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Concentrations of insulin-like growth factor-I in serum of sheep with different ovulation rates: Changes during the estrous cycle
THERIOGENOLOGY CONCENTRATIONS OF INSULIN-LIKE GROWTH FACTOR-I IN SERUM OF SHEEP WITH DIFFERENT OVULATION RATES: CHANGES DURING THE ESTROUS CYCLE L.J. Spice&l"
and M.T. Zavy2
of Animal Science, Oklahoma State University Stillwater, OK 74078 and 2U.S. Department of Agriculture, Agricultural Research Service, El Reno, OK 73036
'Department
Received
for publication: Accepted:
ApriZ 2, September
1991 15, 1991
ABSTRACT To determine if concentrations of IGF-I in serum are rate or if with ovulation IGF-I associated the concentrations change during an estrous cycle, blood was collected from ewes of three genotypes in two experiments. In Experiment 1, blood was collected once during the midluteal phase of an estrous cycle in nulliparous ewes. In Experiment 2, blood was collected every other day from Day 1 Three genotypes of to 19 postestrus in primiparous ewes. Dorset x Rambouillet, ewes were used in both experiments: Finn x Rambouillet and Booroola Merino x Rambouillet. Ovulation rates in Experiment 1 were 1.35, 1.88 and 2.50 for Dorset x Rambouillet (n=17), Finn x Rambouillet (n=49) and (n=za) ewes, respectively. Booroola Merino x Rambouillet IGF-I concentrations in serum were greater (PcO.05) in Finn x Rambouillet ewes than for Dorset x Rambouillet or Booroola Merino x Rambouillet ewes. In Experiment 2, ovulation rates were 1.57, 2.00 and 2.75 for Dorset x Rambouillet, Finn x Rambouillet and Booroola Merino x Rambouillet, respectively. with concentrations increased age in Dorset x IGF-I Rambouillet and Booroola Merino x Rambouillet ewes (but not Finn x Rambouillet ewes) such that they did not differ between crossbred ewe groups. IGF-I concentrations decreased between Days 1 and 5, then increased (PcO.05) between Days a that IGF-I suggesting concentrations and 17 postestrus increase during estrus in ewes. Key words:
insulin-like
growth factor-I, ovulation
rate
Acknowledgments in part by the Oklahoma This work was supported Agricultural Experiment Station (Journal Article Number 5959). Mention of a trademark or product does not constitute The its endorsement by the U.S. Department of Agriculture. authors thank the National Hormone and Pituitary Program (Baltimore, MD) for the IGF-I antiserum; Drs. D.L. Von Tungeln, S. Helmer and B. Hughes of the USDA, El Reno, OK for assistance in data collection; and Dr. L. Young, USDA, MARC, Clay Center, NE for supplying Finn and Merino rams. aTo whom correspondence should be addressed.
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INTRODUCTION Several experiments have attempted to determine the endocrine basis for high ovulation rates in sheep. Booroola (ovulation rate = 3 to 5) have and Romanov ewes significantly higher pituitary and plasma FSH concentrations than control ewes (ovulation rate = 1 to 2) during various periods of their respective estrous cycles (l-3). It has also been shown that inhibin content of ovaries in Booroola gene was ewes having a copy of the Fecundity (F) significantly lower than in ewes not having a copy of the F gene (4). Thus, reduced inhibin production may be at least partially responsible for the increased FSH secretion observed. However, in other high fecundity breeds of sheep, differential FSH secretion has not been observed. For example, FSH secretion in Finn (ovulation rate = 2 to 4) and Suffolk (ovulation rate = 1 to 2) ewes did not differ (5-6). Thus, factors other than differential inhibin production by the ovary must be invoked. Recently, we have observed that cows selected for twinning have significantly higher levels of insulin-like growth factor-I (IGF-I) in serum and follicular fluid than control cows (7). IGF-I has been shown to have dramatic stimulatory effects on steroidogenesis and mitogenesis in cultured granulosa and thecal cells (8-11). In addition, peripheral blood concentrations of IGF-I appear to be genetically determined in several mammalian species (12-14). Thus, one likely endocrine regulator of multiple ovulations among breeds of sheep may be IGF-I. The objective of the present investigation was to determine if the difference in ovulation rate between low and high fecund breeds of sheep with a difference in associated serum IGF-I is concentrations. MATERIALS AND METHODS Experiment 1 Three genotypes of the ewes were used in Experiment 1: Dorset x Rambouillet (n=17), Finn x Rambouillet (nr49) and Booroola Merino x Rambouillet (n=28), the last of which were heterozygous carriers of the F gene (F+) as determined by genetic pedigree. All ewes were maintained in a common flock at El Reno on grass pasture until 4 weeks prior to breeding and subsequent laparoscopy. During this period, the ewes were supplemented with corn (0.45 kg/head/day) and medium quality grass hay ad libitum. All ewes were approximately 18 months of age (nulliparous) at the time of breeding. Estrous cycles of ewes were monitored from September Ewes exhibiting regular estrous through November, 1988. cycles were bred, after which they underwent a mid-ventral
