Interleukin-la Increases Thecal Progesterone Production of Preovulatory Follicles in Cyclic Hamsters

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1 BIOLOGY OF REPRODUCTION 43, (199) EXCEPTIONAL PAPER Interleukin-la Increases Thecal Progesterone Production of Preovulatory Follicles in Cyclic Hamsters Y. NAKAMURA1 2 H. KATO,2 and P. F. TERRANOVA3 Department of Obstetrics and Gynecology2 Yamaguchi University School of Medicine, Yamaguchi, Japan Department of P/ysiology3 University of Kansas Medical Center, Kansas City, Kansas 6613 ABSTRACT Adult cyclic hamsters were used to study the effects of interleukin-la (IL-la) on in vitro steroidogenesis in preovulatory follicles. IL-la increased progesterone secretion by preovulatory fofficles during a 24-h incubation in RPMI-l64 medium containing hcg (1 mu/mi) (progesterone levels: 17.5 ± 2.2 vs. 1.6 ± 1.9 ng/fofficle/ml, p <.5). IL-lu alone bed no effect on follicular steroidogenesis. The source of increased progesterone secretion was the thecae (9.8 ± 1. vs. 5.8 ±.4 ng/2 thecae/ml, p <.1) and not the granulosa cells (6.6 ±.2 vs. 6.8 ±.5 ng/2 viable granulosa cells/mi). IL-la also stimulated production of testosterone in thecae of preovulatory follicles. The follicular progesterone increase was dependent on the time of incubation and dose of IL-la. IL-la at 5-5 U/mi maximally stimulated progesterone production in the preovulatory follicles, and no significant effect of IL-lu was observed until the 12th hour of incubation. The effects of IL-la on in vitro steroidogenesis in preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutes were examined. IL-lu in the presence of hcg also significantly increased progesterone secretion by atretic preovulatory follicles. In the incubation of preantral follicles or newly formed corpora lute however, IL-la did not alter steroidogenesis. These results indicate that IL-Ia stimulates progesterone secretion by preovulatory follicles and that the target tissue for this effect is the thecal layer. Moreover, the effect of IL-lu apparently depends on the stage of follicular development since preantral fofficles do not respond to IL-Ia. INTRODUCTION The existence of macrophages in the ovary has been demonstrated in rats (Bulmer, 1964), mice (Kirsh et xi, 1981), and humans (Katabuchi et al., 1988). Furthermore, it has been shown that macrophages increase progesterone production from cultured granulosa-luteal cells in mice (Kirsh et xi., 1981, 1983) and humans (Halme et al., 1985). Thus, the macrophages appear to play an important role in the control of ovarian function. Macrophages produce a variety of substances, among which interleukin-1 (IL-i) has been most extensively investigated. It is well known that IL-i plays an important role in immune-network (Dinarello and Mier, 1987) and also has various physiological effects including endogenous pyrogen activity, prostaglandin (PG) E2 induction (Baracos et xi., 1983), initiation of the acute phase response, and stimulation of collagenase (Dinarello, 1984). Others have shown that ILla inhibits LH-induced luteinization of porcine granulosa cells in culture and inhibits basal progesterone production without LH in cultured porcine granulosa cells (Fukuoka et xi., 1988, 1989). Gottschall et al. (1987) also reported that IL-I f3 inhibits FSH-induced LH receptor formation and reduces progesterone secretion in cultured rat granulosa cells. Accepted March 21, 199. Received December 18, Reprint requests: Dr. Y. Nakamur Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, 1144 Kogushi Ube, 755 Japan. These reports revealed direct effects of IL-i on cultured granulosa cells. Recently, Rivier and Vale (1989) reported that, in vivo, IL-Ia has inhibitory effects on the reproductive function at the level of brain and gonads. Also recently, Khan et xi. (1988) revealed