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Influence of human chorionic gonadotropin in vivo on steroid formation and gonadotropin responsiveness of isolated human preovulatory follicular cells*
Vol. 39, No.1, January 1983
FERTILITY AND STERILITY Copyright 0 1983 The American Fertility Society
Printed in U.8A.
Influence of human chorionic gonadotropin in vivo on steroid formation and gonadotropin responsiveness of isolated human preovulatory follicular cells*
Bo L. Dennefors, M.D., Ph.D.t Lars Hamberger, M.D., Ph.D. Lars Nilsson, M.D., Ph.D. Department of Obstetrics and Gynecology, University of Gothenburg, Gothenburg, Sweden
Granulosa and thecal cells of preovulatory follicles taken from 12 women were isolated and incubated separately for 2 hours in the presence and absence of human chorionic gonadotropin (heG). To six of these women, an ovulatory dose of heG (9000 1U) had been given 24 to 30 hours before excision of the follicle. Following incubation, cellular cyclic adenosine 3/:5/ monophosphate (cAMP) levels and the medium content of progesterone (P), androstenedione (A), and 1713-estradiol (E:d were determined. All follicles appeared healthy and well developed, and the oocytes recovered were morphologically normal and mature. Exposure to heG in vivo caused a shift in steroidogenesis from A toward P formation in isolated thecal cells and a marked increase in the P production by the granulosa cells of the preovulatory follicles. Furthermore, the thecal cells, but not the granulosa cells, developed refractoriness to further stimulation with heG in vitro. Fertil Steril 39:56, 1983 ;
A single intramuscular injection of human chorionic gonadotropin (hCG) (4000 to 10,000 IV) to induce ovulation in anovulatory women, either primed with clomiphene citrate or human menopausal gonadotropin, has been the standard procedure in our unit for several years. Also, in women with normal ovulatory cycles undergoing in vitro fertilization (lVF), exogenous hCG has been used for precise timing of the oocyte collection (ovulation occurs 34 to 38 hours after the injec-
Received May 5, 1982; revised and accepted August 24, 1982. *Supported by grants from the Swedish Medical Research Council (MRF No. 17x-05978 and 17x-02873), Magnus Bergvall's Foundation, Stockholm, and "Forenade Liv" Mutual Group Life Insurance Company, Stockholm, Sweden. tReprint requests: Dr. Bo L. Dennefors, Kvinnokliniken, Sahlgrenska sjukhuset, S-413 45 Gothenburg, Sweden.
56
tion).l In this connection, it has not been settled whether such treatment with hCG causes a physiologic maturation of the follicles. Stimulation of the ovaries with exogenous gonadotropins has been reported to be associated with shortening of the luteal phase 2 ; and, furthermore, premature administration ofhCG in the late follicular phase has been shown to cause follicular atresia in monkeys.3 We undertook the present study to investigate the influence of an ovulatory in vivo dose of hCG on steroid formation by isolated granulosa and thecal cells of human preovulatory follicles during short-term incubations. In addition, the responsiveness to hCG in vitro, as judged by cellular cyclic adenosine 3/:5/ monophosphate (cAMP) and steroid formation, was studied. Follicles were obtained from women treated with a single dose of hCG (9000 IV) 24 to 30 hours before laparot-
Dennefors et aI. heG and steroids in human follicular cells
Fertility and Sterility
omy. Preovulatory follicles from normal, untreated cycles were used as controls. MATERIALS AND METHODS SOURCE OF FOLLICLES
From each of the 12 women (33 to 43 years of age) undergoing legal sterilization via mini-laparotomy, a small wedge resection of one ovary containing the largest visible follicle was performed. All patients had regular menstrual cycles and had given their informed consent before entering the study. Surgery was undertaken on days 12 to 15 of the cycles as based on menstrual history. A single injection of 9000 IU hCG (Pregnyl, Leo Ltd., Helsingborg, Sweden) was given intramuscularly to six of the patients 24 to 30 hours before the operation. DATING OF THE MENSTRUAL CYCLE