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laparoscopy to assess ovulation rate (i.e., number of corpora lutea) during diestrus (Days 8 to 11 of the estrous cycle or pregnancy; Day 0 = day of estrus) of one or two estrous cycles. A second laparoscopy was conducted on ewes (n=25) which did not become pregnant and had exhibited another estrus. Only data from the first laproscopy were used for analysis. All ewes undergoing laparoscopies were given a local anesthetic (2% lidocaine) and were not allowed access to feed for 12 hours before the procedure. Body weights were measured and blood samples were collected via jugular venipuncture at time of laparoscopy. Serum was harvested and frozen at -70°C until analyzed for IGF-I. Experiment 2 Ewes (n=23) in Experiment 2 were previously used in Experiment 1: Dorset x Rambouillet (n=7), Finn x Rambouillet (n-8) and Booroola Merino x Rambouillet (n=8). Experiment 2 was conducted 12 months after Experiment 1. Thus, all ewes were primiparous and were approximately 30 months of age. The ewes were managed as described in Experiment 1. Estrous cycles were monitored as described in Experiment 1. Blood samples were collected by venipuncture (between 1100 and 1200 hours) from Day 1 to 19 postestrus in the seven or eight ewes from each crossbred group. Blood was collected from half of each group of ewes every other day starting on Day 1 postestrus, and from the other half every other day starting on Day 2 postestrus. Serum was collected and stored as described in Experiment 1. IGF-I RIA Serum samples were stored frozen at -70°C until concentrations of IGF-I were determined by RIA after an acidethanol extraction as described previously (7, 15). The intra-assay coefficient of variation was 8.9f0.6%. Briefly, aliquots of serum were diluted 1~4 with 87.5% acidic ethanol (0.25 N HCl final concentration) and incubated for 16 hours at 4OC. Samples were then centrifuged for 30 minutes at 1200 x g at 4OC and neutralized with 0.855 M Tris. This procedure resulted in parallelism between the human IGF-I standard (Amgen, Thousand Oaks, CA) and ovine serum. The human IGF-I standard was believed to be suitable for assay as Francis et al. (16) have reported the sequence of ovine IGF-I and human IGF-I to be identical except for the substitution in the sheep of Ala for Pro at residue 66. Statistical Analysis For Experiment 1, serum IGF-I and ovulation rate data were subjected to least squares analysis of variance with breed as the main effect. Mean differences were assessed
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using Fisher's Protected test. LSD mean Ovulation frequencies were compared using Chi-square analysis. For Experiment 2, serum IGF-I data were subjected to split-plot analysis of variance with breed as the main effect and period of estrous cycle as the subplot. Data from each of the two groups (within breed) of 3 or 4 ewes collected every other day was combined by a-day intervals to form 9 periods during the estrous cycle (i.e., Period l=Days 1 and 2, Period 2=Days 3 and 4, etc.), each containing data from 7 to 8 ewes from each of three breeds. RESULTS Experiment
1
Body weights (means + SEM) did not significantly differ between Dorset x Rambouillet and Finn x Rambouillet ewes (46.Okl.l vs 46.1kO.7 kg, respectively). However, Booroola Merino x Rambouillet ewes weighed less (39.2kO.6 kg) than either Dorset x Rambouillet or Finn x Rambouillet ewes (PO.lO, n=17). The frequency of multiple ovulations (12/ewe) did not differ (P>O.lO) between Finn x Rambouillet (79.6%) and Booroola Merino x Rambouillet (82.1%) ewes. However, the frequency of multiple ovulations was lower (PcO.05) in Dorset x Rambouillet (29.4%) than in Finn x Rambouillet or Booroola Merino x Rambouillet ewes. Concentrations of IGF-I (mean + SEM) in serum were greater (PcO.05) in Finn x Rambouillet ewes (150 & 9 ng/ml) than in either Dorset x Rambouillet (114 f 18 ng/ml) or Booroola Merino x Rambouillet (106 + 11 ng/ml) ewes. When Booroola Merino x Rambouillet ewes were subgrouped on the basis of the ovulation rate (1 or 2 ovulations, n=13; or 13 ovulations, n=15), no difference (P>O.lO) in IGF-I concentrations was observed: 1.62 + 0.14 ovulations and 98 + 17 ng/ml IGF-I and 3.27 + 0.18 ovulations and 113 + 15 ngfml IGF-I, respectively. Concentrations of IGF-I in serum correlated positively with body weight in Finn x Rambouillet ewes (r=0.41, PO.lO; r=0.22, PBO.10, respectively). The ovulation rate did not correlate (P>O.lO) with serum concentrations of IGF-I in any of the crossbred ewe groups.