high concentrations of IL-i-like factors in ovarian follicular fluid in humans. Thus, it is possible that IL-i plays a role in ovarian function. The present studies were designed to investigate whether IL-ia can alter in vitro steroidogenesis in preovulatory, preantral, and experimentally induced atretic follicles as well as in newly formed corpora lutea in hamsters. Animals and Materials MATERIALS AND METHODS Adult female golden hamsters weighing 8-11 g were maintained in a 23-25#{176}C room with a daily lighting schedule of 14 L:iO D (lights-on: 5 h). Food and water were available ad libitum. Hamsters were used in the experiments only after exhibiting 3 consecutive 4-day estrous cycles. Day i is the day of ovulation, defined by the characteristic vaginal discharge, and Day 4 corresponds to proestrus. RPMI-i64 medium was obtained from Flow Laboratories Inc. (McLean, VA). Recombinant human IL-la (i8 UI mg) was purchased from Genzyme Co. (Boston, MA). HCG was from Sigma Chemical Co. (St. Louis, MO). Falcon tissue culture plates (24 wells) were purchased from Nippon Becton Dickinson Co., Ltd. (Tokyo, Japan). on 25 July

2 i7 NAKAMURA ET AL. Follicle Preparation Cyclic hamsters were decapitiated under light ether anesthesia on Day 4 at 9 h. The ovaries were removed immediately, and the large preovulatory follicles were dissected and cleaned of adhering tissue in a watch glass containing RPMI-i64 medium. For thecae and granulosa cell incubations, preovulatory follicles were ruptured with fine forceps and granulosa cells were removed by gentle pressure. The ruptured follicles were then partially bisected in another dish containing fresh medium and agitated back and forth through a fire-polished medicine dropper. This procedure removed most of the adhering granulosa cells from the thecae after 3-4 repetitions and changes of medium (Terranova et al., i982). Cyclic hamsters were studied on Day i at 9 h, at which time the healthy preantral follicles and newly formed corpora lutea were dissected and used for experiments with the same procedures as described for preovulatory follicles. To obtain experimentally induced atretic preovulatory follicles, ovulation was blocked with phenobarbital for 2 days (Terranov 198). Cyclic hamsters received an injection of 6.5 mg/1 g B.W. phenobarbital s.c. at i3 h on proestrus and of i3 mg/ioo g B.W. phenobarbital s.c. at i2 h the next day. Hamsters were killed at 9 h on the following morning. Ovaries were removed and checked for blockage of ovulation and absence of corpora lutea. Large, delayed atretic follicles were then dissected and used for incubation as described for preovulatory follicles. In Vitro Incubations Each follicle or corpus luteum was preincubated in RPMIi64 medium (one follicle or corpus luteum/mi/well) at 37#{176}C for i h under 95% air: 5% CO2. After preincubation, the medium was exchanged for fresh medium containing IL-ia (.1-5 U/mI) and hcg (ioo miu/ml), and the incubation was continued for up to 24 h. At the end of the incubation, media were saved at - 2#{176}C for steroid assays (progesterone, testosterone, and estradiol). To check for time-dependent responses to IL-i 6 pa incubation medium was removed from each well at the third, sixth, and 12th hours of incubation and the remaining medium was collected at the 24th hour of incubation; all samples were saved at -2#{176}Cfor steroid assays. Thecae incubations were carried out with the same procedures as described for follicles except that two thecae/ ml/well were incubated. The granulosa cells were washed by centrifugation at 7 rpm for 5 mm at 4#{176}C. Fresh medium was added to the pellet of granulosa cells and the sample was preincubated I h. Cell viability was determined by trypan blue exclusion after i-h preincubation. Granulosa cells (2 viable cells) were incubated for 24 