The existence of a preovulatory follicle was in all cases predicted by ultrasonic scanning of the ovaries; the operation was conducted on the following day. Serum samples were taken 1 day before the operation, on the day of the operation, and 4 days postoperatively, for the determination of luteinizing hormone (LH) and estradiol (E 2) levels. Commercially available radioimmunoassays (RIAs) were used for the assays. An endometrial biopsy was taken at operation for histologic examination. By combining these methods, we could estimate the stage of the menstrual cycle within 48 hours. 4 All follicles of the present study were estimated to be within 2 days before ovulation. INCUBATION PROCEDURES
Immediately after removal, the follicles were placed in oxygenated (95% O 2 + 5% CO 2) Krebs-Ringer bicarbonate buffer (KRB) and transported to the laboratory for dissection under a stereomicroscope. Each follicle was isolated from adhering tissues, and its diameter was measured. Subsequently, the follicle was punctured to release the follicular fluid and the cumulus-surrounded oocyte, the latter being cultured for histologic studies. The granulosa cells were harvested by gentle scraping from the follicular wall, after which they were centrifuged in glass tubes at 1000 x g for 20 seconds. The cell pellets were resuspended in 0.5 Vol. 39, No.1, January 1983
ml oxygenated KRB containing glucose (5.5 mM) and bovine serum albumin (BSA) 0.1 %. We counted aliquots of this cell suspension in a hemocytometer to determine the number of granulosa cells. Cell viability was tested with 0.4% trypan blue vital stain and was found to be 60% to 70%. The granulosa cell suspension from each follicle was then separated into several aliquots, each containing approximately 0.5 x 106 granulosa cells/0.5 ml KRB. These aliquots were preincubated for 5 minutes at 37° C under continuous shaking in a gyrating water bath. Thereafter, 10 J.ll of either plain KRB (controls) or KRB containing hCG (1 to 100 IU/ml) was added to the aliquots, which were then incubated for 2 hours at 37° C. The remaining thecal capsule of each follicle was dissected free from surrounding cells under the microscope and cut into small sheets of uniform size (approximately 4 x 4 mm). These theca specimens were transferred to vials containing 0.5 ml KRB, preincubated, and incubated as described for the granulosa cells. Further methodologic details concerning the recovery of follicular cells, the incubation technique, and the purity of the theca cell preparation have been described elsewhere. 5 In certain experiments on follicles exposed to hCG in vivo, granulosa cells and specimens from the thecal capsule were fixed in neutral 4% formaldehyde solution and examined in the light microscope. ANALYSES
Following incubation, the granulosa cells were centrifuged into a pellet, and the supernatant was decanted and stored at - 80° C for later steroid analyses. The granulosa cell pellets were homogenized in 1.2 ml 5% trichloracetic acid, and aliquots of the homogenates were taken for protein determination according to Lowry et al. 6 The tissue content of cAMP was determined by means of the protein-binding assay of Gilman. 7 The incubation media from the theca preparations were frozen at - 80° C immediately after incubation. The pieces of thecal tissue also were homogenized and analyzed for protein and cAMP content, as described for the granulosa cells. After extraction of the incubation media with ether, the concentrations of progesterone (P), androstenedione (A), and E2 were measured by RIA with well-characterized antisera. These antisera
Dennefors et aI. heG and steroids in human follicular cells
57
o
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Comparisons between the two groups (control and experimental) were performed using Student's t-test. Multiple comparisons were performed with analysis of variance followed by the Student-Neuman-Keul test. A P value of less than 0.05 was considered significant.
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FOLLICULAR MORPHOLOGY C
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Figure 1 Effects of hCG (100 IU/mI) on tissue cAMP formation of isolated granulosa and thecal cells after a 2-hour incubation period. Each bar represents the mean of 19 to 28 observations. Vertical lines indicate the standard error of the mean (SEM). A, Cells from normal preovulatory follicles. B, Cells from preovulatory follicles exposed to hCG in vivo. The effects of hCG versus control are significant for granulosa cells from both types of follicles (P < 0.001) and for thecal cells from normal preovulatory follicles (p < 0.01).
