DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 9(2):151-159,1992
THE EFFECT OF EXOGENOUS INSULIN-LIKE GROWTH FACTOR-I (IGF-I) ON THE REPRODUCTIVE PERFORMANCE OF FEMALE RATS, AND ON SERUM CONCENTRATIONS OF ENDOGENOUS IGF-I AND IGF-I BINDING PROTEINS D.E. Kerr 1 and R.N. Kirkwood Department of Animal and Poultry Science University of Saskatchewan Saskatoon, Saskatchewan, CANADA S7N 0W0 Received November 6, 1991
ABSTRACT The effect of exogenous IGF-I on the reproductive performance of female rats was examined by infusing either recombinant human IGF-I (400 lag/d; n = 19) or vehicle (n = 18) over a four-day period (the time of one reproductive cycle) beginning on the day following estrus. The females were exposed to male rats one day after the infusions had commenced, and were euthanized 15 d later. There was no treatment effect on serum progesterone levels at this time or on the number of fetuses. Furthermore, the number of corpora lutea were not different between the IGF-I and vehicle infused groups (15.8 vs. 14.8; P=0.09). Total serum IGF-I concentrations, as determined with a polyclonal antiserum based RIA, were increased approximately three-fold in samples obtained 20 hr after commencing the IGF-I infusion. These samples were also analyzed for IGF-I with a monoclonal antibody based RIA previously shown to detect human, but not rat, IGF-I. By subtraction, the concentration of endogenous rat IGF-I was found to be approximately 60% higher in IGF-I infused rats than in control rats. This increase was likely due to a reduced clearance rate of IGF-I from the circulation, caused by a marked induction of 42-46 kDa and 30-34 kDa IGF-I binding proteins observed in these samples with a ligand blot technique. The binding protein induction indicates that the infused IGF-I was bioactive. This induction may have attenuated the effects of IGF-I on ovarian function. INTRODUCTION In litter beating species, the principal constraint on the number of young produced is the ovulation rate at the estrus of breeding. Mechanisms controlling ovarian function, and the number of ova shed, have received considerable attention. Recently, interest has focused on the role of metabolic hormones such as growth hormone (GH), which has been shown to increase ovulation rate in domestic pigs (1) and in mice (2). The mechanism whereby GH influences ovarian function is believed to involve the mediation of an increase in local ovarian concentrations of insulin-like growth factor-I (IGF-I) (1). Evidence derived from in vitro studies from several laboratories indicates that IGF-I will significantly augment the differentiation and steroidogenic response of ovarian granulosa cells to gonadotrophic stimulation (3). Furthermore, IGF-I messenger RNA is relatively abundant in rat ovaries (4) and specific receptors for IGF-I are present in rat granulosa cells (5). In vivo studies also provide evidence of a role for IGF-I in the regulation of ovarian function. In women, the ovulatory response to exogenous gonadotrophin therapy is improved by co-treatment with GH, an effect believed to be mediated by increased follicular concentrations of IGF-I (6). Also, concentrations of IGF-I are higher in the fluid of domiCopyright © 1992 Butterworth-Heinemann
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KERR AND KIRKWOOD
nant compared to cohort follicles of women (7) and in the follicular fluid of cattle selected for twining compared to unselected animals (8). The literature evidence strongly suggests an important role for IGF-I in mediating normal ovarian function in several species. The mechanism whereby this role is effected is unclear. However, developing follicles within the ovulatory pool are subject to selection, with some follicles becoming atretic (9). It is possible that IGF-I rescues follicles within the ovulatory pool that otherwise may become atretic. If this were to occur, it is possible that it would be manifest as an increased ovulation rate and, potentially, an increase in litter size. To further examine this suggestion, an experiment was undertaken to examine the influence of exogenous IGF-I on the reproductive performance of female rats.
