Regression of Induced Comora Lutea in Mature Cyvclic Gilts by Human CLorionic Gonadotropin' G. B. Rampacek"v2, R. R. Kraelingt, C. R. Barb+, C. E. Estienne*, and M. J. Estienne*J
This study was conducted to determine whether chronic hCG treatment would cause regression of induced corpora lutea (CL) in mature cyclic gilts. Thirtytwo mature gilts that had displayed one or more estrous cycles of 18 to 22 d were used. Sixteen gilts were hysterectomized (HYSTX) on d 6 to 9 (d 0 = onset of estrus) and their CL were marked with charcoal (spontaneous group). Sixteen gilts (induced group) were injected with 1,500 IU of pregnant mare's serum gonadotropin (PMSG) on d 0 and 500 IU of hCG on d 9 (day of hCG = d 0 of the induced cycle). Ovulation was assumed to occur on d 2 of the induced cycle. Induced gilts were HYSTX on d 8 to 9 (d 17 to 18 of the original spontaneous cycle) and their CL were marked with charcoal. Only gilts (n = 14) in which induced CL were present and in which the original CL had regressed were then subjected to treatment with saline or hCG. From d 10 to 29, gilts with spontaneous CL were injected daily with 500 IU of hCG (n = 81 or saline (n = 8). From d 10 to 29 of the induced cycle, induced gilts were injected daily with 500 IU of hCG (n = 01 or ABSTRACT:
saline (n = 8). Jugular blood samples were collected every other day from all gilts beginning on the 1st d of daily hCG treatment and quantified for estradiol and progesterone by RIA. On the day after the last hCG injection, the number of charcoal-marked CL and charcoal-marked corpora albicantia (CAI were determined. All spontaneous CL were maintained. Six of eight gilts with induced CL that received saline maintained CL, whereas only one of six (P c .05) gilts with induced CL that received daily hCG maintained CL. All gilts that were treated with hCG had numerous accessory CL. Serum estradiol concentrations were -c 5 pg/mL throughout the sampling period in gilts receiving daily saline injections. Serum estradiol concentrations in the hCG-treated gilts with spontaneous CL increased by d 12 and remained elevated. In the hCG-treated gilts with induced CL, serum estradiol concentrations were elevated from d 12 to d 22 but returned to control concentrations on d 24. These results indicate that daily administration of hCG caused regression of induced CL of mature cyclic gilts.
Key Words: Luteolysis, hCG, Gilts
J. Anim. Sci. 1992. 70:3144-3148
Introduction 'This research was supported by State and Hatch funds allocated to the Georgia Agric. Exp. Sta. and by USDA funds. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or yarranty by the USDA or Univ. of Georgia and does not imply its @pprovalto the exclusion of other products that may be syitable. The authors thank Bennett Johnson and Elizabeth Taras for their expert technical assistance. 2To whom correspondence should be addressed: Anim. and Dairy Sci. Dept., Livestock-Poultry Bldg. 3Present address: Dept. of Agric. Univ. of Maryland Eastern Shore, Princess Anne 21853. Received Q e c p b e r 13, 1991. Accepted ?4ay 20, 1992.
Prepuberal gilts induced to ovulate form corpora lutea (CL) that function abnormally. The induced CL of prepuberal gilts were more susceptible to the uterine luteolysin than were spontaneous CL of mature gilts (Puglisi et al., 1978, 1979). In addition, luteal cell response in vitro to various stimulators of progesterone synthesis was less (Kineman et al., 1987) and the concentration of LH receptors was lower for induced CL of prepuberal gilts than for spontaneous CL of mature gilts (Est!eqne et al., 1988). Also, daily injections of hCG
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*Animal and Dairy Science Department, University of Georgia, Athens 30602 and +Richard B. Russell Agricultural Research Center, ARS, USDA, Athens, GA 30613
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injected daily with 500 IU of hCG (n = 6) or saline 01 = 8). Blood samples were obtained from all gilts every other day by jugular venipuncture beginInduced Corpora Lutea Corpora Lutea ning on d 10 in the spontaneous group and on d 10 I of the induced cycle. Serum was stored a t -20°C Day 0 2 4 6 8 1 0 1 2 1 4 16 18 2 0 until the RIA of estradiol (Barb et al., 1988) and progesterone (Kraeling et al., 1981). Ovaries were O vtu l P M tS G H C t G Ot v u l collected from all gilts on the day after the last 4 42 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 28 3 0 injection ~ a 0y of hCG and all CL and accessory luteal u I_; :H 1 structures were dissected out of the ovarian tissue. HYSTX Saline The numbers of CL and corpora albicantia (CAI marked with charcoal were recorded. The CL were Figure 1. Experimental procedure for gilts with distinguished