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Regulation of in vitro progesterone release from caprine luteal tissues by prostaglandins E2 and F2a
Theriogenology
38:63-71,
1992
REGULATION OF IN VITRO PROGESTERONE RELEASE FROM CAPRINE LUTEAL TISSUES BY PROSTAGLANDINS E2 AND F2a B. A. Akinlosotu and G. Guraya Department of Physiology and Pharmacology School of Veterinary Medicine Tuskegee University Tuskegee, AL 36088
Received for publication: Accepted:
December
May 5,
3, 1992 1992
ABSTRACT Luteal slices obtained from Day-10 cyclic, sexually mature, mixed-breed, superovulated goats were used to study the effects of prostaglandins E2 and Fna (PGE2 and PGF2a) on the release of progesterone. The goats were synchronized for estrus using a single intramuscular injection of 5 mg PGF2a given during the mid-luteal phase of the estrous cycle. Multiple follicular growth and superovulation were induced using a treatment regimen of follicle stimulating hormone (FSH) and luteinizing hormone releasing hormone (LHRH) previously standardized in our laboratory (1). The luteal slices were treated with PGE2 or PGF2a at concentrations of 1 and 10 ng/ml each. Untreated luteal slices continued to release significant amounts of progesterone over the entire period of incub’ation (30 to 360 minutes). There was a progressive increase in progesterone accumulation following treatment with PGE2 at both concentrations. The mean progesterone values were significantly higher in the PGEZ-treated groups at all incubation periods than in the controls. Progesterone values at 10 ng/ml were higher (PcO.05) than at 1 ng/ml. Treatment with PGF2a decreased (PcO.05) progesterone release at 60 to 360 minutes of incubation compared with tliat of the corresponding controls for each incubation period. However, there appeared to be no differences (P>O.O5) in mean progesterone values between the two concentrations of PGFza. The results of this study showed that PGEp enhanced the release of progesterone by caprine luteal tissues, whereas PGF2a inhibited its release. Key words:
prostaglandin luteum
E2, prostaglandin
Fza, progesterone, caprine, corpus
Acknowledgments Supported by NIH / MBRS (# S06-GM08091) and NSF (# 8603832) Grants. This manuscript is dedicated to the late Dr. 0. P. Verma (l/5/31 - 2/29/88), the authors’ mentor.
Copyright 0 1992 Butterworth-Heinemann
64
Theriogenology
INTRODUCTION Physiological roles for prostaglandins E2 and Fna (PGE2 and PGF2a) in the regulation of luteal functions were suggested because specific binding sites for both prostaglandins are present on luteal cell membranes (2,3). Studies have shown that PGF2a plays a luteolytic role (4,5), while PGEz plays a luteotropic role (6,7) in corpus luteum (CL) functions. Prostaglandin E2 stimulated progesterone secretion by the CL, and prevented PGFza-induced luteal regression (7-10). Extensive evidence exists that PGF2a is responsible for functional luteal regression and, consequently, the decline in luteal progesterone production in several species (4,ll); however, studies of the in vitro response of the CL to PGF2a treatments in progesterone production have generated inconsistent results among species. Contrary to the results obtained in vivo, whereby PGF2a treatment caused sharp declines in blood progesterone levels, in vitro treatment of bovine CL with PGFsa either had no effect (12) or enhanced (13,14) progesterone secretions. Similar studies with the ovine CL have shown that PGF2a either inhibited (8,15) or had no effect (16) on the secretion of progesterone. Yet another study, however, reported that PGF2a had no effect on basal progesterone secretion but inhibited LH-stimulated progesterone secretion by the ovine CL (17). Because of the apparent differences among species in the actions of prostaglandins on luteal progesterone production, and the paucity of information available regarding the regulation of luteal function in the goat, luteal tissues obtained from superovulated goats were used to investigate the effects of PGE2 and PGFza in the regulation of luteal progesterone release.