MATERIALS AND METHODS Animals and experimental protocol. Thirty-seven female Wistar rats (approximately 250 g body weight; Charles River Breeding Laboratories, St. Constant, Quebec, Canada) were caged in pairs and allowed ad libitum access to water and a commercial rodent chow (Purina Mills Inc., St. Louis, MO, USA) To facilitate estrous cyclicity, mature male rats (Charles River) were caged individually within 1 m of the females. For a period of 7 d, female rats were each handled for about 5 min daily to familiarize them with human contact. From the end of the handling period, daily vaginal smears were obtained by lavage and the occurrence of normal estrous cycles (4 d) confirmed. The day following the detection of second estrus, a di-estrus condition was confirmed and the rats allocated to one of two treatments. The rats received a subcutaneous implant of an osmotic pump (Alzet, model 1003d; Alza Corporation, Palo Alto CA, USA) designed to release either 400 lag/d of recombinant human IGF-I (hlGF-I; kindly donated by P.J. Swift, Ciba-Giegy Ltd., St-Aubin, Switzerland) or vehicle (0.1 mol acetic acid/l). According to the manufacturer, these pumps delivered 1 ~/hr and thus the 100 lal capacity would have been delivered over approximately 4 d. Osmotic pumps were filled under sterile conditions with hlGF-I (16.67 g/l) or acetic acid (0.1 mold) solutions that had been sterilized by passage through 0.2 larn filters (ACRO LC13; Gelman Sciences, Ann Arbor, MI, USA). The pumps were inserted under general anaesthesia (Metofane; Pitman-Moore Ltd., Mississauga, Ontario, Canada). Following surgery, rats were rehoused in pairs such that each treatment was represented in each cage. After a 24-hr recovery period, one male rat was introduced to each cage and further daily vaginal smears obtained to detect the occurrence of breeding. Male rats were removed from the cages after 4 d and the females euthanized by decapitation 15 d after breeding. At the time of sacrifice, a blood sample was obtained and the reproductive tract recovered. The ovulation rate was determined for each rat, by dissecting individual corpora lutea (CL) from the ovary, and the number of fetuses was recorded. An additional blood sample was obtained from five IGF-I -infused and six vehicle-infused rats 20 hr after pump insertion. These samples were obtained from the orbital sinus under general anaesthesia. Serum was harvested from all blood samples and stored at -20 C. Serum concentrations of IGF-I. Serum samples obtained 20 hr after pump insertion were assayed for concentrations of IGF-I in two parallel radioimmunoassays utilizing either a polyclonal, rabbit anti-serum to IGF-I (batch UBK487; Drs. J. Van Wyk and L. Underwood, distributed by the Hormone Distribution Program of the NIADDK, Baltimore, MD, USA) or a purified monoclonal antibody to IGF-I (82-9A) (10). The polyclonal antiserum detects both rat and human IGF-I in a parallel fashion (11) while the monoclonal antibody detects human IGF-I but does not recognize rat IGF-I (10). Thus, the contribution of rat and human IGF-I to the total concentration of IGF-I in serum of IGF-I -infused rats was determined by difference between the two assays.