from CA by color. A gilt was classiinduced corpora lutea (adapted from Neil1 and Day, as having maintained CL if 50% or more of the fied 1964). Ovul. = ovulation; PMSG pregnant mare's human chorio&c charcoal-marked structures on the ovaries were Serum gonadotropin; HCG CL (Rampacek et al., 1985). The number of gilts gonadotropin; HYSTX = hysterectomized. with maintained and regressed CL were subjected to chi-square analysis using exact probabilities (Steel and Torrie, 1960). Serum estradiol and from d 10 to 29 caused regression of induced CL of progesterone concentrations were subjected to hysterectomized (HYSTX) Prepuberal gilts but not split-plot in time analysis of variance and selected of spontaneous CL of HYSTX mature gilts (Rampa- comparisons were made using least squares concek et al., 1985). It is not known whether this trasts (SAS, 1985). abnormal function of induced CL in prepuberal gilts is a function of physiological age of the gilt (i.e., prepuberal vs mature) or type of CL (i.e., Results induced vs spontaneous). Therefore, the present study was conducted to determine whether CL The number of CL a t HYSTX, before daily induced in mature gilts are functionally abnormal injection of saline or HCG, was greater ( p < .05)in by determining whether chronic hCG treatment gilts with induced CL (18.7 f 2.8) than in gilts with would Cause regression of induced cL in mature spontaneous CL (12.9 k .81. The effects of hCG or gilts as it does with induced CL in prepuberal gilts. on luteal maintenance in gilts with spontaneous or induced CL are shown in Table 1. All gilts with spontaneous CL maintained CL to d 30, Materials and Methods regardless of whether they received daily saline or hCG. Six of eight gilts with induced CL that ThirtY-two crossbred gilts that had received daily saline maintained CL to d 30, estrous Of l8 to 22 displayed one Or whereas only one of six (P < ,051gilts with induced were used. Sixteen gilts were HYSTX on d 8 to Q (d cL that received daily hCG maintained cL to d 30. 0 onset of estrus), and their CL were marked by injection of charcoal (spontaneous group). Corpora lutea were induced in the remaining 16 gilts Table 1. Effect of daily administration of 500 Iu (induced group) by injecting 1,500 Iu of pregnant of human chorionic gonadotropin (hCG) from day mare's Serum gonadotropin (PMSG) i.m. on d 6 and 10 through 29 on maintenance of induced 500 IU of hCG i.m. on d 0. The day of hCG injection and spontaneous corpora lutea (CL) was considered as d 0 of the induced cycle, and in hysterectomized mature gilts ovulation was assumed to occur on d 2 of the induced cycle (Hunter, 1972; Foxcroft and Van de Spontaneous Induced Wiel, 1982). The induced gilts were HYSTX on d 8 CL CL to 9 of the induced cycle (d 17 to 18 of the original spontaneous cycle; Figure 1) and their CL were Item Saline hCG Saline hCG marked by injection of charcoal. Only gilts (n = 14) No,of gilts 8 8 8 6 in which induced CL were present and in which N ~ of . gilts with maintained gb lcd the original CL had regressed at HYSTX were CL on d 30 8 8* then subjected to treatment with saline or hCG. *One gilt had 12 CL and two corpora albicantia (CAI. gilt with regressed CL had 12 CL and 19 CA and one From d 10 to 29, gilts in the spontaneous group were injected (i.m.)daily with 500 I u OfhCG (n = 8) @lt had four CL and 14 CA. One gilt with maintained CL had four CL and one CA and one gilt had 11 CL and 10 CA. or saline (n = 8). From d 10 to 29 of the induced cone gilt had 33 c ~ , cycle (Figure 11, gilts in the induced group were dLess (P c .OS) than all other treatments.
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Serum progesterone concentrations were detesmined for all hCG-treated gilts. A CL type (induced vs spontaneous) x day interaction CP < .OOoP) was detected for serum progesterone concentrations in hCG-treated gilts (Figure 3). Serum progesterone concentrations were similar (P > ,101 from d 10 through 18 in hCG-treated gilts with spontaneous and induced CL. The serum progesterone concentrations in hCG-treated gilts with induced CL increased (P < .01) from d 10 to 14 and then did not change (P > .50) from d 14 through 28. In contrast, serum progesterone concentrations in hCG-treated gilts with spontaneous CL increased (P < .04) from d 10 to 16 and continued to increase, resulting in greater (P < ,003) concentrations from d 20 through 28 compared with those in hCG-treated gilts with induced CL.
Discussion
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Figure 2. Serum estradiol concentrations in mature hysterectomized gilts with induced or spontaneous corpora lutea receiving daily injections of saline or human chorionic gonadotropin (HCG) from d 10 through 29.
