Anna Maria Fulghesu, M.D. Alessandro Caruso, M.D. Salvatore Mancuso, M.D.*

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1 FERTILITY AND STERILITY Copyright e 1992 The American Fertility Society Vol. 57, No.1, January 1992 Printed on acid-free paper in U.S.A. Human growth hormone enhances progesterone production by human luteal cells in vitro: evidence of a synergistic effect with human chorionic gonadotropin Antonio Lanzone, M.D. Nicoletta DiSimone, M.D. Roberta Castellani Anna Maria Fulghesu, M.D. Alessandro Caruso, M.D. Salvatore Mancuso, M.D.* Department of Obstetrics and Gynecology, Catholic University, Rome, Italy Objective: To examine the possible direct effect of human growth hormone (hgh) on basal and human chorionic gonadodotropin (hcg )-stimulated progesterone (P) production by cultured human luteal cells. Design: Cultures of human luteal cells from early and midluteal phase. Setting: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Catholic University, a public care center. Patients, Participants: Twelve nonpregnant women between 35 and 4 7 years of age underwent surgery for various nonendocrine disorders such as leiomyomatosis. Interventions: Corpora lutea were obtained at the time of hysterectomy. Main Outcome Measure: Luteal cells were incubated with or without hcg and/or hgh at different concentrations. Results: Human growth hormone neither at 250 nor at 500 ng/ml increased basal P production, whereas from 1,000 ng/ml P concentration in media was significantly increased (P < 0.05). The concomitant treatment with uneffective doses of hcg (6 and 12 ng/ml) and hgh (250 and 500 ng/ml) enhanced P production similarly to that obtained with the highest doses of hgh (1,000 ng/ml or more) or hcg (25 to 50 ng/ml) alone. Conclusion: These results indicate a direct effect of hgh on the luteal steroidogenesis in vitro. Fertil Steril 1992;57:92-6 It is well known that isolated growth hormone ( G H) deficiency causes delayed puberty in children; furthermore, treatment of these patients with human growth hormone (hgh) induces sexual maturation.1 2 Kulin et al.2 also reported that isolated GHdeficient children showed a deteriorated response in terms of testosterone production to gonadotropin stimulation test and that hgh treatment improved such response in these subjects. These clinical observations suggest that hgh may have a modulatory effect on gonadal development. Received March 11, 1991; revised and accepted August 20, 199L * Reprint requests: Salvatore Mancuso, M.D., Department of Obstetrics and Gynecology, Universita' Cattolica del S. Cuore, Largo Agostino, Gemelli 8, Rome, Italy. 92 Lanzone et al. P production by hgh from luteal cells Jia et al.3 demonstrated that GH is involved in the differentiation of ovarian granulosa cells by augmenting follicle-stimulating hormone (FSH) stimulated luteinizing hormone (LH) receptor formation and by increasing progestin responsiveness to gonadotropins. In another study, 4 it was found that a decrease in the G H levels is associated with a reduction of ovarian LH receptor content and human chorionic gonadotropin (hcg)-stimulated progesterone (P) production. Growth hormone replacement leads to a normalization of ovarian responsiveness. Furthermore, Homburg et al. 5 demonstrated in vivo that hgh augments the response of human ovary to stimulation by gonadotropins. In this study, we have investigated the effect of hgh on the steroidogenesis by cultured human luteal cells.