IGF-I AND REPRODUCTION IN RATS
153
Prior to analysis, IGF-I was separated from serum binding proteins by acid gel chromatography (12, 13). Briefly, 0.1 ml of serum was incubated (5 min) with 0.4 ml of elution buffer (1 mol acetic acid/l, 0.1 mol NaC1/I), applied to a 1 x 30 cm column packed with Sephadex G-75 (Pharmacia Inc., Baie d'Urf6, Quebec, Canada), and eluted. A 0.1 ml aliquot of the IGF-I peak (12 ml) was then frozen (-70 C), lyophilized (SpeedVac; Savant Instruments Inc., Farmingdale, NY, USA), reconstituted with 0.1 ml H20, and diluted to 1 ml with assay buffer (0.03 mol Na2HPO 4 x 7H20/1, 0.01 mol Na2-EDTA/1, 0.2 g NAN3/1, 0.5 g Tween-20/1, pH 7.5). Recombinant hIGF-I (ICN Biomedicals, St. Laurent, Quebec, Canada) was used as assay standard and was iodinated by the Chloramine-T method (14) for use as assay trace. The two IGF-I assays were run simultaneously using the same assay buffer and batch of ~25I-labeled IGF-I. For the monoclonal based assay, 0.05 ml of sample (further diluted 1:10 in assay buffer) or standard were combined with 0.25 ml of assay buffer and 0.1 ml of a monoclonal antibody solution (diluted 1:107). After a 24 hr incubation (4 C), ~25I-Labeled IGF-I was added (0.1 ml, approximately 40 pg or 10,000 c.p.m.), and the tubes were incubated a further 24 hr at 4 C. Assay tubes then received 0.05 ml of a diluted (1:25 in assay buffer), commercially prepared suspension of rabbit anti-mouse IgG covalently linked to polyacrylamide beads (Immunobeads; BioRad, Richmond, CA, USA). After 24 hr at 4 C the tubes were centrifuged (2,000 g, 15 min), decanted, and the remaining radioactivity determined with a gamma counter (Micromedic, Huntsville, A1, USA). Assay conditions were identical for the polyclonal based assay except that 0.05 ml of antiserum (diluted 1:2,000) and 0.05 ml of normal rabbit serum (diluted 1:200) were used in place of the monoclonal antibody solution. Also, 0.1 ml of sheep anti-rabbit antiserum (diluted 1:80) was used instead of the Immunobeads. To characterize the relationship between the two assays, 107 sheep serum samples from an unrelated experiment were also included in the parallel IGF-I assays. Ovine IGF-I differs from human IGF-I at a single amino acid (15) but this does not appear to effect its recognition by either of the antibody preparations used in this study. As with the rat samples, these samples were acid-gel chromatographed, dried and diluted prior to assay. The standard curves from the two assays are presented in Figure 1 (insert). Specific binding of 125ILabeled IGF-I was 27% and 23% for the monoclonal and polyclonal based assays and intra-assay CVs were 5.2% and 8.9%, respectively. The assays had parallel displacement curves with similar log-logit slopes of -1.01 and -0.96, respectively. However, the monoclonal based assay was apparently more sensitive than the polyclonal assay, in that the amount of unlabelled IGF-I required to produce 50% displacement of 125I-labeled IGF-I was 0.040 ng/tube and 0.067 ng/tube, respectively. Based on the 107 sheep samples, there was an excellent correlation between the assays (R=0.96), although consistently higher values were generated using the polyclonal based assay (Figure 1). The equation describing the relationship between the two assays was Y = 1.269X + 11.9 nmol/L, where Y represents the serum IGF-I concentration (nmol/L) obtained with the polyclonal based assay and X represents the values obtained with the monoclonal based assay. This equation was used to adjust the concentration of hlGF-I detected in the IGF-I -infused rats by the monoclonal based assay. This adjusted value was then subtracted from the value obtained with the polyclonal based assay to determine the concentration of rat IGF-I in these samples. IGF-I binding proteins in serum. Serum samples obtained 20 hr after pump insertion were analyzed for IGF-I binding proteins (IGFBPs) by ligand blotting as described by Hossenlopp et al. (16) with some modifications. Serum samples were diluted 1:6.67 in sample buffer, incubated at 85 C for 10 min and then 20 Ixl aliquots (3 lal serum) were electrophoresed through 12.5% polyacrylamide gels. Prestained molecular weight standards (Bio-Rad) were also loaded onto each gel.