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Neil1 and Day (1964) demonstrated that CL could be induced during the luteal phase of the estrous cycle in gilts and that the original C1 and the induced CL possessed their own inherent lifespans. Specifically, they showed that induced CL could be formed by giving PMSG on d 5 or 6 followed 72 to 96 h later with hCG. Ovulation would occur on approximately d 11. Although the spontaneous CL from the original ovulation regressed on approximately d 15 to 16, the induced CL also functioned for a normal period of approximately 15 to 16 d. In the present study, this procedure was used to obtain mature cycling gilts with induced CL. The timing was such that the gilts could be HYSTX before d 10 of the induced cycle in order to maintain the induced CL for an extended time and yet be able to determine that the CL of the original cycle had regressed. The objective of this study was to determine whether daily injections of hCG would cause regression of induced CL of mature HYSTX gilts as it did with induced CL in prepuberal HYSTX gilts (Rampacek et al., 1985). All HYSTX gilts with spontaneous CL had maintained their CL to d 30 whether they received saline or hCG, which is consistent with results from mature gilts in our previous study (Rampacek et al., 1985).Also, six of eight gilts with induced CL that received saline had CL to d 30. In contrast, hCG ~ I maintained I administration caused luteal regression in five of six gilts with induced CL, which is similar to the effect of hCG on induced CL of prepuberal gilts (Rampacek et al., 1985). Serum estradiol concentrations were different between treatments. Although serum estradiol concentrations remained c 5 pg/mL in salinetreated gilts, hCG caused a n elevation in serum
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All gilts treated with hCG had accessory CL present at d 30 that were not marked with charcoal. The number of accessory CL present at d 30 was greater (P < .01) in the gilts with spontaneous CL (39.4 rt 3.7) than in the gilts with induced CL (20.2 rt 4.41. In addition, four hCG-treated gilts with spontaneous CL had numerous large follicles ( 2 10 mm), whereas only one hCG-treated gilt with induced CL had large follicles, and that gilt was the one that had maintained CL. A treatment (hCG vs saline) x CL type (induced vs spontaneous) x day interaction (P < .0005)was detected for serum estradiol concentrations (Figure 2). Serum estradiol concentrations were similar (P > .50) in saline-treated gilts with spontaneous and induced CL and increased (P c .05) from 2.3 rt .4 pg/mL on d 10 to 3.9 f .4 pg/mL on d 28. Compared with the saline-treated gilts, the hCG-treated gilts with spontaneous CL had elevated serum estradiol concentrations from d 12 to 28 with two secretory peaks at d 12 to 14 and d 26. In the hCG-treated gilts with induced CL, however, serum estradiol concentrations were elevated from d 12 to 22 and returned to control concentrations by d 24.
LUTEOLYSIS BY HCG IN MATURE GILTS
1979; Patek and Watson, 1976; Guthrie et al., 1978; Guthrie and Rexroad, 19801. The results from this study indicate that CL induced in mature gilts are similar to CL induced in prepuberal gilts. Induced CL of prepuberal gilts are more susceptible to the uterine luteolysin (Puglisi et al., 1978) and to exogenous PGF2, (Puglisi et al., 1979) compared with spontaneous CL of mature gilts. We suggest that induced CL in mature gilts also are more susceptible to PGF2, and that the hCG-induced luteolysis of induced CL in mature gilts was due to follicular and luteal production of PGF2,. Therefore, abnormal function of induced CL is not a function of the physiological age of the gilt, but rather is due to the type of CL. Although prepuberal gilts that are induced to ovulate usually fail to maintain pregnancy, there is no indication that mature gilts or sows that are induced to ovulate do not maintain pregnancy. Pinkert et al. (1989) demonstrated that embryos resulting from induced ovulation of prepuberal gilts did not have the same developmental potential in vitro as embryos from mature gilts. Therefore, failure of prepuberal gilts to maintain pregnancy after induced ovulation may be due to a
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Figure 3. Serum progesterone concentrations in mature hysterectomized gilts with induced or spontaneous corpora lutea receiving daily injections of human chorionic gonadotropin (HCG)from d 10 through 29.
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estradiol concentrations. In hCG-treated gilts with spontaneous CL, serum estradiol concentrations increased on d 12 and remained elevated through d 29, which was similar to the estradiol response in hCGtreated mature gilts in our previous study (Rampacek et al., 19851. In hCG-treated gilts with induced CL, serum estradiol concentrations also increased on d 12 but returned to control concentrations on d 24. This response was similar to the estradiol response in hCG-treated prepuberal gilts with induced CL, in which estradiol concentrations also returned to control concentrations on d 24 (Rampacek et al., 1985). Serum estradiol concentrations indicate that there were two major waves of follicular development, on approximately d 12 to 14 and on d 26, in hCG-treated gilts with spontaneous CL, whereas there was only one major wave of follicular development, on approximately d 12, in hCGtreated gilts with induced CL. The serum progesterone concentrations indicate that the second major wave of follicular development in hCGtreated gilts with spontaneous CL resulted in additional accessory CL formation compared with hCG-treated gilts with induced CL. This would account for the greater number of accessory CL a t d 30 in hCG-treated gilts with spontaneous CL than in hCG-treated gilts with induced CL. It would seem that the ovaries of gilts with induced CL became refractory to chronic hCG treatment. The reason for this is not known. The decrease in serum estradiol concentrations in the hCGtreated gilts with induced CL could have contributed to luteal regression because du Mesnil du Buisson and Denamur (1969) indicated that estrogen might be a direct luteotropin a t approximately d 30 of gestation in the pig. Estrogen may have affected luteal function in two ways. First, it may have an influence on luteal LH receptors. Garverick et al. (1982) demonstrated that estradiol increased the number of luteal LH receptors in the gilt and Ziecik et al. (1980) reported that luteal LH receptors increased between d 20 and 30 of gestation in the pig, which is coincident with the time of maximum serum estrone sulphate concentrations (Robertson and King, 1974). Also exogenous LH/ hCG has been demonstrated to reduce the number of luteal LH receptors in the rat (Conti et al., 19771 and ewe (Suter et al., 1980). Therefore, the reduced number of lutea LH receptors that was demonstrated in gilts with induced CL (Estienne et al., 1988) may have been reduced even further by the administration of hCG and lack of stimulation by estrogen. Second, estrogen may have acted as a n antiluteolytic agent to counteract the increased production of prostaglandin Fza (PGFza) by the accessory follicles and CL because follicles and CL of the pig produce PGFz, (Ainsworth et al., 1975,
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combination of abnormally functioning CL and reduced developmental capacity of the subsequent embryos.