MATERIALS AND METHODS Mixed-breed, mature goats that had exhibited at least 2 consecutive estrous cycles of 19 to 22 days in length were used for this study. The study was conducted between late August and October when goats are sexually active. All the goats were synchronized for estrus using a single intramuscular injection of 5 mg PGF2a (Lutalyse, dinoprost tromethamine, Upjohn Company, Kalamazoo, Ml) given during the mid-luteal phase of the estrous cycle. The day of onset of estrus was designated as Day 0 of the treatment estrous cycle. Multiple follicular growth and superovulation were induced using a treatment regimen of FSH and LHRH (1). On Day 10 of the estrous cycle, CL-bearing ovaries were removed through mid-ventral abdominal laparotomies. Immediately after ovariectomy, the CL were carefully dissected out of the ovaries and were trimmed free of loose connective tissue. All CL were washed twice with Hanks’ balance salt solution (HBSS; Gibco, Grand Island NY) and were sliced to a thickness of about 2 mm. The luteal slices (approx. 100 mg each) were pooled, randomly
Theriogenology
65
assigned to 1 of 5 treatment groups, and transferred to individual wells in 6-well culture plates containing 2.95 ml of medium 199 (Sigma Chemical Company, St Louis, MO). The medium contained 20 mM Hepes buffered medium, pH 7.4, with ‘I 00 ug streptomycin sulfate/ml, 100 units of Penicillin G /ml and 0.5% fetal bovirle serum. The culture plates containing luteal slices were placed in a CO:! incubator (5% CO2 in air) at 37°C and 95% humidity. During the first 2 hours of incubation, no treatment was given, thereby allowing the tissues to recover from trauma due to handling. In the control group (Group I) only the vehicle [50 ul Ethyl alcohol (ETOH)] was added. Luteal tissues in Groups II and III were treated with PGE2 at concentrations of 1 and 10 ng/ml, respectively. Tissues in Groups IV and V received PGF2a at concentrations of 1 and 10 ng/ml, respectively. Prostaglandins E2 and F2a (in 50 ul ETOH) were added to the appropriate treatment groups to obtain the respective concentrations. Separate luteal slices were used for each incubation period to avoid residual effects of treatments from the preceding incubation time. Following the various treatments, the tissue slices were incubated for 30, 60, 90, 120, 180 and 360 minutes. After each incubation period, media were pipetted from each well and centrifuged at 3000 xg, for 10 minutes to remove cellular debris. The supernai;ants were aspirated and immediately stored at -20°C until being assayed for progesterone. Progesterone concentrations in the media samples were determined using a specific solid phase 1251 radioimmunoassay kit obtained from Diagnostic Products Corporation (DPC, Los Angeles, CA). Each sample, the controls, and the standards were assayed in duplicate. The standard curve was prep#ared with 6 different concentrations, ranging from 0.1 to 40 ng/ml. The intro and inter-assay coefficients of variation were 4.56 and 10.8%, respectively, with an assay sensitivity of 0.05 ng/ml. The experimental design used in this study was a randomized complete block design with 6 replications (18). Media progesterone concentrations were subjected to analysis of variance. Differences in mean progesterone concentrations among treatment groups at the different sampling periods were compared using least squares linear contrasts (19). Probability values of less than 0.05 were considered significant.
RESULTS The mean progesterone release from luteal slices for the control and for PGE12treatments at different incubation periods are shown in Table 1. The untreated luteal slices (controls) continued to release significant amounts of progesterone over the entire incubation period.
66
Theriogenolog
y
All the tissue slices but the 60- and go-minutes incubations for the control samples and 1 ng/ml of PGE2 in the treated samples showed progressive increase (PO.O5) from those at 90 minutes. The mean values ranged from 21.92 f 4.52 ngiml at 30 minutes of incubation for the control to a high of 117.63 + 12.33 at 360 minutes for the 10 ng/ml PGE2.
Table 1. Mean progesterone release (ng/ml media) from caprine luteal tissues at various incubation periods after prostaglandin E2 treatments Treatment Groups
Incubation period (minutes)
Control
Prostaglandin (1 ngiml)
30 60 90 120 180
21.92 32.30 32.33 48.22 58.29
25.57 46.28 48.92 58.83 71.96
360
81.14 f 10.33ash
2 4.52a,d f 6.07a,e f 5 6gare f 9.91a ,f f. 11 .03a ,9
2 f f & k
E2
5.85bsd 6.35bre 5.37b+ 7.73b.f 10.76b 59
95.60 f 15.00bzh
Prostaglandin (10 ng/ml)
29.77 50.05 61.66 70.14 82.46
f f f f f
117.63;tl
E2
6.51 b,d 5.96b,e 6.09C,f 8.05czg t2.76c,” 2.33csi
Each value is a mean (;t SEM) of 6 observations. a*b*cMeans within the same row with different superscripts differ (P< 0.05). dVe.fj9.hSiMeans within the same column with different superscripts differ (Px 0.05)
Progesterone concentrations for the PGEZ-treated groups were higher (PcO.05) than for the controls at all incubation periods. While mean progesterone concentrations at 30 and 60 minutes of incubation were not different (P>O.O5) between the 1 and 10 ng/ml PGE2 treatments, they were higher at 60to 360-minute incubations for the 10 ng/ml treatment. Table 2 shows the mean progesterone release for the control and for tissues treated with PGF2a (1 and IO ng/ml) at different incubation periods. There was no significant difference in mean progesterone values obtained at 30 minutes of incubation among the 3 treatment groups. Treatment with PGFsa significantly decreased progesterone release at all incubation periods from 60 through 360 minutes of incubation, compared with the corresponding controls. Except for the 90- and 120. minute incubations which showed lower progesterone concentrations for 10 ngiml than 1 nglml of PGFza, the 2 concentrations of PGF2a showed no difference (P>O.O5) in mean progesterone values.