154
KERR AND KIRKWOOD
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Serum IGF-I (nmol/I; polyclonal assay) Figure 1. Concentrations of IGF-I in 107 sheep serum samples from an unrelated experiment as measured in parallel, monoclonal (82-9A) based and polyclonal (UBK 487) based IGF-I assays. The relationship between the assays was Y = 1.269X + 11.9 (R=0.96, P
Vol. 9(2):151-159,1992
THE EFFECT OF EXOGENOUS INSULIN-LIKE GROWTH FACTOR-I (IGF-I) ON THE REPRODUCTIVE PERFORMANCE OF FEMALE RATS, AND ON SERUM CONCENTRATIONS OF ENDOGENOUS IGF-I AND IGF-I BINDING PROTEINS D.E. Kerr 1 and R.N. Kirkwood Department of Animal and Poultry Science University of Saskatchewan Saskatoon, Saskatchewan, CANADA S7N 0W0 Received November 6, 1991
ABSTRACT The effect of exogenous IGF-I on the reproductive performance of female rats was examined by infusing either recombinant human IGF-I (400 lag/d; n = 19) or vehicle (n = 18) over a four-day period (the time of one reproductive cycle) beginning on the day following estrus. The females were exposed to male rats one day after the infusions had commenced, and were euthanized 15 d later. There was no treatment effect on serum progesterone levels at this time or on the number of fetuses. Furthermore, the number of corpora lutea were not different between the IGF-I and vehicle infused groups (15.8 vs. 14.8; P=0.09). Total serum IGF-I concentrations, as determined with a polyclonal antiserum based RIA, were increased approximately three-fold in samples obtained 20 hr after commencing the IGF-I infusion. These samples were also analyzed for IGF-I with a monoclonal antibody based RIA previously shown to detect human, but not rat, IGF-I. By subtraction, the concentration of endogenous rat IGF-I was found to be approximately 60% higher in IGF-I infused rats than in control rats. This increase was likely due to a reduced clearance rate of IGF-I from the circulation, caused by a marked induction of 42-46 kDa and 30-34 kDa IGF-I binding proteins observed in these samples with a ligand blot technique. The binding protein induction indicates that the infused IGF-I was bioactive. This induction may have attenuated the effects of IGF-I on ovarian function. INTRODUCTION In litter beating species, the principal constraint on the number of young produced is the ovulation rate at the estrus of breeding. Mechanisms controlling ovarian function, and the number of ova shed, have received considerable attention. Recently, interest has focused on the role of metabolic hormones such as growth hormone (GH), which has been shown to increase ovulation rate in domestic pigs (1) and in mice (2). The mechanism whereby GH influences ovarian function is believed to involve the mediation of an increase in local ovarian concentrations of insulin-like growth factor-I (IGF-I) (1). Evidence derived from in vitro studies from several laboratories indicates that IGF-I will significantly augment the differentiation and steroidogenic response of ovarian granulosa cells to gonadotrophic stimulation (3). Furthermore, IGF-I messenger RNA is relatively abundant in rat ovaries (4) and specific receptors for IGF-I are present in rat granulosa cells (5). In vivo studies also provide evidence of a role for IGF-I in the regulation of ovarian function. In women, the ovulatory response to exogenous gonadotrophin therapy is improved by co-treatment with GH, an effect believed to be mediated by increased follicular concentrations of IGF-I (6). Also, concentrations of IGF-I are higher in the fluid of domiCopyright © 1992 Butterworth-Heinemann
151
0739-7240192/$3.00
152
KERR AND KIRKWOOD
nant compared to cohort follicles of women (7) and in the follicular fluid of cattle selected for twining compared to unselected animals (8). The literature evidence strongly suggests an important role for IGF-I in mediating normal ovarian function in several species. The mechanism whereby this role is effected is unclear. However, developing follicles within the ovulatory pool are subject to selection, with some follicles becoming atretic (9). It is possible that IGF-I rescues follicles within the ovulatory pool that otherwise may become atretic. If this were to occur, it is possible that it would be manifest as an increased ovulation rate and, potentially, an increase in litter size. To further examine this suggestion, an experiment was undertaken to examine the influence of exogenous IGF-I on the reproductive performance of female rats.
