H. D. Guthrie 1'2 and J. F. Knudsen 3. US Department of Agriculture, Beltsville, MD 20705

Transcription

1 FOLLICULAR GROWTH AND PRODUCTION OF ESTROGEN AND PROGESTERONE AFTER INJECTION OF GILTS WITH HUMAN CHORIONIC GONADOTROPIN ON DAY 12 OF THE ESTROUS CYCLE H. D. Guthrie 1'2 and J. F. Knudsen 3 US Department of Agriculture, Beltsville, MD 275 Summary Twenty cyclic gilts were injected im with either saline (control) or 1, IU of human chorionic gonadotropin (hcg) on d 12 of the estrous cycle to determine the effects of hcg on follicular development and steroidogenesis. Blood was collected when gihs were sacrificed on d 13 or 16. Follicles were classified as medium (3 to 6 mm in diameter) or large (>6 mm diameter), dissected from the ovary, measured and weighed. Pieces of follicle wall were incubated 3 h in Krebs Ringer bicarbonate buffer (KRB) on ice in an atmosphere of air or at 37 C in an atmosphere of 95% 2:5% C2. Unconjugated estrogen and progesterone in blood plasma, follicular fluid and 1, g supernatants of incubated follicular tissue homogenates were quantified by radioimmunoassay. On d 13 follicles on ovaries of control or hcg-injected gilts were <6 mm in diameter. On d 16, one of five control gilts had some large follicles, while all five hcg-treated gilts had large as well as medium follicles. On d 16 follicular fluid of large follicles from hcginjected gilts contained twofold more estrogen and 4-fold more progesterone than medium follicles on the same ovaries. Tissue from large follicles of hcg-injected gilts produced more progesterone in vitro than did tissue from I Reprod. Lab., Anim. Sci. Institute, Behsville Agr. Res. Center, ARS. ZMention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. 3Current address: Good Samaritan Hospital, Lochraven Building, Baltimore, MD. Received June 6, Accepted April 24, medium follicles (P<.5), but estrogen production did not differ. On d 16 medium follicles from control or hcg-injected gilts were larger, contained more estrogen and less progesterone than those recovered on d 13 (P<.1). However, medium follicles on d 13 and 16 from hcginjected gilts were larger and their fluid contained more estrogen and progesterone than medium follicles from control gilts (P<.1). This study demonstrated that 1, IU of hcg injected on d 12 of the estrous cycle in the gilt resulted in development of large follicles, resembling preovulatory follicles at the onset of estrus before the preovulatory luteinizing hormone surge. (Key Words: Gilt, Follicle, Human Chorionic Gonadotropin, Estrogen, Progesterone.) I ntroduction A single injection of human chorionic gonadotropin (hcg) on d 12 of the estrous cycle was found to delay luteolysis in gilts (Guthrie and Rexroad, 1981). After injection of 5 or 1, IU of hcg, plasma estrogen concentration increased during a 3 to 4 d period and then decreased (Guthrie and Bolt, 1983). The transitory increase in plasma estrogen indicated that hcg had some effect on follicular development apart from its ability to induce ovulation of preovulatory follicles (Dziuk and Polge, 1962) or luteinization of granulosa cells (Channing, 197; Channing et al., 198). The discovery of the ability of hcg to increase estrogen secretion in the gilt was important because first, exogenous estrogen delays luteolysis in gilts (Kidder et al., 1955; Gardner et al., 1963) and might contribute to the luteotropic effect of hcg and second, Bogovich et al. (1981) and Carson et al. (1981) have hypothesized that increased secretion of luteinizing hormone (LH) initiates maturation 1295 JOURNAL OF ANIMAL SCIENCE, Vol. 59, No. 5, 1984

2 1296 GUTHRIE AND KNUDSEN of follicles to the preovulatory stage in cyclic rats and during luteolysis in periparturient rats. This experiment was conducted to determine whether an injection of 1, IU of hcg on d 12 of the estrous cycle increased follicle growth and estrogen production. Twenty-four and 96 h after the injection of hcg gilts were slaughtered to determine follicle size, follicular fluid concentration of estrogen and progesterone and follicular tissue production of estrogen and progesterone in vitro. Materials and Methods Twenty crossbred cyclic gilts were assigned at random (1/group) to be injected with vehicle (control) or 1, IU of hcg 4 (hcgtreated) at 8 h on d 12 of an estrous cycle (d = first day of estrus). The hcg was injected im in 2 ml of.9% NaC1 solution. Gilts were slaughtered either 24 or 96 h (d 13 or 16) after injection of.9% NaC1 solution or hcg. One blood sample