(rabbit anti_estradiol-carboxymethyloxime, rabbit anti_progesterone-11a-hemisuccinate, and rabbit anti-androstenedione-7 a-carboxyethylthioether) are of high specificity with low crossreactivity.8 The specificity, sensitivity, and accuracy of the RIAs have been analyzed in earlier experiments in this laboratory and are described elsewhere. 4
The diameter ofthe follicles (determined in two dimensions) varied between 13 and 30 mm. In all follicles an oocyte was recovered, exhibiting various degrees of nuclear maturation and no signs of degeneration. The number of viable granulosa cells varied from 10 to 32 X 106 , depending on the size of the follicles. There were no major differences between the two types of follicles with regard to the number of granulosa cells (related to the follicular diameter) and the appearance of the oocyte. Histologically, both cell types from the follicles exposed to hCG in"vivO showed a marked luteinization and no signs of degeneration. In particular, the theca interna layer was enlarged and made up oflarge, polyhedral, luteinized cells with numerous mitoses. CYCLIC AMP FORMATION
As can be seen in Figure 1, hCG (l00 IUlml) caused a statistically significant increase in
CHEMICALS
ISO,
All reagents (grade A) and essential fatty acidfree BSA" were purchased from Sigma Chemical Company, St. Louis, MO. hCG, 10,000 IV/mg, was used in concentrations varying between 1 and 100 IV/ml of incubation medium. The higher concentration was used in most experiments.
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CALCULATIONS AND STATISTICS
Two types of follicles were collected: normal preovulatory follicles and preovulatory follicles from patients who had been treated with hCG in vivo. Within each experiment, the different cell samples of the follicle were, for each cell type, divided into two subgroups according to the presence or absence ofhCG in the incubation medium: experimental samples and control samples. Each subgroup consisted of four to eight individual observations. The means from each subgroup of the individual follicles were then pooled to form a group representing all follicles of the same type.
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Figure 2 Concentration-response relationship between hCG and cAMP formation in granulosa and thecal cells derived from preovulatory follicles exposed to hCG in vivo. Incubation time, 2 hours. The concentrations of hCG are expressed as IU/ml. Each bar represents the mean of 7 to 8 observations. Vertical lines indicate SEM. In granulosa cells the stimulatory effects of 100 IUlml hCG and 10 IUlml hCG versus control are statistically significant (P < 0.01). Fertility and Sterility
58
Dennefors et aI.
heG and steroids in human follicular cells
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Figure 3 Release of steroids into the incubation medium after a 2-hour incubation period of granulosa cells in the absence and presence of 100 IU/ml hCG. Each bar represents the mean of 11 to 25 observations. Vertical lines indicate SEM. A, Cells from preovulatory follicles. B, Cells from preovulatory follicles exposed to hCG in vivo. No comparisons with control values are significant.
cAMP formation in the granulosa cells from both types offollicles and in the thecal cells from spontaneously matured follicles. However, no significant increase in cAMP formation was observed in thecal cells from follicles exposed to heG in vivo. In two experiments on cells from follicles exposed to heG in vivo, the effects of various concentrations of heG in vitro on cAMP formation were studied; the results are shown in Figure 2. In granulosa cells heG in vitro caused a concentration-dependent increase in cAMP formation, whereas in thecal cells no stimulatory effects were found.
The thecal cells from follicles exposed to heG in vivo formed predominantly P in vitro, while A was the major steroid formed by thecal cells from follicles of the natural cycles. Thus, the amounts of P formed in vitro were much higher in thecal cells from follicles exposed to heG in vivo than from follicles that developed in the natural cycle. E2 was formed in similar and considerable amounts by the thecal cells from both types of follicles. The addition of heG in vitro elicited a statistically significant increase in A formation and a tendency toward an increase of P formation in the thecal cells from follicles not exposed to heG in vivo. However, in vitro heG had no effect on the formation of any of the three steroids measured in the thecal cells from follicles exposed to heG in vivo (Fig. 4). DISCUSSION
All follicles of the present study contained more than 50% of the optimum number of granulosa cells for their respective diameter, according to McNatty et al.,9 and the oocytes recovered from these follicles appeared healthy and mature. These findings indicate that all of the follicles used in the study were healthy and well developed according to the classification scheme of McNatty et a1. 9
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STEROID FORMATION
The basal steroid formation and the influence of heG (100 IU/ml) in vitro after 2 hours of incubation are shown in Figures 3 and 4. The major steroid formed by the granulosa cells from both types of follicles was P. Granulosa cells from follicles exposed to heG in vivo formed very high amounts of P in vitro, while the P formation by the granulosa cells from follicles of the natural cycles was much lower. There was no difference between the two types of follicles in the amounts of A and E2 formed by the granulosa cells. heG in vitro tended to increase the mean value ofP ofthe granulosa cells from both types of follicles, while the production of A and E2 was not altered (Fig. 3). Vol. 39, No.1, January 1983
THECRL·CELLS
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Figure 4 Release of steroids into the incubation medium after a 2-hour incubation period ofthecal cells in the absence and presence of 100 IU/ml hCG. Each bar represents the mean of 11 to 25 observations. Vertical lines indicate SEM. A, Cells from normal preovulatory follicles. B, Cells from preovulatory follicles exposed to hCG in vivo. Note the different scale on the y-axis as compared with Figure 3. The stimulatory effect of hCG on A formation by thecal cells from normal follicles is significant (P < 0.05). No other comparisons with control values are significant.