Implications
Literature Cited Ainsworth, L., R. D. Baker, and D. T. Armstrong. 1975. Preovulatory changes in follicular prostaglandins F levels in swine. Prostaglandins 9:915. Ainsworth, L., B. K. Tsang, B. R. Downey, R. D. Baker, G. J. Marcus, and D. T. Armstrong. 1979. Effect of indomethacin on ovulation and luteal function in gilts. Biol. Reprod. 21: 401.
Barb, C. R., G. B. Rampacek, R. R. Kraeling, M. J. Estienne, E. Taras, C. E. Estienne, and C. S. Whisnant. 1988. Absence of brain opioid peptide modulation of luteinizing hormone secretion in the prepubertal gilt. Biol. Reprod. 39:803. Conti, M., J. P. Harwood, M. L. Dufau, and K. J. Catt. 1977. Effect of gonadotropin-induced receptor regulation on biological responses of isolated rat lutea cells. J. Biol. Chem. 252:8869.
de Mesnil du Buisson, F., and R. Denamur. 1969. Mecanismes du controle de la fonction luteale chez la truie, la brebis et la vache. Proc. 3rd Int. Congr. Endocrinol., Mexico, D. F. 1968. Excerpta Med. Int. Congr. Ser. 184, p 927. Amsterdam, The Netherlands. Estienne, C. E., G. B. Rampacek, R. R. Kraeling, M. J. Estienne, and C. R. Barb. 1988. Luteinizing hormone receptor number and affinity in corpora lutea from prepuberal gilts induced to ovulate and spontaneous corpora lutea of mature gilts. J. h i m . Sci. 66:917. Foxcroft, G. R., and D.F.M. Van de Wiel. 1982. Endocrine control of the oestrous cycle. I n D.J.A. Cole and G. R. Foxcroft (Ed.) Control of Pig Reproduction. pp 161-177. Butterworth Scientific, London.
13:356.
Kineman, R. D., G. B. Rampacek, R. R. Kraeling, N. A. FiorelloStocks, and R. L. Wilson. 1987. Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: In vitro progesterone production. J. Anim. Sci. 64:526. Kraeling, R. R., G. B. Rampacek, and T. E. Kiser. 1981. Corpus luteum function after indomethacin treatment during the estrous cycle and following hysterectomy in the gilt. Biol. Reprod. 25:511. Neill, J. D., and B. N. Day. 1964. Relationship of developmental stage to regression of the corpus luteum in swine. Endocrinology 74:355. Patek, C. E., and J. Watson. 1976. Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro. Prostaglandins 12:97. Pinkert, C. A,, D. L. Kooyman, A. Baumgartner, and D. H. Keisler. 1989. In-vitro development of zygotes from superovulated prepubertal and mature gilts. J. Reprod. Fertil. 8733.
Puglisi, T. A., G. B. Rampacek, and R. R. Kraeling. 1978. Corpus luteum function following subtotal hysterectomy in the prepuberal gilt. J. Anim. Sci. 46:707. Puglisi, T. A., G. B. Rampacek, R. R. Kraeling, and T. E. Kiser. 1979. Corpus luteum susceptibility to prostaglandin Fza (PGF,) luteolysis in hysterectomized prepuberal and mature gilts. Prostaglandins 18:257. Rampacek, G. B., R. R. Kraeling, and C. A. Pinkert. 1985. Regression of induced corpora lutea by human chorionic gonadotropin in prepuberal gilts. J. Anim. Sci. 60:1040. Robertson, H. A,, and G. J. King. 1974. Plasma concentrations of progesterone, estrone, estradiol-17P and of estrone sulphate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fertil. 40:133. SAS. 1965. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. Steel, R.G.D., and J. H. Tome. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Suter, D. E., P. W. Fletcher, P. M. Sluss, L. E. Reichert, Jr., and G. D. Niswender. 1980. Alterations in the number of ovine luteal receptors for LH and progesterone secretion induced by homologous hormones. Biol. Reprod. 22:205. Ziecik, A., H. J. Shaw, and A.P.F. Flint. 1980. Luteal LH recep tors during the estrous cycle and early pregnancy in the pig. J. Reprod. Fertil. 60:129.
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Daily injections of human chorionic gonadotropin caused regression of induced corpora lutea of mature hysterectomized gilts but not of spontaneous corpora lutea of mature hysterectomized gilts. Because previous work indicated that human chorionic gonadotropin caused regression of induced corpora lutea of hysterectomized prepuberal gilts, abnormal function of induced corpora lutea is not due to the physiological age of the gilts, but rather is due to the type of corpora lutea.
Garverick, H. A., C. Polge, and A.P.F. Flint. 1982. Estradiol administration raises luteal LH receptor levels in intact and hysterectomized pigs. J. Reprod. Fertil. 86:371. Guthrie, H. D., and C. E. Rexroad, Jr. 1980. Blockade of luteal prostaglandin F release in vitro during cloprostenol-induced luteolysis in the pig. Biol. Reprod. 23:358. Guthrie, H. D., C. E. Rexroad, Jr., and D. J. Bolt. 1978. In vitro synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig. Prostaglandins 16 Hunter, R.H.F. 1972. Ovulat n the pig: timing of the response to injection of human chorionic gonadotropin. Res. Vet. Sci.