2 MATERIALS AND METHODS Corpora lutea (CL) were obtained at the time of hysterectomy performed for nonendocrine gynecological diseases (leiomyomatosis), in the early midluteal phase of the menstrual cycle (days 3 to 6 from ovulation). Patients were between 35 and 47 years of age. All of them had a story of regular menstrual cycles. A total of 12 experiments were performed. Five CL were used for parallel portions of the study. Informed consent was obtained from each patient. The study obtained the approval from the internal review board. The age of the CL was determined as follows. All patients were monitored until ovulation by daily measurements of basal body temperature (BBT) and ultrasound (US) examination of the follicular growth. When the maximal follicular diameter had reached 18 mm, daily determinations of plasma P values were made. The time of ovulation (day 0) was detected by the biphasic pattern of BBT, by the typical US disappearance of the dominant follicle or the echographic detection of CL, and by the rise of plasma P levels. At the time of surgery, plasma samples were collected immediately before the anesthesia. The removed luteal tissue was immediately freed from blood vessels and ovarian stroma under a dissecting microscope, dissected, and minced. Human CL cultures were established as previously described.6 7 The luteal tissue was placed in Ham's F-12 medium (Flow Laboratories, Milan, Italy) containing 10 mm Hepes buffer and 5 mg/ml of collagenase, then incubated at 37 C in a shaking water bath for 2 hours. The cell suspension was filtered, centrifuged, and then resuspended in fresh medium. Cells were counted, and the viability was determined by the Tripan Blue test. The cells were diluted to a final concentration of 80,000 to,000 live cells/ml of medium supplemented with 2 mm L-glutamine, IU penicillin, /.Lg streptomycin with and without 10% fetal bovine serum, and cultured in multiwell plates (Flow Laboratories, Irvine, United Kingdom). After 24 hours, the cells attached to the wells; at this time the medium was removed and fresh serum-free medium supplemented with 5 ij.g/ml insulin, 5 ij.g/ml transferrin, and 40 ng/ml hydrocortisone was added to the cultures. Cells were cultured for 24 hours in 5% C02, 95% air at 37 C in the presence and absence ofhcg (from 6 ng/ml to ng/ml), with or without hgh at different concentrations (from 250 ng/ml to 4,000 ng/ml), or for up to 5 days with or without hcg (50 ng/ml) or hgh (2,000 ng/ml). Human chorionic gonadotropin and hgh were obtained from Serono, Rome, Italy. To investigate a possible effect of hg H or hcg on cellular growth, at the end of the incubation time, the cells were counted again by a trypsin treatment (0.25%). Culture media were changed every day and stored at -20 C until assay for P. Commercial radioimmunoassay kits were used (Radim, Rome, Italy). The intra-assay and interassay coefficients of variation were 4% and 10%, respectively. Statistical analyses were performed by using ANOV A for multiple comparisons and paired Student's t-test for comparison within groups. RESULTS Progesterone levels before the anesthesia were 7.0 ± 2.2 ng/ml. First of all we investigated the effect of different doses of hcg or hgh on P production by human luteal cells cultured for 24 hours. The addition of hcg significantly increased P production by twofold as compared with the controis; the stimulation was dose-dependent starting at 25 ng/ml and reaching its maximum at 50 ng/ml (Fig. 1). The dose-response curve of hgh is also shown in Figure 1. Human growth hormone did not significantly increase basal P production neither at 250 nor at 500 ng/ml, whereas starting from 1,000 ng/ml P production was significantly increased (P < 0.05). The percent increase was 168%, 196%, and 209% at 1,000, 2,000 and 4,000 ng/ml hgh, respectively. To evaluate the possible synergistic effect of hgh and hcg on steroidogenesis, human luteal cells were cultured for 24 hours with or without increasing concentrations of hgh (250 to 1,000 ng/ml) in the presence or absence of hcg (6, 12, and 25 ng/ml) (Table 1). The concomitant treatment with the uneffective doses of hgh (250 and 500 ng/ml) and hcg (6 and 12 ng/ml) enhanced P production, and the levels were similar to those obtained with the highest doses ofhgh (1,000 ng/ml or more) or hcg (25 to 50 ng/ml) alone. We also wanted to investigate the possible effects of high doses of hcg (50 ng/ml) and hgh (2,000 ng/ml) in long-term cultured human luteal cells. Figure 2 shows P production by luteal cells during 5 days of incubation with or without serum in the media of cultured cells. The maximal values were generally observed on day 1 (with serum) or 2 (without serum) of incubation. One day of culture with Vol. 57, No. 1, January 1992 Lanzone et al. P production by hgh from luteal cells 93