h in i ml RPMI-i64 medium containing io U IL-la and ioo mitj hcg. After incubation, samples were centrifuged and the supernatants were saved at - 2#{176}C for steroid assays. Each group consisted of four to six replicate incubations. Assay Progesterone, testosterone, and estradiol concentrations in media samples were determined by unextracted RIA (Terranova and Garz i983). Progesterone antiserum (GDN 337), testosterone antiserum (GDN 25), and estradiol antiserum (GDN 244) were provided by Dr. Gordon D. Niswender (Colorado State University, Fort Collins, CO). The lower limits of these assay sensitivities were 3 pg/tube for progesterone, 1.5 pg/tube for testosterone, and i.5 pg/tube for estradiol. Characterizations of these antisera were described previously (Korenman et a!., i974; Gibori et al., 1977; Gay and Kerlan, i978). Statistics The N in each Figure and Table represents the number of replicate incubations in each group. Data were analyzed by ANOVA and Duncan s Multiple Range Test; where appropriate, Student s t-test was used. Differences were considered significant if p <.5. Effect of IL-la on ogenesis in Preovulatory Follicles RESULTS IL-la (1 U/follicle/ml) alone did not show any effects on the productions of progesterone, testosterone, or estradiol in preovulatory follicles. HCG significantly increased production of these three hormones and the addition of ILia with hcg further increased progesterone production (i7.5 ± 2.2 vs. 1.6 ± 1.9 ng/ml;p <.5) (Table 1). The effect of IL-ia on progesterone secretion by preovulatory follicles was dependent on the time of incubation (Fig. 1). Only at the i 2th and 24th hours of incubation were progesterone levels in media from the hcg + IL-Ia group significantly higher than in the hcg-alone group. At the 24th hour of incubation, the level of estradiol was also significantly (p <.5) higher in the hcg + IL-ia group than in the hcgalone group. Addition of various doses of IL-la (.1-5 U/ follicle/ml) to incubation media in the presence of hcg (1 miu/ml) resulted in a dose-dependent increase of the progesterone levels in the medium (Fig. 2). The minimum effective dose of IL-ia was 5. U/mI (p <.5). To determine the source of progesterone induced by ILia in preovulatory follicles, the follicles were divided into thecae and granulosa cells and were incubated individually for 24 h. IL-ia significantly increased production of progesterone (9.8 ± 1. vs. 5.8 ±.4 ng/ml, p <.1) and testosterone (534 ± 76 vs. 257 ± 75 pg/ml,p <.5) from thecae, whereas IL-ia did not show any stimulatory effects on production of steroids in granulosa cells (Table 2). on 25 July 218

3 IL-la INCREASES THECAL PROGESTERONE PRODUCTION 171 TABLE 1. Effect of IL-la (1 U/follicle/mi) on progesterone, testosterone, and estradiol secretion by preovulatory follicles in the presence and absence of hcg (1 mlu/follicie/ml) during a 24-h incubation in vitro (N = 6; Mean ± SE). Witho ut hcg With hcg Control IL-la Control IL-la Progesterone (ng/ml) Testosterone (pg/mi) Estradiol (pg/mi) 1.4 ± ± ± ± 188 ± 57 ± ± ± ± ± ± ± 386 p <.5, compared with hcg alone. Effects of IL-i a on Progesterone Secretion by Preantral Follicles, Atretic Preovulatoty Follicles, and Newly Formed Corpora Lutea IL-ia (1 U/mi) in the presence of hcg (1 miu/ml) significantly increased progesterone secretion only in the atretic preovulatory follicles (p <.1) but not in preantral follicles or corpora lutea (Table 3). In the absence of hcg, IL-ia tended to stimulate progesterone production in the atretic preovulatory follicles but the level of stimulation was not statistically significant. DISCUSSION IL-ia in the presence of hcg significantly increased progesterone secretion in preovulatory follicles. There are several possible mechanism through which ILla increases progesterone production in preovulatory fo!