This study was conducted to determine whether chronic hCG treatment would cause regression of induced corpora lutea (CL) in mature cyclic gilts. Thirtytwo mature gilts that had displayed one or more estrous cycles of 18 to 22 d were used. Sixteen gilts were hysterectomized (HYSTX) on d 6 to 9 (d 0 = onset of estrus) and their CL were marked with charcoal (spontaneous group). Sixteen gilts (induced group) were injected with 1,500 IU of pregnant mare's serum gonadotropin (PMSG) on d 0 and 500 IU of hCG on d 9 (day of hCG = d 0 of the induced cycle). Ovulation was assumed to occur on d 2 of the induced cycle. Induced gilts were HYSTX on d 8 to 9 (d 17 to 18 of the original spontaneous cycle) and their CL were marked with charcoal. Only gilts (n = 14) in which induced CL were present and in which the original CL had regressed were then subjected to treatment with saline or hCG. From d 10 to 29, gilts with spontaneous CL were injected daily with 500 IU of hCG (n = 81 or saline (n = 8). From d 10 to 29 of the induced cycle, induced gilts were injected daily with 500 IU of hCG (n = 01 or ABSTRACT:
saline (n = 8). Jugular blood samples were collected every other day from all gilts beginning on the 1st d of daily hCG treatment and quantified for estradiol and progesterone by RIA. On the day after the last hCG injection, the number of charcoal-marked CL and charcoal-marked corpora albicantia (CAI were determined. All spontaneous CL were maintained. Six of eight gilts with induced CL that received saline maintained CL, whereas only one of six (P c .05) gilts with induced CL that received daily hCG maintained CL. All gilts that were treated with hCG had numerous accessory CL. Serum estradiol concentrations were -c 5 pg/mL throughout the sampling period in gilts receiving daily saline injections. Serum estradiol concentrations in the hCG-treated gilts with spontaneous CL increased by d 12 and remained elevated. In the hCG-treated gilts with induced CL, serum estradiol concentrations were elevated from d 12 to d 22 but returned to control concentrations on d 24. These results indicate that daily administration of hCG caused regression of induced CL of mature cyclic gilts.
Key Words: Luteolysis, hCG, Gilts
J. Anim. Sci. 1992. 70:3144-3148
Introduction 'This research was supported by State and Hatch funds allocated to the Georgia Agric. Exp. Sta. and by USDA funds. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or yarranty by the USDA or Univ. of Georgia and does not imply its @pprovalto the exclusion of other products that may be syitable. The authors thank Bennett Johnson and Elizabeth Taras for their expert technical assistance. 2To whom correspondence should be addressed: Anim. and Dairy Sci. Dept., Livestock-Poultry Bldg. 3Present address: Dept. of Agric. Univ. of Maryland Eastern Shore, Princess Anne 21853. Received Q e c p b e r 13, 1991. Accepted ?4ay 20, 1992.
Prepuberal gilts induced to ovulate form corpora lutea (CL) that function abnormally. The induced CL of prepuberal gilts were more susceptible to the uterine luteolysin than were spontaneous CL of mature gilts (Puglisi et al., 1978, 1979). In addition, luteal cell response in vitro to various stimulators of progesterone synthesis was less (Kineman et al., 1987) and the concentration of LH receptors was lower for induced CL of prepuberal gilts than for spontaneous CL of mature gilts (Est!eqne et al., 1988). Also, daily injections of hCG
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*Animal and Dairy Science Department, University of Georgia, Athens 30602 and +Richard B. Russell Agricultural Research Center, ARS, USDA, Athens, GA 30613
LUTEOLYSIS BY HCG I N MATURE GILTS
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injected daily with 500 IU of hCG (n = 6) or saline 01 = 8). Blood samples were obtained from all gilts every other day by jugular venipuncture beginInduced Corpora Lutea Corpora Lutea ning on d 10 in the spontaneous group and on d 10 I of the induced cycle. Serum was stored a t -20°C Day 0 2 4 6 8 1 0 1 2 1 4 16 18 2 0 until the RIA of estradiol (Barb et al., 1988) and progesterone (Kraeling et al., 1981). Ovaries were O vtu l P M tS G H C t G Ot v u l collected from all gilts on the day after the last 4 42 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 28 3 0 injection ~ a 0y of hCG and all CL and accessory luteal u I_; :H 1 structures were dissected out of the ovarian tissue. HYSTX Saline The numbers of CL and corpora albicantia (CAI marked with charcoal were recorded. The CL were Figure 1. Experimental procedure for gilts with distinguished from CA by color. A gilt was classiinduced corpora lutea (adapted from Neil1 and