3 P ng/ml ** HCG ng/ml add * * HGH ng/ml a a a 4000 Figure 1 Dose-response curve of hcg (top panel) and hgh (bottom panel) on the P production by cultured human luteal cells for 24 hours. (l!ll, controls). Significance versus controls: *, P < 0.05; **, P < Data represent means ± SD of five experiments. media containing serum determined in both control and hgh/hcg-stimulated cells a three to fourfold increase in P production when compared with that found in serum-free media-cultured cells. The stimulation of P production by cells, in response to hcg and hgh was sustained at least for 5 days of culture. Only on day 1 the P production stimulated by hcg was significantly greater than that induced by hgh. However, the sum of P production over 5 days of culture showed no difference between hgh (with serum: 568 ± 398 ng/ml; without serum: 179 ± 110 ng/ml) and hcg (with serum: 684 ± 470 ng/ml; without serum: 190 ± 105 ng/ml). Coincubation of maximal effective concentrations of hgh plus hcg did not further increase the P production when compared with the same concen- Table 1 Synergistic Effect of HGH and HCG on P Production by 24-Hours Cultured Human Luteal Cells" HGH 0 ng/ml 250 ng/ml 500 ng/ml 1,000 ng/ml HCG (ng/ml) ± ± ± ± ± 26d ± 30c 208 ± 37d CTR = 97.6 (25 to ) ng/ml Values are expressed as percent increase of P production in respect to those detected in control (untreated) luteal cells (CTR). b Significance versus controls: P < Lanzone et al. P production by hgh from luteal cells 125 ± ± 33b 194 ± 25c 227 ± 32d c Significance versus controls: P < d Significance versus controls: P < ± 22b 189 ± 18c 213 ± 29d 238 ± 33d

4 Png/ml Png/ml with aerum withauc rum I deya Figure 2 Progesterone production by human luteal cells during 5 days of incubation with and without serum in the media of cultured cells. Tissues were processed as described in Materials and Methods, and luteal cells were incubated in the presence and absence of hgh (2,000 ng/ml) with or without hcg (50 ng/ml). Data represent the means± SD of 10 experiments. (control: 0; hcg: A; hgh:.6.; hcg + hgh: e). Significance: controls versus hcg/hgh-treated cells (with serum P < 0.02 day 1 and P < 0.05 days 2, 3, and 4; without serum P < 0.02 days 1 and P < 0.05 days 2 and 3). Human chorionic gonadotropin versus hgh-treated cells (day 1 with and without serum P < 0.02). trations of the two hormones incubated separately (with serum: 641 ± 401 ng/ml; without serum: 186 ± 112 ng/ml). No difference in the cell number or morphology has been found before and after the treatment with hcg and/or hgh. The different age of patients did not affect the luteal responsiveness. DISCUSSION The present study demonstrates, by using an in vitro luteal cell culture system, that hgh has modulatory effects on the luteal steroidogenesis. The data show that: (1) hgh is able to augment P production by human luteal cells; (2) the hgh effect on luteal steroidogenesis is dose-dependent; (3) hgh and hcg have a synergistic effect on P production because uneffective doses of such hormones, when incubated together, determine a remarkable increase of P production. However, these findings are in disagreement with those obtained by Hsu and Hammond, 8 who found that the main action of GH (30 to 600 ng/ml) on porcine granulosa cell function was to facilitate gonadotropin action. The G H alone was able to induce only a modest stimulation of P secretion. Studies in vitro 9 on Leydig cell steroidogenesis indicate that G H may modulate the mechanism of action of hcg, but it does not increase steroidogen- esis by itself. On the other hand, Jia et al. 3 observed that GH stimulated in a dose-dependent manner the progestin production in FSH-treated rat granulosa cells, probably by enhancing LH receptor induction. It is possible that our different culture system (human luteal cells) and the doses of hgh used (from 1,000 to 4,000 ng/ml) may explain these discrepancies. In fact, the doses of hgh used in our study were greater than its physiological circulating levels, although we obtained a maximal P production by using 10- to 20-fold lower doses in the presence of low hcg doses. Therefore, it can be suggested that hgh may contribute to sustaining physiologically the CL secretory activity. The maximum effect obtained with hgh was of the same entity as that obtained with hcg. No further P stimulation was seen by combining the highest hcg and hgh concentrations. These observations suggest a potential role of hgh on ovarian function, confirmed.by clinical and experimental data. Several lines of evidences involve GH in the onset of puberty in mammals. 4 Suppression of endogenous G H secretion by either a tapeworm growth factor or by the implantation of GH in the median eminence resulted in delay ofpuberty. Administration ofgh to GH-deficient animals corrects the modifications induced by G H deficiency. 4 Furthermore, a decrease in the GH Vol. 57, No. 1, January 1992 Lanzone et al. P production by hgh from luteal cells 95