Dennefors et aI. heG and steroids in human follicular cells
59
The pattern of steroidogenesis of the isolated cells from naturally developed follicles found in the present study is in accordance with earlier reports 5, 10 and demonstrates that steroidogenesis is not strictly compartmentalized between the two cell types in the normal human preovulatory follicle. In follicles exposed to hCG in vivo, the amounts of P formed by the isolated granulosa cells were more than 15 times higher than those of A or E2 (Fig. 3). This finding is compatible with the observed histologic appearance of marked luteinization of the granulosa cells. Other studies l l have shown that the P formation of cultured human granulosa cells from follicles exposed to hCG in vivo is several times higher than the formation of any other steroid measured. From studies in the rat in our laboratory it has been shown that a prolonged influence of LH in vivo induces a block in the formation of A and estrogens in the granulosa cells of preovulatory follicles. 12 The results of the present study support the existence of a similar block by hCG in vivo on certain steps in steroidogenesis in isolated human preovulatory granulosa cells. In this context it should be emphasized that the total steroidogenic capacity of the granulosa cells of this study was not completely assessed, since no androgen substrate was added to the incubation medium. Consequently, the results obtained may not be fully representative for the intact follicle. Also, thecal cells from human follicles exposed in vivo to hCG predominantly formed P in vitro, whereas the amounts of A and E2 were lower. The thecal cells from both types of follicles in the present study formed E2 in similar amounts, indicating their preserved aromatizing capacity after hCG treatment in vivo. These patterns of in vitro steroid formation by the two cell types from hCG-treated follicles are thus different from one another and, furthermore, are different from those of isolated cells from human preovulatory follicles of the natural cycle. In this latter type of follicle, both cell types produced approximately one-tenth of the amount of P as those in follicles exposed to hCG in vivo. It must be emphasized, however, that human preovulatory follicles of the natural cycle may have been exposed to varying amounts of LH in vivo. For such reasons, comparisons of the in vitro steroid formation between these two types of follicles must be interpreted with care. Nevertheless, it 60
seems evident from this study that exogenous hCG in "ovulatory" doses creates a higher degree of luteinization of the follicular cells than the endogenous gonadotropin surge. Since all follicles of the present study contained oocytes that were morphologically normal and mature, it may be assumed that this "hyperluteinization" does not interfere with the viability of the oocyte, although the fertilizability has not been tested. The granulosa cells from both types of follicles in this study responded to hCG in vitro in terms of increased cAMP formation. This is in agreement with the finding that follicular maturation in the human is associated with a dynamic increase in the number of LHIhCG receptors on the granulosa cells. 13 It thus seems that an "ovulatory" dose of hCG in vivo does not substantially interfere with the responsiveness to hCG in vitro, as far as the granulosa cells are concerned. On the other hand, thecal cells exposed to hCG in vivo did not respond to hCG in vitro, in terms of cAMP or steroid formation, and this is in contrast to the thecal cells from preovulatory follicles derived from the natural cycle, which were highly sensitive to the in vitro hCG (100 IUfml). It has been reported in earlier studies on rats that after an injection of hCG in vivo the intact preovulatory follicle becomes completely desensitized to hCG or LH in vitro for several days.14 Furthermore, experiments from our laboratory on the same species indicate that the presence of follicular fluid is a prerequisite for the development of LH refractoriness in isolated granulosa cells. 15 The reason for the nonrefractoriness of the human granulosa cells observed in the present study may be the fact that follicular fluid was removed by washing the cells prior to incubation. Experiments are in progress to investigate the importance of human follicular fluid in the development of refractoriness in this type of granulosa cell. No animal or human data have been reported as yet concerning development of gonadotropic refractoriness in isolated thecal cells. In conclusion, the results of the present study demonstrate that a single injection of hCG, in doses often used in clinical practice to induce ovulation, causes a marked luteinization of both follicular cell types with a high production of P in vitro. Furthermore, the isolated thecal cells, but not the granulosa cells, develop complete refractoriness to further stimulation with hCG in vitro for at least 24 hours.