67
Theriogenology
There was no difference (P>O.O5) in mean progesterone accumulation in media during the first 90 minutes of incubation following treatment with either 1 or 10 ng/ml PGF2a. The mean progesterone values increased at 120 to 360 minutes of incubation for both PGF2a treated groups, but with no significant difference between values at 120 and 160 minutes for the 1 ng/ml treatment. Although higher concentrations of progesterone were obtained over time at 120, 160 and 360 minutes of incubation following PGFsa treatments, these values were significantly lower than those of the corresponding controls.
Table 2.
Mean progesterone release (ng/ml media) from caprine luteal tissues at various incubation periods after prostaglandin Fza treatments Treatment Groups
Prostaglandin Fp,
Incubation period (minutes)
Control
30 60 90 120 180 360
21.92 32.30 32.33 48.22 58.29 81.14
f f f f f f
(1 Wmt)
4.52asd 6.07ave 5.6gase 9.91asf 11 .03a,9 10.33aph
22.19 21.96 23.23 32.31 35.67 52.21
f k f f 2 f
5.08a,d 5.17b,o 4.48b,d 7.82bne 8.58bne 7.8gbsf
Prostaglandin (10 ng/ml)
20.24 19.00 19.38 29.48 34.21 48.36
F2a
f 4.76 a,d f 4.57h,d f 4.08crd f 7.5oo+ f 9.60bsf h8.8Sbp9
Each value is a mean (i SEM) of 6 observations. a,b,cMeans within the same row with different superscripts differ (PC 0.05). d@,f,g,hMeans within the same column with different superscripts differ (PC 0.05).
DISCUSSION This study reports for the first time the effects of PGE2 and PGF2a on in vitro progesterone release by caprine luteal tissue. Untreated, cultured luteal slices obtained from Day 10 cyclic, superovulated goats continued to release progesterone throughout the entire incubation period (Tables 1 and 2). This observation agrees with those from previous studies in the sheep, which showed that ovine luteal tissues produced a time-dependent increase in the accumulation of progesterone in media (15,17). The results also indicate that treatment of caprine luteal tissues with PGE2 (1 and 10 ng/ml) stimulated progesterone release after 30 to 360 minutes of incubation, compared with that of the corresponding controls. The lo-ng/ml treatment also resulted in higher progesterone release at 90 to 360 minutes of incubation than treatment with 1 ng/ml. These findings are consistent with previous observations in sheep in which PGE2 (0.1 to 10 ng/ml) enhanced luteal progesterone secretion in vitro. (8). These findings also agree with the results
68
Theriogenology
of studies in the human (7) and monkey (10) in which PGE2 stimulated progesterone production by the CL Compared with the corresponding controls for each incubation period, treatment with PGFsa at 1 and 10 ng/ml inhibited the release of progesterone by the luteal slice samples at 60 to 360 minute of incubation. With the exception of the 90- and 120- minutes incubations in which 10 ng/ml of PGF2a caused lower (PcO.05) progesterone release, there were no significant differences in progesterone values between the 2 PGFna treatments. These findings are similar to the results of other studies in the sheep and cattle, in which PGF2a treatment inhibited in vitro progesterone release by the CL (2,8,15,20,21). The significantly higher concentrations of progesterone obtained over time from 120 to 360 minutes of incubation following PGF2a (1 and 10 ng/ml) treatments could be due to the accumulation of progesterone in the media over time. In contrast to our findings, varying results have been reported from studies in sheep and cattle concerning the actions of PGF2a on in vitro luteal progesterone production. Treatment with PGF2a had no effect on basal in vitro progesterone production, but it was able to inhibit LH-induced increase in progesterone by the ovine and bovine CL (2,17,20, 22,23). While some studies found no effect of PGF2a on basal or LH-stimulated progesterone production by the ovine or bovine CL (14,15), PGF2a has been shown to increase progesterone release by the bovine CL (13). It has been suggested that the variation in the in vitro luteal responses to PGF2a treatment among species may be due to the variations in the relative number of large and small luteal cells at particular stages of the reproductive cycle (24). While PGF2a is found at high levels in early and late luteal phases, its expected luteolytic effects appear only during mid- and late luteal phases (25). The receptors for PGF2a are found exclusively on large luteal cells (26). Studies with ovine CL have shown that there is 1 large cell for every 5 small luteal cells during the mid-luteal phase (27), but large cells produce most of the basal luteal progesterone (28). Results of this study showed that PGEs enhanced the release of progesterone by caprine luteal tissues, whereas PGF2a inhibited its release. The significant increase in progesterone release following treatment with PGE2 may be a result of steroidogenic, luteotropic or antiluteolytic effects of PGF2a on CL (2, 7-9). Prostaglandin Fsa may be exert its luteolytic or antisteroidogenic effects through direct action on large luteal cells; therefore, the coordination of PGE;! and PGF2a production as well as luteal responsiveness to these prostaglandins may be the key factor in the regulation of luteal progesterone Droduction.