- licles. IL-la might induce atresia of the preovulatory follicles during the 24-h culture period. Fukuoka et al. (1988) showed that IL-la inhibits the LH-induced morphological luteinization of cultured porcine granulosa cells, and, re- cently, Gottschail et al. (i989) presented evidence aromatase activity is suppressed by ll-l. Both studies indicate that IL-i may play an important role in steroidogenic changes associated with atresia. However, the mechanism by which IL-i would cause atresia is unknown. Experimentally induced atretic preovulatory follicles are much more sensitive to LH in the stimulation of progesterone secretion than preovulatory follicles and thecae are the source of the increased progesterone secretion (Terranova et a!., 1982), similar to IL-ia in the present study. The source of increased progesterone secretion by IL-ia appeared to be thecae. It is possible that IL-ia increases hcg receptors in thecae, which subsequently increases progesterone secretion, especially since IL-ia alone does not stimulate progesterone secretion. It is also known that IL-i stimulates PGE2 production in some tissues (Baracos et a!., 1983; Katsuura et xi., i989) and PGE2 is known to increase progesterone secretion by rat Graaflan follicles by increasing camp synthesis. This mechanism is different from the LH stimulation of steroidogenesis in the ovary (Lamprecht et a!., 1973). However, there are several, indirect, lines of evidence that E 15. Ot C C C,. 1. * P<O.5 ** p<o.ol VS 5CC alone dot tine : 5CC aba. solid line t,cg #{149} IL-I #{149}1 4-J 3 2 (ng/mi) PTE 11 I a w 1 C #{149} 5, Prog..t.rons El Estradlol * p<o.5 is po.o1 Vs ** #{149} Incubation Time (hrs.) FIG. 1. Effects of IL-la (1 U/follicle/mI) on progesterone, testosterone, and estradiol accumulation in the medium by preovulatory follicles at the third, sixth, 12th, and 24th hours of incubation in the presence of hcg (1 miu/follicle/ml). Each Mean ± SE represents the six follicle incubations from which the media were serially sampled (6 tl) at the third, sixth, and 12th hours of incubation. Data were analyzed by Duncan s Multiple Range Test IL-i a (U/mi) FIG. 2. Effects of various doses of IL-la (.1-5 U/follicle/mI) on progesterone, testosterone, and estradiol secretion by preovulatory follicles in the presence of hcg (1 mlu/follicle/mi) during a 24-h incubation. Each bar represents the Mean ± SE of five follicle incubations. Data were analyzed by Duncan s Multiple Range Test. on 25 July 218

4 172 NAKAMURA ET AL. TABLE 2. Effects of IL-la (1 U/mI) in the presence of hcg (1 mlu/ml) on progesterone, testosterone, and estradiol secretion in thecae and granulosa cells of preovulatory follicles during a 24-h incubation in vitro (Mean ± SE). 2 thecae/ml 2 viable granulosa cells/mi) Control IL-la Co ntrol IL-la (N = 5) (N = 6) (N = 4) (N = 4) Progesterone (ng/ml) Testosterone (pg/mi) Estradiol (pg/mi) p <.5, compared with hcg alone. #{149}p<.1, compared with hcg alone. tnd = not detectable (less than 75 pg/mi). 5.8 ± ± 1.** 6.8 ± ± ± ± 76* NDt ND 853 ± ± 3 ND ND IL-la may not stimulate progesterone production through PGE2. First of all, IL-ia alone did not increase progesterone production in preovulatory follicles. Second, in rat Graafian follicles, PGE2 significantly increases camp levels in incubation medium within 2 mm (Lamprecht et al., 1973). IL- 1 increases PGE2 levels in the culture medium of astrocytes within 3 h (Katsuura et al., i989) and in rat muscles within 2 h (Baracos et a!., 1983). In the present study, however, IL-ia did not increase progesterone production until the i 2th hour of incubation. The time course of action is too long for IL-ia to act through PGE2 and camp. A similar time lag in progesterone synthesis has been observed when rat follicles are stimulated by tumor necrosis factor-a in vitro (Roby and