MATERIALS AND METHODS Animals and experimental protocol. Thirty-seven female Wistar rats (approximately 250 g body weight; Charles River Breeding Laboratories, St. Constant, Quebec, Canada) were caged in pairs and allowed ad libitum access to water and a commercial rodent chow (Purina Mills Inc., St. Louis, MO, USA) To facilitate estrous cyclicity, mature male rats (Charles River) were caged individually within 1 m of the females. For a period of 7 d, female rats were each handled for about 5 min daily to familiarize them with human contact. From the end of the handling period, daily vaginal smears were obtained by lavage and the occurrence of normal estrous cycles (4 d) confirmed. The day following the detection of second estrus, a di-estrus condition was confirmed and the rats allocated to one of two treatments. The rats received a subcutaneous implant of an osmotic pump (Alzet, model 1003d; Alza Corporation, Palo Alto CA, USA) designed to release either 400 lag/d of recombinant human IGF-I (hlGF-I; kindly donated by P.J. Swift, Ciba-Giegy Ltd., St-Aubin, Switzerland) or vehicle (0.1 mol acetic acid/l). According to the manufacturer, these pumps delivered 1 ~/hr and thus the 100 lal capacity would have been delivered over approximately 4 d. Osmotic pumps were filled under sterile conditions with hlGF-I (16.67 g/l) or acetic acid (0.1 mold) solutions that had been sterilized by passage through 0.2 larn filters (ACRO LC13; Gelman Sciences, Ann Arbor, MI, USA). The pumps were inserted under general anaesthesia (Metofane; Pitman-Moore Ltd., Mississauga, Ontario, Canada). Following surgery, rats were rehoused in pairs such that each treatment was represented in each cage. After a 24-hr recovery period, one male rat was introduced to each cage and further daily vaginal smears obtained to detect the occurrence of breeding. Male rats were removed from the cages after 4 d and the females euthanized by decapitation 15 d after breeding. At the time of sacrifice, a blood sample was obtained and the reproductive tract recovered. The ovulation rate was determined for each rat, by dissecting individual corpora lutea (CL) from the ovary, and the number of fetuses was recorded. An additional blood sample was obtained from five IGF-I -infused and six vehicle-infused rats 20 hr after pump insertion. These samples were obtained from the orbital sinus under general anaesthesia. Serum was harvested from all blood samples and stored at -20 C. Serum concentrations of IGF-I. Serum samples obtained 20 hr after pump insertion were assayed for concentrations of IGF-I in two parallel radioimmunoassays utilizing either a polyclonal, rabbit anti-serum to IGF-I (batch UBK487; Drs. J. Van Wyk and L. Underwood, distributed by the Hormone Distribution Program of the NIADDK, Baltimore, MD, USA) or a purified monoclonal antibody to IGF-I (82-9A) (10). The polyclonal antiserum detects both rat and human IGF-I in a parallel fashion (11) while the monoclonal antibody detects human IGF-I but does not recognize rat IGF-I (10). Thus, the contribution of rat and human IGF-I to the total concentration of IGF-I in serum of IGF-I -infused rats was determined by difference between the two assays.