5 levels is associated with reduction of both ovarian LH receptor content and hcg-stimulated P production.4 However, the mechanism by which hgh could act on the ovary is still unclear. Different mechanisms could be involved. Human growth hormone may act: (1) directly by binding to its own receptors or (2) by increasing intraovarian insulin-like growth factor I (IGF-I) levels so that this resultant peptide could then directly affect ovarian function. It is also well known that IGF-I is involved in ovarian steroidogenesis. Adashi et al found that the steroidogenetic responses of rat granulosa cells to FSH and LH stimulation were augmented by coincubation with IGF-1. Recently, several authors12 13 have showed in human granulosa cells the presence of IGF-I-receptors. Furthermore, Hsu and Hammond14 demonstrated that IGF-I could be synthesized by cultured porcine granulosa cells. Insulin -like growth factor I enhances rat granulosa cell differentiation in vitro, supporting the hypothesis that this substance mediates hgh-enhanced ovarian development. The finding that hgh directly augments granulosa cell LH receptor content and P production in vitro, as does IGF-I, further suggests that the ovary itself may be a source of hgh-dependent IGF-1. These data are entirely consistent with IGF-I as a mediator of hgh effects, but they do not exclude the possibility that hgh could act through IGF-Iindependent mechanisms. On the other hand, it has been found that hgh and its relating factor (growth hormone-releasing hormone) were able to increase in vivo steroidogenesis from gonadal tissues.4 We have demonstrated, for the first time, an effect of hgh alone on luteal steroidogenesis. Clearly, further studies are required to clarify the mechanism(s) of hgh action. The cultured luteal cell system may provide an interesting model for elucidating possible intraovarian hgh mechanism involved in the control and steroidogenesis of ovarian function. REFERENCES L Tanner JM, Whitehouse RH: A note on the bone age at which patients with true isolated growth hormone deficiency enter puberty. J Clin Endocrinol Metab 36:55, Kulin HE, Samojlik E, Santen R, Santner S: The effect of gonadotropin in boys with hypopituitarism. Clin Endocrinol (Oxf) 15:463, Jia X, Kalmijn J, Hsueh AJW: Growth hormone enhances follicle stimulating hormone-induced differentiation of cultured rat granulosa cells. Endocrinology 118:1401, Advis JP, White SS, Ojeda SR: Activation of growth hormone short loop negative feedback delays puberty in the female rat. Endocrinology 108:1343, Homburg R, West C, Torresani T, Jacobs HS: Cotreatment with human growth hormone and gonadotropins for induction of ovulation: a controlled clinical trial. Fertil Steril 53:254, Lanzone A, Panetta V, DiSimone N, Arno' E, Fulghesu AM, Caruso A, Mancuso S: Effect of gonadotropin releasing-hormone and related analogue on human luteal cell function in vitro. Hum Reprod 4:906, Alila HW, Rogo KO, Gombe S: Effect of prolactin on steroidogenesis by human luteal cells in culture. Fertil Steril47: 947, Hsu CJ, Hammond JM: Concomitant effects of growth hormone on secretion of insulin-like growth factor I and progesterone by cultured porcine granulosa cells. Endocrinology 121:1343, Horikawa R, Asakawa K, Hizuka N, Takano K, Shizume K: Growth hormone and insulin-like growth factor I stimulate Leydig cell steroidogenesis. Eur J Pharmacol166:87, Adashi EY, Resnick CE, Svoboda ME, VanWyk JJ: A novel role for somatomedin-c in the cytodifferentiation of the ovarian granulosa cell. Endocrinology 115:1227, L Adashi EY, Resnik CE, Svoboda ME, VanWyk JJ: Somatomedin -C enhances induction of luteinizing hormone receptors by follicle-stimulating hormone in cultured rat granulosa cells. Endocrinology 116:2369, Gates GS, Bayer S, Seibel M, Poretsky L, Flier JS, Moses AC: Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization. J Rec Res 7:885, Balboni GC, Vannelli GB, Barni T, Orlando C, Serio M: Transferrin and somatomedin C receptors in the human ovarian follicles. Fertil Steril 48:706, Hsu CJ, Hammond JM: Concomitant effects of growth hormone on secretion of insulin-like growth factor I and progesterone by cultured porcine granulosa cells. Endocrinology 121:1343, Davoren JB, Hsueh AJW, Li CH: Somatomedin C augments FSH-induced differentiation of cultured rat granulosa cells. Am J Physiol249:E26, Lanzone et al. P production by hgh from luteal cells