Terranov i988). IL-ia stimulated testosterone secretion in isolated thecae (Table 2) but not in whole preovulatory follicles. The reason for this difference may be that in the whole follicles testosterone is metabolized by the granulosa cells to another steroid such as estradiol. The conversion of testosterone to estradiol, however, would not be stimulated direedy by IL-la because IL-ia did not alter aromatase activity in hamster preovulatory follicles in the presence of hcg (Nakamur unpublished data). It is also interesting to note in the present study that the early preantral stages of follicular development (stages III and IV according to Greenwald, 1974) did not respond to hcg + IL-la whereas preovulatory follicles were quite responsive. The reasons for these differences are unknown. Possibly, the IL-ia receptor does not appear until the later stages of follicular development. On the other hand, maybe the LH/FSH surges on proestrus alter the response of the preantral follicles to IL-l since the newly formed corpora lutea also not respond to hcg + IL-ia but the preovulatory follicles from which they were derived 24 h earlier were quite responsive to hcg + IL-ia. Previous studies have shown that on the morning of estrus preantral follicles and newly formed corpora lutea are quite responsive to U-I in vitro (Terranova and Garz 1983). Fukuoka et a!. (1989) also reported the different responses of the granulosa cells to IL-i showing the inhibition of basal progesterone production in moderately differentiated cells, but no effect in granulosa cells already luteinized in vivo. It appears that the effect of IL-la is dependent on the stage of differentiation of follicles. The reason for the differences between the present study and Fukuoka et xi. (1989) might be due to our use of thecae and their use of granulosa cells. At this point, it would be pure speculation as to why IL-i would augment gonadotropmn-stimulated progesterone secretion in thecal cells and inhibit gonadotropin-stimulated progesterone secretion in granulosa cells. It is our belief that IL- 1 may be causal in atresia. During follicular atresi it is well known that thecal responsiveness to hcg increases and that granulosa cells undergo pyknosis; this is evidenced by increased 1251-hCG binding to thecae and increased progesterone secretion in thecae (Schwall and Erickson, i981; Terranova et al., 1982). The present study showed that ILla augmented the response of the thecae to hcg for progesterone secretion. Thus, if IL-i is released at the time of atresi it is possible that IL-i mediates some of these changes in steroidogenesis. In summary, this is the first study revealing an effect of IL-ia on thecae of the preovulatory follicles. IL-ia en- TABLE 3. Effect of IL-la (1 U/follicle or corpus luteum/mi) on progesterone secretion by preantral follicles, experimentally induced atretic preovulatory follicles, and newly formed corpora lutea in the presence and absence of hcg (1 mlu/ml) during a 24-h incubation in vitro (Mean ± SE). Without hcg With hcg Preantral follicles Atretic follicles Corpora lutes (N) Control IL-la (N) Control IL-la (6) 174 ± ± ± ± 677 ± 1.9 ± 23 (pg/mi) 176 (pg/mi).4 (ng/ml) 4.2 ± ± ± ±.5 (ng/mi) 37.8 ± 2.* (ng/ml) 5.5 ± 1.1 (ng/ml) p <.1, compared with hcg alone. on 25 July 218

