and endogenous binding protein by cation exchange chromatography on SP-Sephadex the material was passed through a column packed with pure human amniotic fluid binding protein covalently coupled to Sepharose. The bound insulin-like growth factors I and II were eluted by 1M acetic acid and separated on a Mono S cation exchange column by use of a salt gradient. The 30 micrograms insulin-like growth factor I and 18 micrograms insulin-like growth factor II recovered from 1 liter plasma gave an overall recovery of 30% for insulin-like growth factor I but only 2.5% for insulin-like growth factor II.     

Experimental assumption of dominance by a smaller follicle and associated hormonal changes in mares.

A two-follicle model was used to study the nature of selection of the dominant follicle in mares by ablating neither or one of the two follicles on the day the larger follicle reached >/= 20 mm (Day 0). The larger follicle became the dominant follicle in all mares in which both follicles (n = 8) or only the larger follicle (n = 10) was retained. When only the smaller follicle (n = 9) was retained, it became dominant and ovulated in six mares and became atretic in three mares; the difference in diameter between the two follicles on Day 0 was less (p < 0.01) in mares in which the retained smaller follicle grew and ovulated (2.2 +/- 0.6 mm) than in the mares in which the follicle became atretic (5.9 +/- 1.2 mm). A decline (p < 0. 0001) in FSH concentrations occurred over Days -4 (8.4 +/- 0.7 ng/ml) to 0 (5.9 +/- 0.3 ng/ml), averaged over all groups, and the decline continued for several more days in the groups with both follicles or with only the larger follicle retained. In the group with only the smaller follicle retained, compared to the group with both follicles retained, FSH concentrations and diameter of the smaller follicle increased between Days 0 and 1 (significant interaction for each end point). After Day 1, FSH concentrations continued to increase when the smaller retained follicle became atretic; concentrations decreased when the smaller retained follicle became dominant. An increase (p < 0.0001) in LH concentrations occurred over Days -4 (12.2 +/- 1.1 pg/ml) to 0 (21.1 +/- 2.0 pg/ml), averaged over the three groups. In 23 of 27 mares, a transient peak in LH concentrations occurred within 2 days of Day 0. In the groups with both follicles or with only the larger follicle retained, an increase (p < 0.0001) in systemic estradiol concentrations occurred between Day 0 (5.3 +/- 0.6 pg/ml) and Day 2 (7.5 +/- 0.4 pg/ml). When only the smaller follicle was retained, estradiol did not begin to increase until Day 2, and it increased only when the retained follicle grew and became dominant. The beginning of an increase in estradiol and continued decrease in FSH at the expected beginning of deviation were attributable to the future dominant follicle; there was no indication that the smaller follicle was involved.     

Mosaic evolution of the insulin-like growth factors. Organization, sequence, and expression of the rat insulin-like growth factor I gene

Insulin-like growth factor I (IGF-I) plays a major role in mammalian growth and regenerative processes as a mediator of many of the biological effects of growth hormone. We have demonstrated recently that the human IGF-I gene is transcribed and processed into distinct messenger RNA molecules, each of which directs the synthesis of unique IGF-I-containing peptides. As a means to determine whether a similar model of IGF-I gene organization and expression is the paradigm in mammals and as an initial step in devising experimental approaches to the study of regulation of IGF-I biogenesis, we have isolated and characterized the rat IGF-I gene. The rat gene, like its human counterpart, is very large, extending over at least 73 kilobases, and is composed of five exons subdivided by four introns. As in the human example, the rat IGF-I gene hybridizes to several messenger RNAs: 0.8-1.2, 1.6-2.1, and 7.8 kilobases. There is extensive nucleotide and amino acid sequence conservation between the two genes. The predicted mature rat IGF-I protein is identical to the human peptide in 67 of 70 residues. A comparably high degree of amino acid sequence identity is also found for both the amino- and carboxyl-terminal extension peptides, suggesting that, like mature IGF-I, the extension molecules may have physiological function.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic variations of insulin-like growth factor I in Italy

Four Italian populations were examined for a HindIII RFLP associated with the human insulin-like growth factor I gene. No differences were observed among the four samples. The allele frequencies in Italy were: IGF-I HF = 0.834; IGF-I HS = 0.166. The polymorphism appears to be due to a 400-bp sequence insertion-deletion mechanism.