from each gilt was collected into a heparinized tube during slaughter. Ovaries were excised and placed on ice. Eight to 15 follicles, at least 3 mm in diameter and transparent in appearance, were dissected from the ovaries of each gilt by use of small forceps, a scalpel with a number 12 blade and iris scissors. The excised follicles were classified according to their diameter as being medium (3 to 6 mm) or large (>6 ram). Each follicle was weighed, then the follicle was bisected and follicular fluid drained into a 12 x 75 mm glass culture tube. Two to 12 tubes of follicular fluid were collected from the bisected follicles of each gilt. Fluid from as many as 12 medium follicles of 3 to 4 mm in diameter was collected in two tubes; fluid from each large follicle was collected in a separate tube. The total amount of follicular fluid in each tube ranged from 44 to 695 rag. The follicular fluid in each tube was diluted tenfold with phosphate buffered saline, ph 7.4, and centrifuged at 1, g for 2 min. The supernatant was stored at -2 C. After follicular fluid was collected, the remaining follicular tissue was weighed and follicular fluid weight for each follicle was calculated. Corpora lutea were counted and their gross appearance was recorded and compared to the description of ovarian morphology 4APL, Ayerst Research Laboratories, Nu s Leitz, Wetzlar, West Germany. by Akins and Morrissette (1968). Pieces of follicle wall, approximately 5 mg total, were placed in 17 1 mm plastic Falcon tubes containing 4 ml of Krebs Ringer bicarbonate buffer solution, ph 7.4. The tissue was incubated for 3 h in either an ice water bath with an atmosphere of air to estimate tissue hormone content prior to incubation or in a water bath at 37 C with an atmosphere of 95% 2:5% CO2 to study production of estrogen and progesterone. After incubation, the contents of each Falcon tube were homogenized and centrifuged 2 rain at 1, g at 4 C. Incubation supernatant was aspirated and stored at -2 C. Pieces of unincubated follicle wall from 11 gilts (two or three/treatment combination) and samples of luteal tissue were fixed in Bouin's solution, imbedded in paraffin, sectioned and stained with hematoxylin and eosin. The size of cells of theca interna, membrane granulosa and luteal tissue was estimated by counting the number of nuclei in 1 locations in four different sections of the same tissue. Analysis of the stained sections was made with a Laborlux 12 microscope 5 by projecting the image on a television screen. Data were expressed as the number of nuclei in an area of.2 mm z. Progesterone and estrogen (unconjugated estradiol-1713 and estrone) in plasma and in tenfold dilutions of follicular fluid and incubation supernatant of follicular tissue were quantified by radioimmunoassays described in detail previously (Guthrie, 1977; Guthrie and Bolt, 1983). Hormone content of follicular fluid was expressed as ng/follicle and ng/ml fluid. The intraasay and interassay coefficients of variation were 12.4 and 13.1%, respectively, for seven progesterone and were 9.2 and 11.7%, respectively, for seven estrogen radioimmunoassays. The assay sensitivity, defined as pg/tube at 9% of zero hormone binding (1%), was 12 and 2, respectively, for the progesterone and estrogen radioimmunoassays. Statistical Analysis. Means for follicle diameter, follicle weight, follicular fluid weight, follicular tissue weight, hormone content of follicular fluid and follicle cell size for medium and large follicles (when present) were calculated for each gilt. The gilt means provided the data set for calculation of treatment means and statistical analysis (SAS, 1979) so each gilt was an experimental unit. The statistical model for data from medium follicles and plasma hormones was a completely random design with

3 INDUCED FOLLICULAR DEVELOPMENT 1297 a 2 x 2 factorial arrangement of dose of hcg ( or 1, IU) and day of the cycle when the follicles were recovered (d 13 or 16). Estrogen and progesterone accumulation during incubation of tissue from medium follicles was analyzed with the same statistical model with incubation temperature ( or 37 C) as a splitplot for repeat sampling of follicular tissue of each gilt. Follicle cell size was also analyzed statistically by this model with tissue type (theca interna or membrana granulosa) as a split-plot for repeat sampling. In addition, the effect of follicle size (medium or large) on estrogen and progesterone accumulation in follicular fluid and in follicular tissue incubations