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THERIOGENOLOGY A second laparoscopy was conducted on ewes which did Blood not become pregnant and had exhibited another estrus. was collected during the mid-luteal phase of this subsequent cycle on 10 Dorset x Rambouillet and 15 Finn estrous the similar to first X ewes. Results Rambouillet The Dorset x Rambouillet obtained: laparoscopy were ovulation rate = 1.40+0.15, serum IGF-I = 96f19 ng/ml and the Finn x Rambouillet ovulation rate = 1.87kO.09, IGF-I = 143f14 ng/ml. Experiment
2
As in Experiment 1, body weights (mean f SEM) did not differ (P>O.lO) between Dorset x Rambouillet and Finn ewes (53.6 + 1.8 vs 49.2 f 1.7 kg, Rambouillet Merino x Rambouillet and Booroola ewes Zespectively) les$ either Dorset weighed (43.0 + 1.7 than kg) Mean (+ x Rambouillet or Finn x Rambouillet ewes (PeO.05). ovulation rates were slightly increased but not SEM) different (P>O.lO) over those observed in Experiment 1, and were 2.75 f -21, 2.00 + .21 and 1.57 + .23 for Booroola Rambouillet, Finn x Rambouillet and Merino x Dorset x Rambouillet ewes, respectively.
Day Figure 1.
of
Cycle
Least squares means of concentrations of IGF-I in the serum of Dorset x Rambouillet, Finn x Rambouillet and Booroola Merino x Rambouillet $wes during an estrous cycle in Experiment 2. Mean differs from Day 1 to 2 (PcO.05); **Mean differs from Day 7 to 8 (PcO.05). Pooled SEM=9.1 ng/ml.
Concentrations of IGF-I in serum were not affected by genotype (P>O.lO) during the estrous cycle. In addition, was no there sianificant breed-by-period interaction Thus, daGa from all three crossbred ewe groups (P>O.lO). were pooled and are shown in Figure 1. Serum concentrations
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of IGF-I were greater (PcO.05) on Days 1 to 2 than on Days 5 to 6 of the estrous cycle, and were greater (PcO.05) on Days 13 to 14 than on Days 7 to 8 of the cycle (Figure 1). For comparison with Experiment 1, concentrations of IGF-I in serum averaged from samples collected on Days 7 through 12 of the estrous cycle were 155 -+ 14, 164 + 15 and 152 + 16 ng/ml for Dorset x Rambouillet, Finn x Rambouillet and Merino x Rambouillet Booroola ewes, respectively. Concentrations of IGF-I increased between 18 and 30 months of age in Dorset x Rambouillet and Booroola Merino x Rambouillet ewes but not Finn x Rambouillet ewes. Among crossbred ewe groups, concentrations of IGF-I in serum correlated with body weight (r=0.40, PcO.06) but not with the ovulation rate (r=0.25, PBO.10). DISCUSSION Recently, we reported that ovariectomy causes a decrease in serum IGF-I in cattle, and exogenous estradiol given to ovariectomized cows causes a significant increase in serum IGF-I concentrations (17). Furthermore, ovarian concentrations of IGF-I in rats are greatest during estrus (18). In the present study, serum IGF-I decreased significantly after estrus and increased prior to next expected estrus, suggesting that follicular estrogens may Thus, it regulate hepatic IGF-I production in sheep. appears that estrogens may acutely regulate IGF-I production in several species. The IGFs have been implicated as potential regulators of follicular growth and differentiation (8,9). Therefore, our present study was undertaken to examine the possible relationship between serum concentrations of IGF-I and the ovulation rate in sheep. We observed that concentrations of IGF-I in serum were significantly greater (32%) in more prolific Finn x Rambouillet nulliparous, 18-month-old ewes (ovulation rate = 1.88 and frequency of multiple ovulations = 80%) than less prolific Dorset x Rambouillet 18-month-old ewes (ovulation rate = 1.35 and frequency of multiple ovulations = 29%). Also, 18-month-old Booroola Merino x Rambouillet ewes had a higher ovulation rate than Finn rambouillet ewes, but had serum IGF-I concentrations X similar to Dorset x Rambouillet ewes. However, in the older (30-month-old) ewes in Experiment 2, no significant differences in serum IGF-I were observed between crossbred ewe groups. Concentrations of IGF-I in serum increased between 18 and 30 months of age in Dorset x Rambouillet and Booroola Merino x Rambouillet ewes but not in Finn Associated with these increases in x Rambouillet ewes. serum IGF-I with age were 10 to 20% increases in ovulation rates in Dorset x Rambouillet and Booroola Merino x Rambouillet ewes. In comparison, Finn x Rambouillet ewes had c 10% increase in ovulation rate and no change in serum IGF-I concentration with age. Finn ewes reach their mature