69
Theriogenology
REFERENCES 1.
Akinlosotu, B. A. and Verma, O.P. LHRH induced superovulation in FSHtreated anestrous goats. Proc. 68th Annual Meeting CRWAD Ii8:19 abstr (1987).
2.
Alila, H.W., Corradino, R.A., and Hansel, W. A comparison of the effects of cycle-oxygenase prostanoids on progesterone production by small and large bovine luteal cells. Prostaglandins X:259-270 (1988a).
3.
McArdle, C.A.and Holtorf, A.P. Oxytocin and progesterone release from bovine corpus luteal cells in culture: effects of insulin-like growth factor 1, insulin and prostaglandins. Endocrinology j&l 278-l 286 (1989).
4.
Horton, E.W. and Poyser, N.L. Uterine Luteolytic hormone: a physiologic role for prostaglandin F2a. Physiol. Rev. %:595-651 (1976).
5.
Cooke, R.G. and Homeida, A.M. Delayed luteolysis and suppression of the pulsatile release of oxytocin after indomethacin treatment in the goat. Res. Vet. Sci. X:48-51 (1984).
6.
Akinlosotu, B.A., Diehl, J.R. and Gimenez, T. Sparing effect of intrauterine treatment with prostaglandin E2 on luteal function in cycling gilts. Prostaglandins =:291-299 (1986).
7.
Zelinski-Wooten, M.B., Sargent, E.L., Molskness, T.A. and Stouffer, R.L. Disparate effects of the prostaglandin synthesis inhibitors, meclofenamate and flurbiprofen on monkey luteal tissue in vitro. Endocrinology 126:1380-l 387 ( 1990).
a.
Fitz, T.A., Mock, E.J., Mayan, M.H. and Niswender, G.D. Interactions of prostaglandins with subpopulations of ovine luteal cells. II. Inhibitory effects of PGF2a and protection by PGEz. Prostaglandins 3&l 27-l 38 (1984).
9.
Ford, S. P. and Christenson, L. K. Role of Prostaglandin E2 in protecting individual pig CL from prostaglandin F2a. Biol. Reprod. &(Suppl. 1): p.76 abstr.(l990).
10.
Hahlin, M., Lindblom, B., Schuurs, A., Kloosterboer, H. and Hamberger, L. Characterization of a monoclonal antibody which inhibits the biological activity of human chorionic gonadotropin in human corpora lutea. Human Reprod. A:1 52-l 57 (1989).
11.
Knickerbocker, J.J., Wiltbank, M.C. and Niswender G.D. Mechanisms of luteolysis in domestic livestock. Domest. Anim. Endocrinol. 5:91-l 07 (1988).
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12.
Litch, S.J. and Condon, W.A. interaction of hCG and lutalyse on steroidogenesis of bovine luteal cells. Mol. Cell Endocrinol. Z:81-85 (1988).
13.
Weston, P.G. and Hixon, J.E. Effects of In vitro prostaglandin Fza administration on in vitro progesterone synthesis by bovine corpora lutea. Biol. Reprod. 22:259-268 (1980).
14.