was analyzed using the data from the five hcg-treated gilts sacrificed on d 16. Results Two-hundred-thirteen follicles (8 to 15/gilt) were dissected from the ovaries of 2 gilts. The follicles recovered from or observed on ovaries of control gilts and hcg-injected gilts were <6 mm in diameter on d 13. One of five control gilts sacrificed on d 16 had large as well as small and medium follicles. The follicle variables and estrogen and progesterone accumulation in follicular fluid from the large follicles of this one gilt are included in tables 1 and 2. Twenty-one medium and 31 large follicles were recovered on d 16 from hcg-injected gilts. Follicular fluid estrogen (ng/folliele) and progesterone (rig/follicle) were two- and 4-fold greater (P<.5), respectively, for large follicles than for medium follicles (table 2). The concentration of estrogen in follicular fluid (ng/ml) did not differ between large and medium follicles from the same ovaries, but progesterone concentration (ng/ml) in large follicles was tenfold greater than in the medium follicles (P<.5). During a 3 h incubation, tissue from large and medium follicles did not differ significantly in accumulation of estrogen during incubation (table 3). However, more progesterone accumulated during incubation of tissue from large follicles than from medium follicles (P<.OS). Medium follicles from control and hcginjected gilts were larger on d 16 than d 13 in terms of follicle diameter, follicle weight, follicular fluid weight and residual tissue weight (table 1, P<.1). Fluid from medium follicles from control and hcg-injected gilts contained more estrogen and less progesterone on d 16 than on d 13 (table 2, P<.5). However, medium follicles from hcg-injected gilts were larger and contained more estrogen and progesterone than medium follicles from control gilts on both d 13 and 16 (P<.1). Pieces of follicular wall from medium follicles of control and hcg-treated gilts accumulated more estrogen during a 3 h incubation at 37 C (P<.1) than at C (table 3). Follicular tissue from medium follicles accumulated more estrogen on d 16 than on d 13 (P=.6). Tissue from medium follicles of hcg-injected gilts accumulated more estrogen on d 13 and 16 than tissue from medium follicles of control gilts (P<.1). Progesterone accumulation during incubation of tissue from medium follicles was not significantly affected by incubation temperature, day of the cycle or dose of hcg. The mean number of corpora lutea/gilt was 13.2 for the 2 gilts and did not vary significantly by dose of hcg or day of the cycle. The corpora lutea of control gilts on d 13 were 8 to 1 mm in diameter with a characteristic liver color and had an extensive network of blood vessels on the luteal surface. On d 16 the corpora lutea of control gilts were more ischemic, pale in color and in three of five gilts luteal diameter was reduced to 6 mm with few blood vessels visable on the luteal surface. Corpora lutea from hcg-injected gilts on d 13 and 16 were indistinguishable in appearance from those of control gilts on d 13. Plasma progesterone concentration was less (P<.1) on d 16 in control gilts (3.6 ng/ml) as compared with d 13 in control gilts and d 13 and 16 in hcg-injected gilts (29 ng/ml; table 2). The ovaries from three of five hcg-treated gilts on d 16 had 11, 5 and 3 accessory structures of 4 to 5 mm in diameter in addition to 14, 16 and 12 corpora lutea, respectively. After dissection and visual observation these accessory ovarian structures were found to consist of what appeared to be luteal tissue, and did not contain clotted blood or show evidence of hemorrhage. The ceil size (number of nuclei/.2 mm 2) in histological sections from three of the accessory ovarian structures and from seven corpora lutea were similar with means of 78 and 73, respectively. For medium follicles the cells of the theea interna were larger than those of the membrana granulosa (mean -+ SE of and nuclei/.2 mm 2, respectively, P<.1). The day of the cycle and dose of hcg had no significant effect on cell size in medium follicles. The cell size for the theca interna and

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6 13 GUTHRIE AND KNUDSEN, I +1 +l +~ V 6 "..1 o~ ~'~ t",. ~ L' ~.,,-u Oe'l ~--I at.. o~ ~ a ~ +~ +1 ~ _~ ~.~ ~8 6 ~ ~.~ ~R z~ d M < ~3e~ ~ ~".~ ~, ~oo d o u ='o S ~ ~ ~