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THERIOGENOLOGY body weight at an earlier age than Dorset or Booroola Merino lambing rates in Dorset and Booroola ewes, and maximal Merino ewes are achieved at an older age than in Finn ewes Thus, IGF-I may play a role in mediating age(19-21). rates observed in some associated changes in ovulation Further studies will be required to breeds of sheep. establish the mechanism by which IGF-I regulates follicular growth and ovulation rate in ewes. Several other theories have been proposed to explain increased ovulation rates in 1) greater number of large antral various breeds of sheep: follicles with lower atresia rates (22); 2) prolonged recruitment and low intensity of selection of follicles destined to ovulate (23); 3) a need for more follicles to estradiol to initiate produce the quantity of same surges larger preovulatory gonadotropin (24); and 4) secondary preovulatory surges of FSH caused by a lower production of inhibin by the ovary (l-4). Previous studies have not found a difference in ovarian steroid levels or in FSH secretion between Finn ewes and less prolific ewes In contrast, it is thought that an (e.g., Suffolk; 5,6). important regulator of the ovulation rate in Booroola Merino ewes is that of increased FSH secretion caused by a decrease in inhibin secretion by the ovary (regulated by the F gene; l-4, 24). We observed that serum concentrations of IGF-I in Booroola Merino x Rambouillet ewes regardless of ovulation rate, were similar to levels seen in Dorset x Rambouillet ewes. Thus, our results would support the notion that factor(s) other than IGF-I (e.g., inhibin) may regulate the ovulation rate in Booroola Merino x Rambouillet ewes. Previous studies have emphasized the potential intraor paracrine role of the IGFs in regulating ovarian follicular growth and differentiation (8,9) without concern Such a paracrine role has been for blood-derived IGFs. supported by the observations that granulosa cells can secrete immunoreactive IGF-I andjor IGF-II in vitro (25, 26) and that ovarian tissues contain mRHA for IGFs (27, 28). However, because the level of mRWA for IGF-I is nearly lOOfold greater in the liver than in the ovary (28) and concentrations of IGF-I are significantly lower in ovarian follicular fluid than in serum of pigs (29, 30), cattle (7, 31) and horses (32), IGF-I derived from blood serum must be source of intraovarian IGFs. considered a likely In addition, serum and follicular fluid IGF-I concentrations are highly correlated (7, 32). Thus, further research will be required to determine whether hepatic IGF-I or ovarian IGF-I (despite high serum levels) is responsible for stimulating ovulatory events. In addition, further studies will be needed to determine if intraovarian levels of IGF-I differ among crossbred ewe groups. Previous studies have demonstrated that there may be of blood concentrations of IGF-I in genetic determinants mice (14), pigs (13) and humans (12, 33). Our studies also
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provide evidence for a genetic and age determinant of blood IGF-I concentrati@ns in sheep. Previous studies in mice (14, 34) have demonstrated a link between body weight and IGF-I concentrations. Similarly, we observed significant correlations between serum IGF-I concentrations and body weights in ewes in Experiments 1 and 2. However, to establish a definitive role for IGF-I in growth in ruminants will require further study. In summary, results from the present study suggests that 1) breed differences in serum concentrations of IGF-I may be dependent upon age of the ewe and 2) concentrations of IGF-I in serum change significantly during the estrous cycle in ewes. Thus, these data support the hypothesis that the ovary may acutely regulate production of IGF-I. REFERENCES 1.
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McNatty, K.P., Hudson, N., Henderson, K.M., Gibb, M., Morrison, L., Ball, K. and Smith, P. Differences in gonadotrophin concentrations and pituitary responsiveness to GnRH between Booroola ewes which were homozygous (FF), heterozygous (F+), and non-carriers (++) of a major gene influencing their ovulation rate. J. Reprod. Fertil. =:577-588 (1987).
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Robertson, D.M., Ellis, S., Foulds, L.M., Findlay, J.K. and Bindon, B.M. Pituitary gonadotrophins in Booroola and control Merino sheep. J. Reprod. Fertil. =:189197 (1984).
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Cummins, L.J., O'Shea, T., Bindon, B-M., Lee, V.W.K. and Findlay, J.K. Ovarian inhibin in Booroola and control strain Merino ewes. J. Reprod. Fertil. 67:1-7 (1983).
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Webb, R. and England, B.G. Identification of the ovulatory follicle in the ewe: associated changes in follicular size, thecal and granulosa cell luteinizing hormone receptors, antral fluid steroids, and circulating hormones during the preovulatory period. Endocrinology m:873-881 (1984).
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Wheaton, J.E., Marche, J.M., Hamra, H.A. and Al-Raheem, S.N. Plasma gonadotropin and progesterone concentrations during the estrous cycle of Finn, Suffolk and Targhee ewes. Theriogenology x:99-106 (1988).
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Spicer, L.J., Gregory, K.E., Echternkamp, S.E., Concentrations of Canning, S.F. and Hammond, J.M. insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins. Biol. e:a-I4 (1990). Reprod.
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Adashi, E.Y., Resnick, C.E., D'Ercole, A.J., Svoboda, Insulin-like growth factors as M.E. and Van Wyk, J.J. intraovarian regulators of granulosa cell growth and function. Endocrine Rev. 6:400-420 (1985).