Day, as having maintained CL if 50% or more of the fied 1964). Ovul. = ovulation; PMSG pregnant mare's human chorio&c charcoal-marked structures on the ovaries were Serum gonadotropin; HCG CL (Rampacek et al., 1985). The number of gilts gonadotropin; HYSTX = hysterectomized. with maintained and regressed CL were subjected to chi-square analysis using exact probabilities (Steel and Torrie, 1960). Serum estradiol and from d 10 to 29 caused regression of induced CL of progesterone concentrations were subjected to hysterectomized (HYSTX) Prepuberal gilts but not split-plot in time analysis of variance and selected of spontaneous CL of HYSTX mature gilts (Rampa- comparisons were made using least squares concek et al., 1985). It is not known whether this trasts (SAS, 1985). abnormal function of induced CL in prepuberal gilts is a function of physiological age of the gilt (i.e., prepuberal vs mature) or type of CL (i.e., Results induced vs spontaneous). Therefore, the present study was conducted to determine whether CL The number of CL a t HYSTX, before daily induced in mature gilts are functionally abnormal injection of saline or HCG, was greater ( p < .05)in by determining whether chronic hCG treatment gilts with induced CL (18.7 f 2.8) than in gilts with would Cause regression of induced cL in mature spontaneous CL (12.9 k .81. The effects of hCG or gilts as it does with induced CL in prepuberal gilts. on luteal maintenance in gilts with spontaneous or induced CL are shown in Table 1. All gilts with spontaneous CL maintained CL to d 30, Materials and Methods regardless of whether they received daily saline or hCG. Six of eight gilts with induced CL that ThirtY-two crossbred gilts that had received daily saline maintained CL to d 30, estrous Of l8 to 22 displayed one Or whereas only one of six (P < ,051gilts with induced were used. Sixteen gilts were HYSTX on d 8 to Q (d cL that received daily hCG maintained cL to d 30. 0 onset of estrus), and their CL were marked by injection of charcoal (spontaneous group). Corpora lutea were induced in the remaining 16 gilts Table 1. Effect of daily administration of 500 Iu (induced group) by injecting 1,500 Iu of pregnant of human chorionic gonadotropin (hCG) from day mare's Serum gonadotropin (PMSG) i.m. on d 6 and 10 through 29 on maintenance of induced 500 IU of hCG i.m. on d 0. The day of hCG injection and spontaneous corpora lutea (CL) was considered as d 0 of the induced cycle, and in hysterectomized mature gilts ovulation was assumed to occur on d 2 of the induced cycle (Hunter, 1972; Foxcroft and Van de Spontaneous Induced Wiel, 1982). The induced gilts were HYSTX on d 8 CL CL to 9 of the induced cycle (d 17 to 18 of the original spontaneous cycle; Figure 1) and their CL were Item Saline hCG Saline hCG marked by injection of charcoal. Only gilts (n = 14) No,of gilts 8 8 8 6 in which induced CL were present and in which N ~ of . gilts with maintained gb lcd the original CL had regressed at HYSTX were CL on d 30 8 8* then subjected to treatment with saline or hCG. *One gilt had 12 CL and two corpora albicantia (CAI. gilt with regressed CL had 12 CL and 19 CA and one From d 10 to 29, gilts in the spontaneous group were injected (i.m.)daily with 500 I u OfhCG (n = 8) @lt had four CL and 14 CA. One gilt with maintained CL had four CL and one CA and one gilt had 11 CL and 10 CA. or saline (n = 8). From d 10 to 29 of the induced cone gilt had 33 c ~ , cycle (Figure 11, gilts in the induced group were dLess (P c .OS) than all other treatments.
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3 146
RAMPACEK ET AL.
Serum progesterone concentrations were detesmined for all hCG-treated gilts. A CL type (induced vs spontaneous) x day interaction CP < .OOoP) was detected for serum progesterone concentrations in hCG-treated gilts (Figure 3). Serum progesterone concentrations were similar (P > ,101 from d 10 through 18 in hCG-treated gilts with spontaneous and induced CL. The serum progesterone concentrations in hCG-treated gilts with induced CL increased (P < .01) from d 10 to 14 and then did not change (P > .50) from d 14 through 28. In contrast, serum progesterone concentrations in hCG-treated gilts with spontaneous CL increased (P < .04) from d 10 to 16 and continued to increase, resulting in greater (P < ,003) concentrations from d 20 through 28 compared with those in hCG-treated gilts with induced CL.
Discussion
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Figure 2. Serum estradiol concentrations in mature hysterectomized gilts with induced or spontaneous corpora lutea receiving daily injections of saline or human chorionic gonadotropin (HCG) from d 10 through 29.