7 INDUCED FOLLICULAR DEVELOPMENT 131 membrana granulosa of seven large follicles recovered on d 16 from three hcg-injected gilts was and nuclei/.2 mm 2 (mean -+ SE), respectively. Discussion We conclude that the transitory increase in plasma estrogen following an injection of hcg on d 12 of the cycle (Guthrie and Bolt, 1982) was a result of estrogen production by some medium follicles as they grew to preovulatory size. However, follicles were not marked prior to the hcg injection in this study to prove conclusively that the large follicles present on d 16 were from the population of medium sized follicles present on d 12. Bogovich et al. (1981) and Carson et al. (1981) showed that preovulatory follicles could be developed in pregnant rats before plasma progesterone and had decreased by injection of hcg 9 d before expected time of parturition. From these results, they proposed that gradual increases in plasma LH associated with decreasing progesterone concentrations in preparturient rats initiated growth of preovulatory follicles. Goodman et al. (1981) and McNatty et al. (1981) have provided evidence in the ewe of an active role for LH and a permissive role for follicle-stimulating hormone (FSH) in follicular growth and maturation to the preovulatory state. Progesterone withdrawal during luteolysis may enhance the follicular response to LH (Goodman et al., 1981). A similar relationship for LH and FSH may exist in the gilt. Secretion of LH in terms of pulse amplitude and(or) pulse frequency was reported to increase or persist during the first 1 to 3 d of luteolysis (Foxcroft and Van de Wiel, 1982; Kopf et al., 1983) or after withdrawal of the progestogen, Altrenogest (Jones et al., 1983). Plasma FSH decreased during luteolysis and remained low until the preovulatory LH surge in gilts (Foxcroft and Van de Wiel, 1982; Guthrie and Bolt, 1983). Follicles destined to ovulate are selected between d 14 to 17 of the cycle in the gilt (Clark et al., 1973; Clark et al., 1975; Daily et al., 1975), corresponding to the time when the transient pulsatile secretion of LH was reported. The large follicles recovered on d 16 from hcg-injected gilts in this study were similar to large follicles developed in other experiments. Typically, follicular fluid estrogen and pro- gesterone were found to increase in large follicles as they developed prior to the preovulatory LH surge in sows after withdrawal of methallibure (Daguet, 1979), in gilts after luteolysis (Eiler and Nalbandov, 1977) and in prepuberal gilts after injection of pregnant mares serum gonadotropin (Ainsworth et al., 198). Theca ceils of large follicles were larger than the theca cells of medium follicles, however, granulosa cells from large or medium follicles did not show evidence of luteinization (cellular hypertrophy). The increase in plasma estrogen following the hcg injection (Guthrie and Bolt, 1983) did not elicit a preovulatory LH surge. The large follicles induced by hcg treatment in the present study would probably have regressed as has been reported for preovulatory follicles in rats (Uilenbroek et al., 198) and gilts (Guthrie and Bolt, 1982) when the prevoulatory LH surge mechanism was blocked. Medium follicles recovered from control gilts were slightly, but significantly larger and had been producing more estrogen and less progesterone in vivo on d 16 than 13. The cause for this shift in the pattern of steroidogenesis is not known, however, Eiler and Nalbandov (1977) reported similar changes in follicular fluid estrogen and progesterone in gilts between d 11 and 14 of the estrous cycle. Medium follicles from hcg-injected gilts were similar to those from control gilts with respect to changes in size and follicular fluid hormone content, except follicles from hcg-treated gilts and were larger and contained more steroid on d 13 and 16 than follicles recovered on the same days from control gilts. Follicles of 1 to 5 mm in diameter present during the estrous cycle of the gilt were reported not to ovulate to an ovulatory dose of hcg (Channing and Kammerman, 1974). The accessory ovarian structures found on d 16 in three of five hcg-injected gilts in our study did not show evidence of ovulation, however, study of histological sections of these accessory ovarian structures indicated that they had luteinized with hypertrophied cells similar in size to those of tissue from the corpora lutea of the estrous cycle. Thus a single injection of hcg on d 12 in the gilt caused some follicles to grow to preovulatory size and produce estrogen and progesterone in quantities similar to that produced by large follicles present at the onset of natural estrus before the preovulatory LH surge.