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Hammond, J.M., HSU, C.J., Mondschein, J.S. and Canning, autocrine functions of Paracrine and growth S.F. factors in the ovarian follicle. J. Anim. Sci. 66 (Suppl. 2):21-31 (1988).
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Hernandez, E.R., Resnick, C.E., Svoboda, M.E., Van Wyk, Payne, D.W. Adashi, E.Y. SomatomedinJ.J., and C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells. Endocrinology U:1603-1612 (1988).
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Merimee, T.J., Zapf, J. and Froesch, E.R. Insulin-like growth factors (IGFs) in Pygmies and subjects with the pygmy trait: characterization of the metabolic actions J. Clin. Endocrinol. of IGF-I and IGF-II in man. Metab. 55:ioai-1089 (1982).
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Buonomo, F.C., Lauterio, T.J., Baile, C.A. and Campion, D.R. Determination of insulin-like growth factor-I (IGF-I) and IGF binding protein levels in swine. Domestic Anim. Endocrinol. 3:23-31 (1987).
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Blair, H.T., McCutcheon, S.N., Mackenzie, D.S., Ormsby, J.E., Siddiqui, R-A., Breier, B.H. and Gluckman, P.D. Genetic selection for insulin-like growth factor-I in mice is growing associated with altered growth. Endocrinology U:1690-1692 (1988).
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S.F. and Spicer, L.J., Echternkamp, S.E., Canning, Relationship between concentrations of Hammond, J.M. immunoreactive insulin-like growth factor-I ’ follicular fluid and various biochemical markers $: differentiation in bovine antral follicles. Biol. Reprod. 39:573-580 (1988).
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Francis, G.L., McNeil, K.A., Wallace, J.C., Ballard, F.J. and Owens, P.C. Sheep insulin-like growth factors
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Carlsson, B., Carlsson, L. and Billig, H. Estrous cycle-dependent co-variation of insulin-like growth factor-I (IGF-I) messenger ribonucleic acid and protein 64:271-276 in the rat ovary. Mol. Cell. Endocrinol. (1989).
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Driancourt, M.A., and Bindon, B.M. Cahill, L.P. populations Ovarian follicular and preovulatory enlargement in Booroola and control Merino ewes. J. Reprod. Fertil. n:93-107 (1985).
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McNatty, K.P., Henderson, K.M., Lun, S., Heath, D.A., Ball, K., Hudson, N.L., Fannin, J., Gibb, M., Kieboom, Ovarian activity in Booroola X L.E. and Smith, P. Romney ewes which have a major gene influencing their ovulation rate. J. Reprod. Fertil. =:109-120 (1985).
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Hammond, J.M., HSU, C-J., Klindt, J.M., Ainsworth, L., Gonadotropins increase Downey, B.R. and Tsang, B.K. concentrations of immunoreactive insulin-like growth Biol. factor-I in porcine follicular fluid in vivo. Reprod. x:304-308 (1988).
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Bryan, K.A., Hammond, J.M., Canning, S., Mondschein, Carbaugh, D.E., Clark, A.M. and Hagen, D.R. J Reproductive and growth responses of gilts to exogenous porcine pituitary growth hormone. J. Anim. Sci. fl:196-205 (1989).
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Spicer, L.J., Tucker, K.E., Henderson, K.A. and Duby, R.T. Concentrations of insulin-like growth factor-I in follicular fluid and blood plasma of mares during early Anim. Reprod. and late oestrus. Sci. =:57-65 (1991).
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Blair, H.T., McCutcheon, S.N., Mackenzie, D.D.S., Gluckman, P.D. and Ormsby, J.E. Variation in plasma concentration of insulin-like growth factor-I and its covariation with liveweight in mice. Aust. J. Biol. SCi. &Q:287-293 (1987).
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and M.T. Zavy2
of Animal Science, Oklahoma State University Stillwater, OK 74078 and 2U.S. Department of Agriculture, Agricultural Research Service, El Reno, OK 73036
'Department
Received
for publication: Accepted:
ApriZ 2, September
1991 15, 1991
ABSTRACT To determine if concentrations of IGF-I in serum are rate or if with ovulation IGF-I associated the concentrations change during an estrous cycle, blood was collected from ewes of three genotypes in two experiments. In Experiment 1, blood was collected once during the midluteal phase of an estrous cycle in nulliparous ewes. In Experiment 2, blood was collected every other day from Day 1 Three genotypes of to 19 postestrus in primiparous ewes. Dorset x Rambouillet, ewes were used in both experiments: Finn x Rambouillet and Booroola Merino x Rambouillet. Ovulation rates in Experiment 1 were 1.35, 1.88 and 2.50 for Dorset x Rambouillet (n=17), Finn x Rambouillet (n=49) and (n=za) ewes, respectively. Booroola Merino x Rambouillet IGF-I concentrations in serum were greater (PcO.05) in Finn x Rambouillet ewes than for Dorset x Rambouillet or Booroola Merino x Rambouillet ewes. In Experiment 2, ovulation rates were 1.57, 2.00 and 2.75 for Dorset x Rambouillet, Finn x Rambouillet and Booroola Merino x Rambouillet, respectively. with concentrations increased age in Dorset x IGF-I Rambouillet and Booroola Merino x Rambouillet ewes (but not Finn x Rambouillet ewes) such that they did not differ between crossbred ewe groups. IGF-I concentrations decreased between Days 1 and 5, then increased (PcO.05) between Days a that IGF-I suggesting concentrations and 17 postestrus increase during estrus in ewes. Key words:
insulin-like
growth factor-I, ovulation
rate
Acknowledgments in part by the Oklahoma This work was supported Agricultural Experiment Station (Journal Article Number 5959). Mention of a trademark or product does not constitute The its endorsement by the U.S. Department of Agriculture. authors thank the National Hormone and Pituitary Program (Baltimore, MD) for the IGF-I antiserum; Drs. D.L. Von Tungeln, S. Helmer and B. Hughes of the USDA, El Reno, OK for assistance in data collection; and Dr. L. Young, USDA, MARC, Clay Center, NE for supplying Finn and Merino rams. aTo whom correspondence should be addressed.