Dennefors et al. heG and steroids in human follicular cells
Fertility and Sterility
Acknowledgments. Human chorionic gonadotropin was a gift from Leo Ltd., Helsingborg, Sweden. The RIAs of serum hormones were performed by Prof. Goran Lindstedt, Department of Clinical Chemistry, Sahlgrens Hospital, Gothenburg, Sweden. Antisera were donated by Drs. Hans Lindner and Sara Bauminger, Department of Hormone Research, Weizman Institute, Rehovot, Israel. REFERENCES 1. Edwards RG, Steptoe PC: Induction of follicular growth, ovulation and luteinization in the human ovary. J Reprod Fertil (Suppl) 22:121, 1975 2. Black WP, Martin BT, Whyte WG: Plasma progesterone concentration as an index of ovulation and corpus luteum function in normal and gonadotrophin-stimulated menstrual cycles. J Obstet Gynaecol Br Commonw 79:363, 1972 3. Williams RF, Hodgen GD: Disparate effects of human chorionic gonadotropin during the late follicular phase in monkeys: normal ovulation, follicular atresia, ovarian acyclicity, and hypersecretion of follicle-stimulating hormone. Fertil Steril 33:64, 1980 4. Dennefors BL: Studies on the endocrine function of the human ovary. Experiments conducted on isolated ovarian compartments. Thesis ISBN 91-7222-423-1, Goteborg, 1981 5. Dennefors BL, Nilsson L, Hamberger L: Steroid and 3'5'monophosphate formation in granulosa and thecal cells from human preovulatory follicles in response to human chorionic gonadotropin. J Clin Endocrinol Metab 54:436, 1982 6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folinphenol reagent. J Bioi Chern 193:265, 1951 7. Gilman AG: A protein binding assay for adenosine 3'5'cyclic monophosphate. Proc Natl Acad Sci USA 67:305, 1970
Vol. 39, No.1, January 1983
8. Lindner HR, Bauminger S: Production and characterization of antisera to steroid hormones. In Recent Advances in Reproductive Endocrinology, Edited by PG Crosignani, VHTJames. New York, Academic Press Inc., 1974, p 193 9. McNatty KP, Smith DM, Makris A, Osathanondh R, Ryan KJ: The microenvironment of the human antral follicle: interrelationships among the steroid levels in antral fluid, the population of granulosa cells, and the status of the oocyte in vivo and in vitro. J Clin Endocrinol Metab 49:851, 1979 10. McNatty KP, Makris A, De Grazia C, Osathanondh R, Ryan KJ: The production of progesterone, androgens and estrogens by granulosa cells, thecal tissue, and stromal tissue from human ovaries in vitro. J Clin Endocrinol Metab 49:687, 1979 11. Fowler RE, Fox NL, Edwards RG, Walters DE, Steptoe PC: Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors. J Endocrinol 77:171, 1978 12. Hamberger L, Hillensjo T, Ahren K: Steroidogenesis in isolated cells of preovulatory rat follicles. Endocrinology 103:771, 1978 13. Rajaniemi HJ, Ronnberg L, Kauppila A, Ylostalo R, Jalkanen M, Saastamoinen J, Selander K, Pystynen P, Vihko R: Luteinizing hormone receptors in human ovarian follicles and corpora lutea during menstrual cycle and pregnancy. J Clin Endocrinol Metab 52:307, 1981 14. Lamprecht SA, Zor U, Tsafriri A, Lindner HL: Action of prostaglandin E2 and of luteinizing hormone on ovarian adenylate cyclase, protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat. J Endocrinol 57:217, 1973 15. Nordenstrom K, Sjogren A, Hamberger L: Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid. Acta Endocrinol (Copenh) 95:84, 1980
Dennefors et al.
heG and steroids in human follicular cells
61
FERTILITY AND STERILITY Copyright 0 1983 The American Fertility Society
Printed in U.8A.