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Neil1 and Day (1964) demonstrated that CL could be induced during the luteal phase of the estrous cycle in gilts and that the original C1 and the induced CL possessed their own inherent lifespans. Specifically, they showed that induced CL could be formed by giving PMSG on d 5 or 6 followed 72 to 96 h later with hCG. Ovulation would occur on approximately d 11. Although the spontaneous CL from the original ovulation regressed on approximately d 15 to 16, the induced CL also functioned for a normal period of approximately 15 to 16 d. In the present study, this procedure was used to obtain mature cycling gilts with induced CL. The timing was such that the gilts could be HYSTX before d 10 of the induced cycle in order to maintain the induced CL for an extended time and yet be able to determine that the CL of the original cycle had regressed. The objective of this study was to determine whether daily injections of hCG would cause regression of induced CL of mature HYSTX gilts as it did with induced CL in prepuberal HYSTX gilts (Rampacek et al., 1985). All HYSTX gilts with spontaneous CL had maintained their CL to d 30 whether they received saline or hCG, which is consistent with results from mature gilts in our previous study (Rampacek et al., 1985).Also, six of eight gilts with induced CL that received saline had CL to d 30. In contrast, hCG ~ I maintained I administration caused luteal regression in five of six gilts with induced CL, which is similar to the effect of hCG on induced CL of prepuberal gilts (Rampacek et al., 1985). Serum estradiol concentrations were different between treatments. Although serum estradiol concentrations remained c 5 pg/mL in salinetreated gilts, hCG caused a n elevation in serum
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All gilts treated with hCG had accessory CL present at d 30 that were not marked with charcoal. The number of accessory CL present at d 30 was greater (P < .01) in the gilts with spontaneous CL (39.4 rt 3.7) than in the gilts with induced CL (20.2 rt 4.41. In addition, four hCG-treated gilts with spontaneous CL had numerous large follicles ( 2 10 mm), whereas only one hCG-treated gilt with induced CL had large follicles, and that gilt was the one that had maintained CL. A treatment (hCG vs saline) x CL type (induced vs spontaneous) x day interaction (P < .0005)was detected for serum estradiol concentrations (Figure 2). Serum estradiol concentrations were similar (P > .50) in saline-treated gilts with spontaneous and induced CL and increased (P c .05) from 2.3 rt .4 pg/mL on d 10 to 3.9 f .4 pg/mL on d 28. Compared with the saline-treated gilts, the hCG-treated gilts with spontaneous CL had elevated serum estradiol concentrations from d 12 to 28 with two secretory peaks at d 12 to 14 and d 26. In the hCG-treated gilts with induced CL, however, serum estradiol concentrations were elevated from d 12 to 22 and returned to control concentrations by d 24.
LUTEOLYSIS BY HCG IN MATURE GILTS
1979; Patek and Watson, 1976; Guthrie et al., 1978; Guthrie and Rexroad, 19801. The results from this study indicate that CL induced in mature gilts are similar to CL induced in prepuberal gilts. Induced CL of prepuberal gilts are more susceptible to the uterine luteolysin (Puglisi et al., 1978) and to exogenous PGF2, (Puglisi et al., 1979) compared with spontaneous CL of mature gilts. We suggest that induced CL in mature gilts also are more susceptible to PGF2, and that the hCG-induced luteolysis of induced CL in mature gilts was due to follicular and luteal production of PGF2,. Therefore, abnormal function of induced CL is not a function of the physiological age of the gilt, but rather is due to the type of CL. Although prepuberal gilts that are induced to ovulate usually fail to maintain pregnancy, there is no indication that mature gilts or sows that are induced to ovulate do not maintain pregnancy. Pinkert et al. (1989) demonstrated that embryos resulting from induced ovulation of prepuberal gilts did not have the same developmental potential in vitro as embryos from mature gilts. Therefore, failure of prepuberal gilts to maintain pregnancy after induced ovulation may be due to a
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Figure 3. Serum progesterone concentrations in mature hysterectomized gilts with induced or spontaneous corpora lutea receiving daily injections of human chorionic gonadotropin (HCG)from d 10 through 29.
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estradiol concentrations. In hCG-treated gilts with spontaneous CL, serum estradiol concentrations increased on d 12 and remained elevated through d 29, which was similar to the estradiol response in hCGtreated mature gilts in our previous study (Rampacek et al., 19851. In hCG-treated gilts with induced CL, serum estradiol concentrations also increased on d 12 but returned to control concentrations on d 24. This response was similar to the estradiol response in hCG-treated prepuberal gilts with induced CL, in which estradiol concentrations also returned to control concentrations on d 24 (Rampacek et al., 1985). Serum estradiol concentrations indicate that there were two major waves of follicular development, on approximately d 12 to 14 and on d 26, in hCG-treated gilts with spontaneous CL, whereas there was only one major wave of follicular development, on approximately d 12, in hCGtreated gilts with induced CL. The serum progesterone concentrations indicate that the second major wave of follicular development in hCGtreated gilts with spontaneous CL resulted in additional accessory CL formation compared with hCG-treated gilts with induced CL. This would account for the greater number of accessory CL a t d 30 in hCG-treated gilts with spontaneous CL than in hCG-treated gilts with induced CL. It would seem that the ovaries of gilts with induced CL became refractory to chronic hCG treatment. The reason for this is not known. The decrease in serum estradiol concentrations in the hCGtreated gilts with induced CL could have contributed to luteal regression because du Mesnil du Buisson and Denamur (1969) indicated that estrogen might be a direct luteotropin a t approximately d 30 of gestation in the pig. Estrogen may have affected luteal function in two ways. First, it may have an influence on luteal LH receptors. Garverick et al. (1982) demonstrated that estradiol increased the number of luteal LH receptors in the gilt and Ziecik et al. (1980) reported that luteal LH receptors increased between d 20 and 30 of gestation in the pig, which is coincident with the time of maximum serum estrone sulphate concentrations (Robertson and King, 1974). Also exogenous LH/ hCG has been demonstrated to reduce the number of luteal LH receptors in the rat (Conti et al., 19771 and ewe (Suter et al., 1980). Therefore, the reduced number of lutea LH receptors that was demonstrated in gilts with induced CL (Estienne et al., 1988) may have been reduced even further by the administration of hCG and lack of stimulation by estrogen. Second, estrogen may have acted as a n antiluteolytic agent to counteract the increased production of prostaglandin Fza (PGFza) by the accessory follicles and CL because follicles and CL of the pig produce PGFz, (Ainsworth et al., 1975,
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combination of abnormally functioning CL and reduced developmental capacity of the subsequent embryos.