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INTRODUCTION Several experiments have attempted to determine the endocrine basis for high ovulation rates in sheep. Booroola (ovulation rate = 3 to 5) have and Romanov ewes significantly higher pituitary and plasma FSH concentrations than control ewes (ovulation rate = 1 to 2) during various periods of their respective estrous cycles (l-3). It has also been shown that inhibin content of ovaries in Booroola gene was ewes having a copy of the Fecundity (F) significantly lower than in ewes not having a copy of the F gene (4). Thus, reduced inhibin production may be at least partially responsible for the increased FSH secretion observed. However, in other high fecundity breeds of sheep, differential FSH secretion has not been observed. For example, FSH secretion in Finn (ovulation rate = 2 to 4) and Suffolk (ovulation rate = 1 to 2) ewes did not differ (5-6). Thus, factors other than differential inhibin production by the ovary must be invoked. Recently, we have observed that cows selected for twinning have significantly higher levels of insulin-like growth factor-I (IGF-I) in serum and follicular fluid than control cows (7). IGF-I has been shown to have dramatic stimulatory effects on steroidogenesis and mitogenesis in cultured granulosa and thecal cells (8-11). In addition, peripheral blood concentrations of IGF-I appear to be genetically determined in several mammalian species (12-14). Thus, one likely endocrine regulator of multiple ovulations among breeds of sheep may be IGF-I. The objective of the present investigation was to determine if the difference in ovulation rate between low and high fecund breeds of sheep with a difference in associated serum IGF-I is concentrations. MATERIALS AND METHODS Experiment 1 Three genotypes of the ewes were used in Experiment 1: Dorset x Rambouillet (n=17), Finn x Rambouillet (nr49) and Booroola Merino x Rambouillet (n=28), the last of which were heterozygous carriers of the F gene (F+) as determined by genetic pedigree. All ewes were maintained in a common flock at El Reno on grass pasture until 4 weeks prior to breeding and subsequent laparoscopy. During this period, the ewes were supplemented with corn (0.45 kg/head/day) and medium quality grass hay ad libitum. All ewes were approximately 18 months of age (nulliparous) at the time of breeding. Estrous cycles of ewes were monitored from September Ewes exhibiting regular estrous through November, 1988. cycles were bred, after which they underwent a mid-ventral
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laparoscopy to assess ovulation rate (i.e., number of corpora lutea) during diestrus (Days 8 to 11 of the estrous cycle or pregnancy; Day 0 = day of estrus) of one or two estrous cycles. A second laparoscopy was conducted on ewes (n=25) which did not become pregnant and had exhibited another estrus. Only data from the first laproscopy were used for analysis. All ewes undergoing laparoscopies were given a local anesthetic (2% lidocaine) and were not allowed access to feed for 12 hours before the procedure. Body weights were measured and blood samples were collected via jugular venipuncture at time of laparoscopy. Serum was harvested and frozen at -70°C until analyzed for IGF-I. Experiment 2 Ewes (n=23) in Experiment 2 were previously used in Experiment 1: Dorset x Rambouillet (n=7), Finn x Rambouillet (n-8) and Booroola Merino x Rambouillet (n=8). Experiment 2 was conducted 12 months after Experiment 1. Thus, all ewes were primiparous and were approximately 30 months of age. The ewes were managed as described in Experiment 1. Estrous cycles were monitored as described in Experiment 1. Blood samples were collected by venipuncture (between 1100 and 1200 hours) from Day 1 to 19 postestrus in the seven or eight ewes from each crossbred group. Blood was collected from half of each group of ewes every other day starting on Day 1 postestrus, and from the other half every other day starting on Day 2 postestrus. Serum was collected and stored as described in Experiment 1. IGF-I RIA Serum samples were stored frozen at -70°C until concentrations of IGF-I were determined by RIA after an acidethanol extraction as described previously (7, 15). The intra-assay coefficient of variation was 8.9f0.6%. Briefly, aliquots of serum were diluted 1~4 with 87.5% acidic ethanol (0.25 N HCl final concentration) and incubated for 16 hours at 4OC. Samples were then centrifuged for 30 minutes at 1200 x g at 4OC and neutralized with 0.855 M Tris. This procedure resulted in parallelism between the human IGF-I standard (Amgen, Thousand Oaks, CA) and ovine serum. The human IGF-I standard was believed to be suitable for assay as Francis et al. (16) have reported the sequence of ovine IGF-I and human IGF-I to be identical except for the substitution in the sheep of Ala for Pro at residue 66. Statistical Analysis For Experiment 1, serum IGF-I and ovulation rate data were subjected to least squares analysis of variance with breed as the main effect. Mean differences were assessed