Influence of human chorionic gonadotropin in vivo on steroid formation and gonadotropin responsiveness of isolated human preovulatory follicular cells*
Bo L. Dennefors, M.D., Ph.D.t Lars Hamberger, M.D., Ph.D. Lars Nilsson, M.D., Ph.D. Department of Obstetrics and Gynecology, University of Gothenburg, Gothenburg, Sweden
Granulosa and thecal cells of preovulatory follicles taken from 12 women were isolated and incubated separately for 2 hours in the presence and absence of human chorionic gonadotropin (heG). To six of these women, an ovulatory dose of heG (9000 1U) had been given 24 to 30 hours before excision of the follicle. Following incubation, cellular cyclic adenosine 3/:5/ monophosphate (cAMP) levels and the medium content of progesterone (P), androstenedione (A), and 1713-estradiol (E:d were determined. All follicles appeared healthy and well developed, and the oocytes recovered were morphologically normal and mature. Exposure to heG in vivo caused a shift in steroidogenesis from A toward P formation in isolated thecal cells and a marked increase in the P production by the granulosa cells of the preovulatory follicles. Furthermore, the thecal cells, but not the granulosa cells, developed refractoriness to further stimulation with heG in vitro. Fertil Steril 39:56, 1983 ;
A single intramuscular injection of human chorionic gonadotropin (hCG) (4000 to 10,000 IV) to induce ovulation in anovulatory women, either primed with clomiphene citrate or human menopausal gonadotropin, has been the standard procedure in our unit for several years. Also, in women with normal ovulatory cycles undergoing in vitro fertilization (lVF), exogenous hCG has been used for precise timing of the oocyte collection (ovulation occurs 34 to 38 hours after the injec-
Received May 5, 1982; revised and accepted August 24, 1982. *Supported by grants from the Swedish Medical Research Council (MRF No. 17x-05978 and 17x-02873), Magnus Bergvall's Foundation, Stockholm, and "Forenade Liv" Mutual Group Life Insurance Company, Stockholm, Sweden. tReprint requests: Dr. Bo L. Dennefors, Kvinnokliniken, Sahlgrenska sjukhuset, S-413 45 Gothenburg, Sweden.
56
tion).l In this connection, it has not been settled whether such treatment with hCG causes a physiologic maturation of the follicles. Stimulation of the ovaries with exogenous gonadotropins has been reported to be associated with shortening of the luteal phase 2 ; and, furthermore, premature administration ofhCG in the late follicular phase has been shown to cause follicular atresia in monkeys.3 We undertook the present study to investigate the influence of an ovulatory in vivo dose of hCG on steroid formation by isolated granulosa and thecal cells of human preovulatory follicles during short-term incubations. In addition, the responsiveness to hCG in vitro, as judged by cellular cyclic adenosine 3/:5/ monophosphate (cAMP) and steroid formation, was studied. Follicles were obtained from women treated with a single dose of hCG (9000 IV) 24 to 30 hours before laparot-
Dennefors et aI. heG and steroids in human follicular cells
Fertility and Sterility
omy. Preovulatory follicles from normal, untreated cycles were used as controls. MATERIALS AND METHODS SOURCE OF FOLLICLES
From each of the 12 women (33 to 43 years of age) undergoing legal sterilization via mini-laparotomy, a small wedge resection of one ovary containing the largest visible follicle was performed. All patients had regular menstrual cycles and had given their informed consent before entering the study. Surgery was undertaken on days 12 to 15 of the cycles as based on menstrual history. A single injection of 9000 IU hCG (Pregnyl, Leo Ltd., Helsingborg, Sweden) was given intramuscularly to six of the patients 24 to 30 hours before the operation. DATING OF THE MENSTRUAL CYCLE
The existence of a preovulatory follicle was in all cases predicted by ultrasonic scanning of the ovaries; the operation was conducted on the following day. Serum samples were taken 1 day before the operation, on the day of the operation, and 4 days postoperatively, for the determination of luteinizing hormone (LH) and estradiol (E 2) levels. Commercially available radioimmunoassays (RIAs) were used for the assays. An endometrial biopsy was taken at operation for histologic examination. By combining these methods, we could estimate the stage of the menstrual cycle within 48 hours. 4 All follicles of the present study were estimated to be within 2 days before ovulation. INCUBATION PROCEDURES
Immediately after removal, the follicles were placed in oxygenated (95% O 2 + 5% CO 2) Krebs-Ringer bicarbonate buffer (KRB) and transported to the laboratory for dissection under a stereomicroscope. Each follicle was isolated from adhering tissues, and its diameter was measured. Subsequently, the follicle was punctured to release the follicular fluid and the cumulus-surrounded oocyte, the latter being cultured for histologic studies. The granulosa cells were harvested by gentle scraping from the follicular wall, after which they were centrifuged in glass tubes at 1000 x g for 20 seconds. The cell pellets were resuspended in 0.5 Vol. 39, No.1, January 1983