IGF-I AND REPRODUCTION IN RATS
153
Prior to analysis, IGF-I was separated from serum binding proteins by acid gel chromatography (12, 13). Briefly, 0.1 ml of serum was incubated (5 min) with 0.4 ml of elution buffer (1 mol acetic acid/l, 0.1 mol NaC1/I), applied to a 1 x 30 cm column packed with Sephadex G-75 (Pharmacia Inc., Baie d'Urf6, Quebec, Canada), and eluted. A 0.1 ml aliquot of the IGF-I peak (12 ml) was then frozen (-70 C), lyophilized (SpeedVac; Savant Instruments Inc., Farmingdale, NY, USA), reconstituted with 0.1 ml H20, and diluted to 1 ml with assay buffer (0.03 mol Na2HPO 4 x 7H20/1, 0.01 mol Na2-EDTA/1, 0.2 g NAN3/1, 0.5 g Tween-20/1, pH 7.5). Recombinant hIGF-I (ICN Biomedicals, St. Laurent, Quebec, Canada) was used as assay standard and was iodinated by the Chloramine-T method (14) for use as assay trace. The two IGF-I assays were run simultaneously using the same assay buffer and batch of ~25I-labeled IGF-I. For the monoclonal based assay, 0.05 ml of sample (further diluted 1:10 in assay buffer) or standard were combined with 0.25 ml of assay buffer and 0.1 ml of a monoclonal antibody solution (diluted 1:107). After a 24 hr incubation (4 C), ~25I-Labeled IGF-I was added (0.1 ml, approximately 40 pg or 10,000 c.p.m.), and the tubes were incubated a further 24 hr at 4 C. Assay tubes then received 0.05 ml of a diluted (1:25 in assay buffer), commercially prepared suspension of rabbit anti-mouse IgG covalently linked to polyacrylamide beads (Immunobeads; BioRad, Richmond, CA, USA). After 24 hr at 4 C the tubes were centrifuged (2,000 g, 15 min), decanted, and the remaining radioactivity determined with a gamma counter (Micromedic, Huntsville, A1, USA). Assay conditions were identical for the polyclonal based assay except that 0.05 ml of antiserum (diluted 1:2,000) and 0.05 ml of normal rabbit serum (diluted 1:200) were used in place of the monoclonal antibody solution. Also, 0.1 ml of sheep anti-rabbit antiserum (diluted 1:80) was used instead of the Immunobeads. To characterize the relationship between the two assays, 107 sheep serum samples from an unrelated experiment were also included in the parallel IGF-I assays. Ovine IGF-I differs from human IGF-I at a single amino acid (15) but this does not appear to effect its recognition by either of the antibody preparations used in this study. As with the rat samples, these samples were acid-gel chromatographed, dried and diluted prior to assay. The standard curves from the two assays are presented in Figure 1 (insert). Specific binding of 125ILabeled IGF-I was 27% and 23% for the monoclonal and polyclonal based assays and intra-assay CVs were 5.2% and 8.9%, respectively. The assays had parallel displacement curves with similar log-logit slopes of -1.01 and -0.96, respectively. However, the monoclonal based assay was apparently more sensitive than the polyclonal assay, in that the amount of unlabelled IGF-I required to produce 50% displacement of 125I-labeled IGF-I was 0.040 ng/tube and 0.067 ng/tube, respectively. Based on the 107 sheep samples, there was an excellent correlation between the assays (R=0.96), although consistently higher values were generated using the polyclonal based assay (Figure 1). The equation describing the relationship between the two assays was Y = 1.269X + 11.9 nmol/L, where Y represents the serum IGF-I concentration (nmol/L) obtained with the polyclonal based assay and X represents the values obtained with the monoclonal based assay. This equation was used to adjust the concentration of hlGF-I detected in the IGF-I -infused rats by the monoclonal based assay. This adjusted value was then subtracted from the value obtained with the polyclonal based assay to determine the concentration of rat IGF-I in these samples. IGF-I binding proteins in serum. Serum samples obtained 20 hr after pump insertion were analyzed for IGF-I binding proteins (IGFBPs) by ligand blotting as described by Hossenlopp et al. (16) with some modifications. Serum samples were diluted 1:6.67 in sample buffer, incubated at 85 C for 10 min and then 20 Ixl aliquots (3 lal serum) were electrophoresed through 12.5% polyacrylamide gels. Prestained molecular weight standards (Bio-Rad) were also loaded onto each gel.
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Serum IGF-I (nmol/I; polyclonal assay) Figure 1. Concentrations of IGF-I in 107 sheep serum samples from an unrelated experiment as measured in parallel, monoclonal (82-9A) based and polyclonal (UBK 487) based IGF-I assays. The relationship between the assays was Y = 1.269X + 11.9 (R=0.96, P