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using Fisher's Protected test. LSD mean Ovulation frequencies were compared using Chi-square analysis. For Experiment 2, serum IGF-I data were subjected to split-plot analysis of variance with breed as the main effect and period of estrous cycle as the subplot. Data from each of the two groups (within breed) of 3 or 4 ewes collected every other day was combined by a-day intervals to form 9 periods during the estrous cycle (i.e., Period l=Days 1 and 2, Period 2=Days 3 and 4, etc.), each containing data from 7 to 8 ewes from each of three breeds. RESULTS Experiment
1
Body weights (means + SEM) did not significantly differ between Dorset x Rambouillet and Finn x Rambouillet ewes (46.Okl.l vs 46.1kO.7 kg, respectively). However, Booroola Merino x Rambouillet ewes weighed less (39.2kO.6 kg) than either Dorset x Rambouillet or Finn x Rambouillet ewes (P
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THERIOGENOLOGY A second laparoscopy was conducted on ewes which did Blood not become pregnant and had exhibited another estrus. was collected during the mid-luteal phase of this subsequent cycle on 10 Dorset x Rambouillet and 15 Finn estrous the similar to first X ewes. Results Rambouillet The Dorset x Rambouillet obtained: laparoscopy were ovulation rate = 1.40+0.15, serum IGF-I = 96f19 ng/ml and the Finn x Rambouillet ovulation rate = 1.87kO.09, IGF-I = 143f14 ng/ml. Experiment
2
As in Experiment 1, body weights (mean f SEM) did not differ (P>O.lO) between Dorset x Rambouillet and Finn ewes (53.6 + 1.8 vs 49.2 f 1.7 kg, Rambouillet Merino x Rambouillet and Booroola ewes Zespectively) les$ either Dorset weighed (43.0 + 1.7 than kg) Mean (+ x Rambouillet or Finn x Rambouillet ewes (PeO.05). ovulation rates were slightly increased but not SEM) different (P>O.lO) over those observed in Experiment 1, and were 2.75 f -21, 2.00 + .21 and 1.57 + .23 for Booroola Rambouillet, Finn x Rambouillet and Merino x Dorset x Rambouillet ewes, respectively.
Day Figure 1.
of
Cycle
Least squares means of concentrations of IGF-I in the serum of Dorset x Rambouillet, Finn x Rambouillet and Booroola Merino x Rambouillet $wes during an estrous cycle in Experiment 2. Mean differs from Day 1 to 2 (PcO.05); **Mean differs from Day 7 to 8 (PcO.05). Pooled SEM=9.1 ng/ml.
Concentrations of IGF-I in serum were not affected by genotype (P>O.lO) during the estrous cycle. In addition, was no there sianificant breed-by-period interaction Thus, daGa from all three crossbred ewe groups (P>O.lO). were pooled and are shown in Figure 1. Serum concentrations
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of IGF-I were greater (PcO.05) on Days 1 to 2 than on Days 5 to 6 of the estrous cycle, and were greater (PcO.05) on Days 13 to 14 than on Days 7 to 8 of the cycle (Figure 1). For comparison with Experiment 1, concentrations of IGF-I in serum averaged from samples collected on Days 7 through 12 of the estrous cycle were 155 -+ 14, 164 + 15 and 152 + 16 ng/ml for Dorset x Rambouillet, Finn x Rambouillet and Merino x Rambouillet Booroola ewes, respectively. Concentrations of IGF-I increased between 18 and 30 months of age in Dorset x Rambouillet and Booroola Merino x Rambouillet ewes but not Finn x Rambouillet ewes. Among crossbred ewe groups, concentrations of IGF-I in serum correlated with body weight (r=0.40, PcO.06) but not with the ovulation rate (r=0.25, PBO.10). DISCUSSION Recently, we reported that ovariectomy causes a decrease in serum IGF-I in cattle, and exogenous estradiol given to ovariectomized cows causes a significant increase in serum IGF-I concentrations (17). Furthermore, ovarian concentrations of IGF-I in rats are greatest during estrus (18). In the present study, serum IGF-I decreased significantly after estrus and increased prior to next expected estrus, suggesting that follicular estrogens may Thus, it regulate hepatic IGF-I production in sheep. appears that estrogens may acutely regulate IGF-I production in several species. The IGFs have been implicated as potential regulators of follicular growth and differentiation (8,9). Therefore, our present study was undertaken to examine the possible relationship between serum concentrations of IGF-I and the ovulation rate in sheep. We observed that concentrations of IGF-I in serum were significantly greater (32%) in more prolific Finn x Rambouillet nulliparous, 18-month-old ewes (ovulation rate = 1.88 and frequency of multiple ovulations = 80%) than less prolific Dorset x Rambouillet 