Hoedemaker, M., Grunert, K., Maas, D.H.A. and Grunert, E. Influence of luteinizing hormone and prostaglandin F2a on progesterone secretion in superfused bovine luteal tissue slices. Acta Endocrinol. m:405-412 (1987).
15.
Conley, A.J. and Ford, S.P. Effects of TPA, A231 87, and prostaglandin Fna on progesterone synthesis by dispersed ovine luteal cells. Biol. Reprod. a:1 224-l 230 (1989).
16.
Evrard-Herourard, M., de La Llosa-Hermier, M.P., P., de La Llosa, P. and Hermier, C. LH receptors relation to various physiological states and effects induced steroidogenesis in vitro. J. Reprod. Fertil.
17.
Fletcher, P.W. and Niswender, G.D. Effect of PGF2a on progesterone secretion and adenylate cyclase activity in ovine luteal tissue. Prostaglandins Z:803-819 (1982).
18.
Mendenhall, W. (ed.) Introduction to Linear Models and the Design and Analysis of Experiments. Duxbury Press, Belmont, CA, 1968 pp. 62-82.
19.
Statistical Analysis System,Users Guide: Statistics. SAS Institute, Inc. Cary, N.C, (1987).
20.
Wiltbank, M.C., Diskin, M. G., Flores, J. A. and Niswender, G. D. Regulation of the corpus luteum by protein kinase C. II. Inhibition of lipoprotein-stimulated steroidogenesis by prostaglandin F2a. Biol. Reprod. 42:239-245 (1990).
21.
Pate, J. L. and Condon, W.A. Regulation of steroidogenesis and cholesterol synthesis by prostaglandin F2a and lipoproteins in bovine luteal cells. J. Reprod. Fertil. =:439-446 (1989).
22.
Eurand, M., Leaboulueux, P. and Hermier, C. Role of prostaglandin F2a in modulation of LH-stimulated steroidogenesis in vitro in different types of rat and ewe corpora lutea. Prostaglandins I_!$ 491-502 (1978).
23.
Alila, H. W., Dowd, J. P., Corradino, R.A., Harris, W.V. and Hansel, W. Control of progesterone production in small and large bovine luteal cells separated by flow cytometry. J. Reprod. Fertil. g:645-655 (1988b).
Martinet, J., Mauleon, in ovine corpora lutea in of PGF2a on LH fi:225-233 (1981).
Theriogenology
71
24.
Harrison, L. M., Kenny, N. and Niswender, G. D. Progesterone production, LH receptors, and oxytocin secretion by ovine luteal cell types on Days 6, 10 and 15 of the oestrous cycle and Day 25 of pregnancy J. Fieprod. Fertil. 29:539-548 (1987).
25.
Rogers, R. J., Mitchell, M.D. and Simpson, E. R. Secretion of progesterone and prostaglandins by cells of bovine corpora lutea from three stages of the luteal phase. J. Endocrinol. 118:121-l 26 (1988).
26.
Fitz, T. A., Mayan, M. H., Sawyer, H.R. and Niswender, G. D. Characterization of two steroidogenic cell types in the ovine corpus luteum. Biol. Reprod. Z:703-711 (1982).
27.
Rogers, R.J., O’Shea, J.D. and Bruce, N.W. Analysis of the cellular composition of corpus luteum. J. Anat. U:757-769 (1984).
28.
Niswender, G.D., Schwall, R.H., Fitz, T.A., Farin, C.E. and Sawyer, H.R. Regulation of luteal function in domestic ruminants: new concepts. Recent Prog. Horm. Res. a:1 01 -1 51 (1985).