8 132 GUTHRIE AND KNUDSEN Literature Cited Ainsworth, L., B. K. Tsang, B. R. Downey, G. J. Marcus and D. T. Armstrong Interrelationships between follicular fluid steroid levels, gonadotropic stimuli, and oocyte maturation during preovulatory development of porcine follicles. Biol. Reprod. 23:621. Akins, E. L. and M. C. Morrissette Gross ovarian changes during estrous cycle of swine. Amer. J. Vet. Res. 29:1953. Bogovich, K., J. S. Richards and L. E. Reichert, Jr Obligatory role of luteinizing hormone (LH) in the initiation of preovulatory follicular growth in the pregnant rat: Specific effects of human chorionic gonadotropin and folliclestimulating hormone on LH receptors and steroidogenesis in theca, granulosa, and luteal cells. Endocrinology 19:6. Carson, R. S., J. S. Richards and L. E. Kahn Functional and morphological differentiation of theca and granulosa cells during pregnancy in the rat: Dependence on increased basal luteinizing hormone activity. Endocrinology 19:1433. Channing, C. P Effect of stage of the estrous cycle and gonadotropins upon luteinization of porcine granulosa cells in culture. Endocrinology 87:156. Channing, C. P., H. J. Brinkley and E. P. Young Relationship between serum luteinizing hormone levels and the ability of porcine granulosa cells to luteinize and respond to exogenous luteinizing hormone in culture. Endocrinology 16: 317. Channing, C. P. and S. Kammerman Binding of gonadotropins to ovarian cells. Biol. Reprod. 1:179. Clark, J. R., T. N. Edey, N. L. First, A. B. Chapman and L. E. Casida Effects of four genetic groups and two levels of feeding on ovulation rate and follicular development in puberal gilts. J. Anita. Sci. 36:1164. Clark, J. R., N. L. First, A. B. Chapman and L. E. Casida Ovarian follicular development during the estrous cycle in gilts following electrocautery of follicles. J. Anita. Sci. 44:115. Daguet, M. C Increase of follicle cell LH binding and changes in the LH level of follicular fluid during the preovulatory period in the sow. Ann. Biol. Anita. Biochem. Biophys. 19:1655. Dailey, R. A., J. R. Clark, N. L. First, A. B. Chapman and L. E. Casida Loss of follicles during the follicular phase of the estrous cycle of swine as affected by genetic group and level of feed intake. J. Anita. Sci. 41:835. Dziuk, P. and C. Polge Fertility in swine after induced ovulation. J. Reprod. Fertil. 4:27. Eiler, H. and A. V. Nalbandov Sex steroids in follicular fluid and blood plasma during the estrous cycle of pigs. Endocrinology 1:331. Foxcroft, G. R. and D.F.M. Van de Wiel Endocrine control of the oestrous cycle. In: D.J.A. Cole and G. R. Foxcroft (Ed.) Control of Pig Reproduction. pp Buttersworth, London. Gardner, M. L., N. L. First and L. E. Casida Effect of exogenous estrogens on corpus luteum maintenance in gilts. J. Anita. Sci. 22:132. Goodman, R. L., L. E. Reichert, Jr., S. J. Legan, K. D. Ryan, D. L. Foster and F. J. Karsch Role of gonadotropins and progesterone in determining the preovulatory estradiol rise in the ewe. Biol. Reprod. 25:134. Guthrie, H. D Induction of ovulation and fertility in prepuberal gilts. J. Anita. Sci. 45:136. Guthrie, H. D. and D. J. Bolt Effect of allyl trenbolone (AT) treatment on follicular development, preovulatory LH surge, and ovulation in the pig. J. Anim. Sei. 55(Suppl. 1):354. Guthrie, H. D. and D. J. Bolt Changes in plasma estrogen, luteinizing hormone, follicle-stimulating hormone and 13,14-dihydro-15-keto-prostaglandin F2~ after human chorionic gonadotropin to block luteolysis in pigs. J. Anita. Sci. 57:993. Guthrie, H. D. and C. E. Rexroad Endometrial prostaglandin F release in vitro and plasma 13,14-dihydro-15-keto-prostaglandin Faa in pigs with luteolysis blocked by pregnancy, estradiol benzoate or human chorionic gonadotropin. J. Anita. Sci. 52:33. Jones, H. R., W. A. Bennett, T. G. Althen and N. M. Cox Effects of dietary energy and exogenous insulin during the period of follicular growth on ovulation rate and LH patterns in gilts. J. Anita. Sci. 57(Suppl. 1): 346. Kidder, H. E., L. E. Casida and R. H. Grummer Some effects of estrogen injections on the estrual cycle of gilts. J. Anim. Sci. 14:47. Kopf, J. D., C. R. Kelly, R. J. Kittok and D. R. Zimmerman Characterization of luteinizing hormone (LH) pattern during luteal and follicular phases of the estrous cycle in swine. J. Anita. Sci. 57(Suppl. 1):351. McNatty, K. P., M. Gibb, C. Dobson and D. C. Thurley Evidence that changes in luteinizing hormone secretion regulate the growth of the preovulatory follicle in the ewe. J. Endocrinol. 9:375. SAS SAS User's Guide. Statistical Analysis System Institute, Inc., CaW, NC. p 237. Uilenbroek, J.Th.J., P.J.A. Woutersen and P. Van der Sehoot Atresia of preovulatory follicles: Gonadotropin binding and steroidogenic activity. Biol. Reprod. 23:219.