18-month-old ewes (ovulation rate = 1.35 and frequency of multiple ovulations = 29%). Also, 18-month-old Booroola Merino x Rambouillet ewes had a higher ovulation rate than Finn rambouillet ewes, but had serum IGF-I concentrations X similar to Dorset x Rambouillet ewes. However, in the older (30-month-old) ewes in Experiment 2, no significant differences in serum IGF-I were observed between crossbred ewe groups. Concentrations of IGF-I in serum increased between 18 and 30 months of age in Dorset x Rambouillet and Booroola Merino x Rambouillet ewes but not in Finn Associated with these increases in x Rambouillet ewes. serum IGF-I with age were 10 to 20% increases in ovulation rates in Dorset x Rambouillet and Booroola Merino x Rambouillet ewes. In comparison, Finn x Rambouillet ewes had c 10% increase in ovulation rate and no change in serum IGF-I concentration with age. Finn ewes reach their mature
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THERIOGENOLOGY body weight at an earlier age than Dorset or Booroola Merino lambing rates in Dorset and Booroola ewes, and maximal Merino ewes are achieved at an older age than in Finn ewes Thus, IGF-I may play a role in mediating age(19-21). rates observed in some associated changes in ovulation Further studies will be required to breeds of sheep. establish the mechanism by which IGF-I regulates follicular growth and ovulation rate in ewes. Several other theories have been proposed to explain increased ovulation rates in 1) greater number of large antral various breeds of sheep: follicles with lower atresia rates (22); 2) prolonged recruitment and low intensity of selection of follicles destined to ovulate (23); 3) a need for more follicles to estradiol to initiate produce the quantity of same surges larger preovulatory gonadotropin (24); and 4) secondary preovulatory surges of FSH caused by a lower production of inhibin by the ovary (l-4). Previous studies have not found a difference in ovarian steroid levels or in FSH secretion between Finn ewes and less prolific ewes In contrast, it is thought that an (e.g., Suffolk; 5,6). important regulator of the ovulation rate in Booroola Merino ewes is that of increased FSH secretion caused by a decrease in inhibin secretion by the ovary (regulated by the F gene; l-4, 24). We observed that serum concentrations of IGF-I in Booroola Merino x Rambouillet ewes regardless of ovulation rate, were similar to levels seen in Dorset x Rambouillet ewes. Thus, our results would support the notion that factor(s) other than IGF-I (e.g., inhibin) may regulate the ovulation rate in Booroola Merino x Rambouillet ewes. Previous studies have emphasized the potential intraor paracrine role of the IGFs in regulating ovarian follicular growth and differentiation (8,9) without concern Such a paracrine role has been for blood-derived IGFs. supported by the observations that granulosa cells can secrete immunoreactive IGF-I andjor IGF-II in vitro (25, 26) and that ovarian tissues contain mRHA for IGFs (27, 28). However, because the level of mRWA for IGF-I is nearly lOOfold greater in the liver than in the ovary (28) and concentrations of IGF-I are significantly lower in ovarian follicular fluid than in serum of pigs (29, 30), cattle (7, 31) and horses (32), IGF-I derived from blood serum must be source of intraovarian IGFs. considered a likely In addition, serum and follicular fluid IGF-I concentrations are highly correlated (7, 32). Thus, further research will be required to determine whether hepatic IGF-I or ovarian IGF-I (despite high serum levels) is responsible for stimulating ovulatory events. In addition, further studies will be needed to determine if intraovarian levels of IGF-I differ among crossbred ewe groups. Previous studies have demonstrated that there may be of blood concentrations of IGF-I in genetic determinants mice (14), pigs (13) and humans (12, 33). Our studies also
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provide evidence for a genetic and age determinant of blood IGF-I concentrati@ns in sheep. Previous studies in mice (14, 34) have demonstrated a link between body weight and IGF-I concentrations. Similarly, we observed significant correlations between serum IGF-I concentrations and body weights in ewes in Experiments 1 and 2. However, to establish a definitive role for IGF-I in growth in ruminants will require further study. In summary, results from the present study suggests that 1) breed differences in serum concentrations of IGF-I may be dependent upon age of the ewe and 2) concentrations of IGF-I in serum change significantly during the estrous cycle in ewes. Thus, these data support the hypothesis that the ovary may acutely regulate production of IGF-I. REFERENCES 1.
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