Implications
Literature Cited Ainsworth, L., R. D. Baker, and D. T. Armstrong. 1975. Preovulatory changes in follicular prostaglandins F levels in swine. Prostaglandins 9:915. Ainsworth, L., B. K. Tsang, B. R. Downey, R. D. Baker, G. J. Marcus, and D. T. Armstrong. 1979. Effect of indomethacin on ovulation and luteal function in gilts. Biol. Reprod. 21: 401.
Barb, C. R., G. B. Rampacek, R. R. Kraeling, M. J. Estienne, E. Taras, C. E. Estienne, and C. S. Whisnant. 1988. Absence of brain opioid peptide modulation of luteinizing hormone secretion in the prepubertal gilt. Biol. Reprod. 39:803. Conti, M., J. P. Harwood, M. L. Dufau, and K. J. Catt. 1977. Effect of gonadotropin-induced receptor regulation on biological responses of isolated rat lutea cells. J. Biol. Chem. 252:8869.
de Mesnil du Buisson, F., and R. Denamur. 1969. Mecanismes du controle de la fonction luteale chez la truie, la brebis et la vache. Proc. 3rd Int. Congr. Endocrinol., Mexico, D. F. 1968. Excerpta Med. Int. Congr. Ser. 184, p 927. Amsterdam, The Netherlands. Estienne, C. E., G. B. Rampacek, R. R. Kraeling, M. J. Estienne, and C. R. Barb. 1988. Luteinizing hormone receptor number and affinity in corpora lutea from prepuberal gilts induced to ovulate and spontaneous corpora lutea of mature gilts. J. h i m . Sci. 66:917. Foxcroft, G. R., and D.F.M. Van de Wiel. 1982. Endocrine control of the oestrous cycle. I n D.J.A. Cole and G. R. Foxcroft (Ed.) Control of Pig Reproduction. pp 161-177. Butterworth Scientific, London.
13:356.
Kineman, R. D., G. B. Rampacek, R. R. Kraeling, N. A. FiorelloStocks, and R. L. Wilson. 1987. Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: In vitro progesterone production. J. Anim. Sci. 64:526. Kraeling, R. R., G. B. Rampacek, and T. E. Kiser. 1981. Corpus luteum function after indomethacin treatment during the estrous cycle and following hysterectomy in the gilt. Biol. Reprod. 25:511. Neill, J. D., and B. N. Day. 1964. Relationship of developmental stage to regression of the corpus luteum in swine. Endocrinology 74:355. Patek, C. E., and J. Watson. 1976. Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro. Prostaglandins 12:97. Pinkert, C. A,, D. L. Kooyman, A. Baumgartner, and D. H. Keisler. 1989. In-vitro development of zygotes from superovulated prepubertal and mature gilts. J. Reprod. Fertil. 8733.
Puglisi, T. A., G. B. Rampacek, and R. R. Kraeling. 1978. Corpus luteum function following subtotal hysterectomy in the prepuberal gilt. J. Anim. Sci. 46:707. Puglisi, T. A., G. B. Rampacek, R. R. Kraeling, and T. E. Kiser. 1979. Corpus luteum susceptibility to prostaglandin Fza (PGF,) luteolysis in hysterectomized prepuberal and mature gilts. Prostaglandins 18:257. Rampacek, G. B., R. R. Kraeling, and C. A. Pinkert. 1985. Regression of induced corpora lutea by human chorionic gonadotropin in prepuberal gilts. J. Anim. Sci. 60:1040. Robertson, H. A,, and G. J. King. 1974. Plasma concentrations of progesterone, estrone, estradiol-17P and of estrone sulphate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fertil. 40:133. SAS. 1965. SAS User’s Guide: Statistics. SAS Inst. Inc., Cary, NC. Steel, R.G.D., and J. H. Tome. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. Suter, D. E., P. W. Fletcher, P. M. Sluss, L. E. Reichert, Jr., and G. D. Niswender. 1980. Alterations in the number of ovine luteal receptors for LH and progesterone secretion induced by homologous hormones. Biol. Reprod. 22:205. Ziecik, A., H. J. Shaw, and A.P.F. Flint. 1980. Luteal LH recep tors during the estrous cycle and early pregnancy in the pig. J. Reprod. Fertil. 60:129.
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Daily injections of human chorionic gonadotropin caused regression of induced corpora lutea of mature hysterectomized gilts but not of spontaneous corpora lutea of mature hysterectomized gilts. Because previous work indicated that human chorionic gonadotropin caused regression of induced corpora lutea of hysterectomized prepuberal gilts, abnormal function of induced corpora lutea is not due to the physiological age of the gilts, but rather is due to the type of corpora lutea.
Garverick, H. A., C. Polge, and A.P.F. Flint. 1982. Estradiol administration raises luteal LH receptor levels in intact and hysterectomized pigs. J. Reprod. Fertil. 86:371. Guthrie, H. D., and C. E. Rexroad, Jr. 1980. Blockade of luteal prostaglandin F release in vitro during cloprostenol-induced luteolysis in the pig. Biol. Reprod. 23:358. Guthrie, H. D., C. E. Rexroad, Jr., and D. J. Bolt. 1978. In vitro synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig. Prostaglandins 16 Hunter, R.H.F. 1972. Ovulat n the pig: timing of the response to injection of human chorionic gonadotropin. Res. Vet. Sci.