38:63-71,
1992
REGULATION OF IN VITRO PROGESTERONE RELEASE FROM CAPRINE LUTEAL TISSUES BY PROSTAGLANDINS E2 AND F2a B. A. Akinlosotu and G. Guraya Department of Physiology and Pharmacology School of Veterinary Medicine Tuskegee University Tuskegee, AL 36088
Received for publication: Accepted:
December
May 5,
3, 1992 1992
ABSTRACT Luteal slices obtained from Day-10 cyclic, sexually mature, mixed-breed, superovulated goats were used to study the effects of prostaglandins E2 and Fna (PGE2 and PGF2a) on the release of progesterone. The goats were synchronized for estrus using a single intramuscular injection of 5 mg PGF2a given during the mid-luteal phase of the estrous cycle. Multiple follicular growth and superovulation were induced using a treatment regimen of follicle stimulating hormone (FSH) and luteinizing hormone releasing hormone (LHRH) previously standardized in our laboratory (1). The luteal slices were treated with PGE2 or PGF2a at concentrations of 1 and 10 ng/ml each. Untreated luteal slices continued to release significant amounts of progesterone over the entire period of incub’ation (30 to 360 minutes). There was a progressive increase in progesterone accumulation following treatment with PGE2 at both concentrations. The mean progesterone values were significantly higher in the PGEZ-treated groups at all incubation periods than in the controls. Progesterone values at 10 ng/ml were higher (PcO.05) than at 1 ng/ml. Treatment with PGF2a decreased (PcO.05) progesterone release at 60 to 360 minutes of incubation compared with tliat of the corresponding controls for each incubation period. However, there appeared to be no differences (P>O.O5) in mean progesterone values between the two concentrations of PGFza. The results of this study showed that PGEp enhanced the release of progesterone by caprine luteal tissues, whereas PGF2a inhibited its release. Key words:
prostaglandin luteum
E2, prostaglandin
Fza, progesterone, caprine, corpus
Acknowledgments Supported by NIH / MBRS (# S06-GM08091) and NSF (# 8603832) Grants. This manuscript is dedicated to the late Dr. 0. P. Verma (l/5/31 - 2/29/88), the authors’ mentor.
Copyright 0 1992 Butterworth-Heinemann
64
Theriogenology
INTRODUCTION Physiological roles for prostaglandins E2 and Fna (PGE2 and PGF2a) in the regulation of luteal functions were suggested because specific binding sites for both prostaglandins are present on luteal cell membranes (2,3). Studies have shown that PGF2a plays a luteolytic role (4,5), while PGEz plays a luteotropic role (6,7) in corpus luteum (CL) functions. Prostaglandin E2 stimulated progesterone secretion by the CL, and prevented PGFza-induced luteal regression (7-10). Extensive evidence exists that PGF2a is responsible for functional luteal regression and, consequently, the decline in luteal progesterone production in several species (4,ll); however, studies of the in vitro response of the CL to PGF2a treatments in progesterone production have generated inconsistent results among species. Contrary to the results obtained in vivo, whereby PGF2a treatment caused sharp declines in blood progesterone levels, in vitro treatment of bovine CL with PGFsa either had no effect (12) or enhanced (13,14) progesterone secretions. Similar studies with the ovine CL have shown that PGF2a either inhibited (8,15) or had no effect (16) on the secretion of progesterone. Yet another study, however, reported that PGF2a had no effect on basal progesterone secretion but inhibited LH-stimulated progesterone secretion by the ovine CL (17). Because of the apparent differences among species in the actions of prostaglandins on luteal progesterone production, and the paucity of information available regarding the regulation of luteal function in the goat, luteal tissues obtained from superovulated goats were used to investigate the effects of PGE2 and PGFza in the regulation of luteal progesterone release.
MATERIALS AND METHODS Mixed-breed, mature goats that had exhibited at least 2 consecutive estrous cycles of 19 to 22 days in length were used for this study. The study was conducted between late August and October when goats are sexually active. All the goats were synchronized for estrus using a single intramuscular injection of 5 mg PGF2a (Lutalyse, dinoprost tromethamine, Upjohn Company, Kalamazoo, Ml) given during the mid-luteal phase of the estrous cycle. The day of onset of estrus was designated as Day 0 of the treatment estrous cycle. Multiple follicular growth and superovulation were induced using a treatment regimen of FSH and LHRH (1). On Day 10 of the estrous cycle, CL-bearing ovaries were removed through mid-ventral abdominal laparotomies. Immediately after ovariectomy, the CL were carefully dissected out of the ovaries and were trimmed free of loose connective tissue. All CL were washed twice with Hanks’ balance salt solution (HBSS; Gibco, Grand Island NY) and were sliced to a thickness of about 2 mm. The luteal slices (approx. 100 mg each) were pooled, randomly
Theriogenology
65