5 IL-la INCREASES THECAL PROGESTERONE PRODUCTION i73 hanced the ability of hcg to stimulate thecal progesterone production in vitro. The effect of IL-la was limited to preovulatory follicles that were healthy or atretic; IL-la had no effect in preantral follicles or newly formed corpora lutea. REFERENCES Baracos V, Rodemann HP, Dinarello CA, Goldberg AL, Stimulation of muscle protein degradation and prostaglandin E2 release by leukocytic pyrogen (Interleukin-1). N EngI J Med 38: Bulmer D, The histochemistiy of ovarian macrophages in the rat J Anat (Lond) 98: Dinarello CA, lnterleukln-1. Rev Infect Dis 6:51-95 Dinarello CA, Mier JW, Current concepts; lymphokines. N Engi J Med 317: Fukuoka M, Mon T, Tan S. Yasuda K, lnterleukin-1 inhibits luteinization of porcine granulosa cells iii culture. Endocrinology 122: Fukuoka M, Yasuda K, Tail S, Takakura K, Mon T, Interleukin-1 stimulates growth and inhibits progesterone secretion in cultures of porcine granulosa cells. Endocrinology 124:884-9 Gay VL, Kerlan JF Serum UI and FSH following passive immunization against circulating testosterone in the intact male rat and orchidectomized rats bearing subcutaneous silastic implants of testosterone. Arch Androl 1: Gibori G, Anctzak E, Rothchild I, The role of estrogen in the regulation of luteal progesterone secretion in the rat after Day 12 of pregnancy. Endocrinology 1: Gooschall FE, Katsuura G, Arimura A, Interleukin-1 suppresses follicle-stimulating hormone-induced estradiol secretion from cultured ovarian granulosa cells. J Reprod Immunol 15:281-9 Gottschall FE, Uehara A, Hoffmann SI, Arimura A, Interleukin-1 inhibits follicle stimulating hormone-induced differentiation in rat granulosa cells in vitro. Biochem Biophys Res Commun 149:52-9 Greenwald GS, Quantitative aspects of follicular development in the untreated and PMS-treated cyclic hamster. AnaL Rec 178: Halme J, Hammond MG, Syrop CII, Talbert LM, Penitoneal macrophages modulate human granulosa-luteal cell progesterone production. J Clin Endocrinol Metab 61: Katabuchi H, Fukumatsu Y, Okamura H, Immunohistochemical and morphological observations of macrophages in the human ovary. \ Il Ovarian Workshop: Paracrine Communication In the Ovary: Ontogenesis and Growth Factors. Randolph, Mk Serono Symposia USA Press, pp Katsuura G, Goctschall FE, DahI RE, Arimura A, lnterleukin-1 beta increases prostaglandin E2 in rat astrocyte cultures: modulatory effect of neuropepudes. Endocrinology 124: Khan SA. Schmidt K, Hahn P, Di Pauli R, De Geyter Ch, Nieschlag E, Human testis cytosol and ovarian follicular fluid contain high amounts of interleukin- 1-like factor(s). Mol Cell Endocrinol 58:221-3 Kirsh list, Friedman AC, Vogel RI, Flickinger GL, Macrophages in corpora lutea of mice: characterization and effects on steroid secretion. Biol Reprod 25: Kirsh TM, Vogel RL, Flickinger Gl, Macrophages: a source of luteocropic cybemins. EndocrInology 113: Korenman 5(1, Stevens RH, Carpenter L*, Robb M, Niswender GD, Sherman BM, Estradiol radioimmunoassay without chromatography: procedure, validation and normal values. J Clin Endocninol Metab 38:718-2 Lamprecht SA, Zor U, Tsafriri A, Lindner BR, Action of prostaglandin in E2 and of luteinizing hormone on ovarian adenylate cyclase, protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat.) Endocninol 57: Rivier C, Vale W, In the rat, Interleukin-1 a acts at the level of the brain and the gonads to interfere with gonadotropin and sex steroid secretion. Endocrinology 124:215-9 Roby KF, Terranova PF, Tumor necrosis factor alpha alters follicular steroidogenesis in vitro. Endocrinology 123: Schwail 8, Erickson G, Functional and morphological changes in rat theca cells during atresia. In: Schwartz NB, Hunzicker-Dunn M (eds.), Dynamics of Ovarian Function. New York: Raven Press, pp Terranova PF, 198. Effects of phenobarbital-induced ovulatory delay on the follicular population and serum levels of steroids and gonadotropins in the hamster: a model for atresla. Biol Reprod 23:92-99 Terranova PF, Garza F, Relationship between the preovulatoty lutelnizing hormone (LII) surge and androstenedione systhesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LII. Biol Reprod 29:63-36 Terranova PF, Martin NC, Chien 5, Theca is the source of progesterone in experimentally induced atretic follicles of the hamater. Biol Reprod 26: on 25 July 218