assigned to 1 of 5 treatment groups, and transferred to individual wells in 6-well culture plates containing 2.95 ml of medium 199 (Sigma Chemical Company, St Louis, MO). The medium contained 20 mM Hepes buffered medium, pH 7.4, with ‘I 00 ug streptomycin sulfate/ml, 100 units of Penicillin G /ml and 0.5% fetal bovirle serum. The culture plates containing luteal slices were placed in a CO:! incubator (5% CO2 in air) at 37°C and 95% humidity. During the first 2 hours of incubation, no treatment was given, thereby allowing the tissues to recover from trauma due to handling. In the control group (Group I) only the vehicle [50 ul Ethyl alcohol (ETOH)] was added. Luteal tissues in Groups II and III were treated with PGE2 at concentrations of 1 and 10 ng/ml, respectively. Tissues in Groups IV and V received PGF2a at concentrations of 1 and 10 ng/ml, respectively. Prostaglandins E2 and F2a (in 50 ul ETOH) were added to the appropriate treatment groups to obtain the respective concentrations. Separate luteal slices were used for each incubation period to avoid residual effects of treatments from the preceding incubation time. Following the various treatments, the tissue slices were incubated for 30, 60, 90, 120, 180 and 360 minutes. After each incubation period, media were pipetted from each well and centrifuged at 3000 xg, for 10 minutes to remove cellular debris. The supernai;ants were aspirated and immediately stored at -20°C until being assayed for progesterone. Progesterone concentrations in the media samples were determined using a specific solid phase 1251 radioimmunoassay kit obtained from Diagnostic Products Corporation (DPC, Los Angeles, CA). Each sample, the controls, and the standards were assayed in duplicate. The standard curve was prep#ared with 6 different concentrations, ranging from 0.1 to 40 ng/ml. The intro and inter-assay coefficients of variation were 4.56 and 10.8%, respectively, with an assay sensitivity of 0.05 ng/ml. The experimental design used in this study was a randomized complete block design with 6 replications (18). Media progesterone concentrations were subjected to analysis of variance. Differences in mean progesterone concentrations among treatment groups at the different sampling periods were compared using least squares linear contrasts (19). Probability values of less than 0.05 were considered significant.
RESULTS The mean progesterone release from luteal slices for the control and for PGE12treatments at different incubation periods are shown in Table 1. The untreated luteal slices (controls) continued to release significant amounts of progesterone over the entire incubation period.
66
Theriogenolog
y
All the tissue slices but the 60- and go-minutes incubations for the control samples and 1 ng/ml of PGE2 in the treated samples showed progressive increase (P
Table 1. Mean progesterone release (ng/ml media) from caprine luteal tissues at various incubation periods after prostaglandin E2 treatments Treatment Groups
Incubation period (minutes)
Control
Prostaglandin (1 ngiml)
30 60 90 120 180
21.92 32.30 32.33 48.22 58.29
25.57 46.28 48.92 58.83 71.96
360
81.14 f 10.33ash
2 4.52a,d f 6.07a,e f 5 6gare f 9.91a ,f f. 11 .03a ,9
2 f f & k
E2
5.85bsd 6.35bre 5.37b+ 7.73b.f 10.76b 59
95.60 f 15.00bzh
Prostaglandin (10 ng/ml)
29.77 50.05 61.66 70.14 82.46
f f f f f
117.63;tl
E2
6.51 b,d 5.96b,e 6.09C,f 8.05czg t2.76c,” 2.33csi
Each value is a mean (;t SEM) of 6 observations. a*b*cMeans within the same row with different superscripts differ (P< 0.05). dVe.fj9.hSiMeans within the same column with different superscripts differ (Px 0.05)
Progesterone concentrations for the PGEZ-treated groups were higher (PcO.05) than for the controls at all incubation periods. While mean progesterone concentrations at 30 and 60 minutes of incubation were not different (P>O.O5) between the 1 and 10 ng/ml PGE2 treatments, they were higher at 60to 360-minute incubations for the 10 ng/ml treatment. Table 2 shows the mean progesterone release for the control and for tissues treated with PGF2a (1 and IO ng/ml) at different incubation periods. There was no significant difference in mean progesterone values obtained at 30 minutes of incubation among the 3 treatment groups. Treatment with PGFsa significantly decreased progesterone release at all incubation periods from 60 through 360 minutes of incubation, compared with the corresponding controls. Except for the 90- and 120. minute incubations which showed lower progesterone concentrations for 10 ngiml than 1 nglml of PGFza, the 2 concentrations of PGF2a showed no difference (P>O.O5) in mean progesterone values.
67
Theriogenology
There was no difference (P>O.O5) in mean progesterone accumulation in media during the first 90 minutes of incubation following treatment with either 1 or 10 ng/ml PGF2a. The mean progesterone values increased at 120 to 360 minutes of incubation for both PGF2a treated groups, but with no significant difference between values at 120 and 160 minutes for the 1 ng/ml treatment. Although higher concentrations of progesterone were obtained over time at 120, 160 and 360 minutes of incubation following PGFsa treatments, these values were significantly lower than those of the corresponding controls.
Table 2.
Mean progesterone release (ng/ml media) from caprine luteal tissues at various incubation periods after prostaglandin Fza treatments Treatment Groups
Prostaglandin Fp,
Incubation period (minutes)
Control
30 60 90 120 180 360
21.92 32.30 32.33 48.22 58.29 81.14
f f f f f f
(1 Wmt)
4.52asd 6.07ave 5.6gase 9.91asf 11 .03a,9 10.33aph
22.19 21.96 23.23 32.31 35.67 52.21
f k f f 2 f
5.08a,d 5.17b,o 4.48b,d 7.82bne 8.58bne 7.8gbsf
Prostaglandin (10 ng/ml)
20.24 19.00 19.38 29.48 34.21 48.36
F2a
f 4.76 a,d f 4.57h,d f 4.08crd f 7.5oo+ f 9.60bsf h8.8Sbp9
Each value is a mean (i SEM) of 6 observations. a,b,cMeans within the same row with different superscripts differ (PC 0.05). d@,f,g,hMeans within the same column with different superscripts differ (PC 0.05).
DISCUSSION This study reports for the first time the effects of PGE2 and PGF2a on in vitro progesterone release by caprine luteal tissue. Untreated, cultured luteal slices obtained from Day 10 cyclic, superovulated goats continued to release progesterone throughout the entire incubation period (Tables 1 and 2). This observation agrees with those from previous studies in the sheep, which showed that ovine luteal tissues produced a time-dependent increase in the accumulation of progesterone in media (15,17). The results also indicate that treatment of caprine luteal tissues with PGE2 (1 and 10 ng/ml) stimulated progesterone release after 30 to 360 minutes of incubation, compared with that of the corresponding controls. The lo-ng/ml treatment also resulted in higher progesterone release at 90 to 360 minutes of incubation than treatment with 1 ng/ml. These findings are consistent with previous observations in sheep in which PGE2 (0.1 to 10 ng/ml) enhanced luteal progesterone secretion in vitro. (8). These findings also agree with the results
68
Theriogenology
of studies in the human (7) and monkey (10) in which PGE2 stimulated progesterone production by the CL Compared with the corresponding controls for each incubation period, treatment with PGFsa at 1 and 10 ng/ml inhibited the release of progesterone by the luteal slice samples at 60 to 360 minute of incubation. With the exception of the 90- and 120- minutes incubations in which 10 ng/ml of PGF2a caused lower (PcO.05) progesterone release, there were no significant differences in progesterone values between the 2 PGFna treatments. These findings are similar to the results of other studies in the sheep and cattle, in which PGF2a treatment inhibited in vitro progesterone release by the CL (2,8,15,20,21). The significantly higher concentrations of progesterone obtained over time from 120 to 360 minutes of incubation following PGF2a (1 and 10 ng/ml) treatments could be due to the accumulation of progesterone in the media over time. In contrast to our findings, varying results have been reported from studies in sheep and cattle concerning the actions of PGF2a on in vitro luteal progesterone production. Treatment with PGF2a had no effect on basal in vitro progesterone production, but it was able to inhibit LH-induced increase in progesterone by the ovine and bovine CL (2,17,20, 22,23). While some studies found no effect of PGF2a on basal or LH-stimulated progesterone production by the ovine or bovine CL (14,15), PGF2a has been shown to increase progesterone release by the bovine CL (13). It has been suggested that the variation in the in vitro luteal responses to PGF2a treatment among species may be due to the variations in the relative number of large and small luteal cells at particular stages of the reproductive cycle (24). While PGF2a is found at high levels in early and late luteal phases, its expected luteolytic effects appear only during mid- and late luteal phases (25). The receptors for PGF2a are found exclusively on large luteal cells (26). Studies with ovine CL have shown that there is 1 large cell for every 5 small luteal cells during the mid-luteal phase (27), but large cells produce most of the basal luteal progesterone (28). Results of this study showed that PGEs enhanced the release of progesterone by caprine luteal tissues, whereas PGF2a inhibited its release. The significant increase in progesterone release following treatment with PGE2 may be a result of steroidogenic, luteotropic or antiluteolytic effects of PGF2a on CL (2, 7-9). Prostaglandin Fsa may be exert its luteolytic or antisteroidogenic effects through direct action on large luteal cells; therefore, the coordination of PGE;! and PGF2a production as well as luteal responsiveness to these prostaglandins may be the key factor in the regulation of luteal progesterone Droduction.
69
Theriogenology
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