ml oxygenated KRB containing glucose (5.5 mM) and bovine serum albumin (BSA) 0.1 %. We counted aliquots of this cell suspension in a hemocytometer to determine the number of granulosa cells. Cell viability was tested with 0.4% trypan blue vital stain and was found to be 60% to 70%. The granulosa cell suspension from each follicle was then separated into several aliquots, each containing approximately 0.5 x 106 granulosa cells/0.5 ml KRB. These aliquots were preincubated for 5 minutes at 37° C under continuous shaking in a gyrating water bath. Thereafter, 10 J.ll of either plain KRB (controls) or KRB containing hCG (1 to 100 IU/ml) was added to the aliquots, which were then incubated for 2 hours at 37° C. The remaining thecal capsule of each follicle was dissected free from surrounding cells under the microscope and cut into small sheets of uniform size (approximately 4 x 4 mm). These theca specimens were transferred to vials containing 0.5 ml KRB, preincubated, and incubated as described for the granulosa cells. Further methodologic details concerning the recovery of follicular cells, the incubation technique, and the purity of the theca cell preparation have been described elsewhere. 5 In certain experiments on follicles exposed to hCG in vivo, granulosa cells and specimens from the thecal capsule were fixed in neutral 4% formaldehyde solution and examined in the light microscope. ANALYSES
Following incubation, the granulosa cells were centrifuged into a pellet, and the supernatant was decanted and stored at - 80° C for later steroid analyses. The granulosa cell pellets were homogenized in 1.2 ml 5% trichloracetic acid, and aliquots of the homogenates were taken for protein determination according to Lowry et al. 6 The tissue content of cAMP was determined by means of the protein-binding assay of Gilman. 7 The incubation media from the theca preparations were frozen at - 80° C immediately after incubation. The pieces of thecal tissue also were homogenized and analyzed for protein and cAMP content, as described for the granulosa cells. After extraction of the incubation media with ether, the concentrations of progesterone (P), androstenedione (A), and E2 were measured by RIA with well-characterized antisera. These antisera
Dennefors et aI. heG and steroids in human follicular cells
57
o
300
control
:;:: 250 OJ
c
hCG
X
.~ c 200
" .~
~
E" ~ ~
Comparisons between the two groups (control and experimental) were performed using Student's t-test. Multiple comparisons were performed with analysis of variance followed by the Student-Neuman-Keul test. A P value of less than 0.05 was considered significant.
150
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a.
RESULTS
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100
u-
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E Q.
50
FOLLICULAR MORPHOLOGY C
C hCG
hCG
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GRANULOSA CELLS
Figure 1 Effects of hCG (100 IU/mI) on tissue cAMP formation of isolated granulosa and thecal cells after a 2-hour incubation period. Each bar represents the mean of 19 to 28 observations. Vertical lines indicate the standard error of the mean (SEM). A, Cells from normal preovulatory follicles. B, Cells from preovulatory follicles exposed to hCG in vivo. The effects of hCG versus control are significant for granulosa cells from both types of follicles (P < 0.001) and for thecal cells from normal preovulatory follicles (p < 0.01).
(rabbit anti_estradiol-carboxymethyloxime, rabbit anti_progesterone-11a-hemisuccinate, and rabbit anti-androstenedione-7 a-carboxyethylthioether) are of high specificity with low crossreactivity.8 The specificity, sensitivity, and accuracy of the RIAs have been analyzed in earlier experiments in this laboratory and are described elsewhere. 4
The diameter ofthe follicles (determined in two dimensions) varied between 13 and 30 mm. In all follicles an oocyte was recovered, exhibiting various degrees of nuclear maturation and no signs of degeneration. The number of viable granulosa cells varied from 10 to 32 X 106 , depending on the size of the follicles. There were no major differences between the two types of follicles with regard to the number of granulosa cells (related to the follicular diameter) and the appearance of the oocyte. Histologically, both cell types from the follicles exposed to hCG in"vivO showed a marked luteinization and no signs of degeneration. In particular, the theca interna layer was enlarged and made up oflarge, polyhedral, luteinized cells with numerous mitoses. CYCLIC AMP FORMATION
As can be seen in Figure 1, hCG (l00 IUlml) caused a statistically significant increase in
CHEMICALS
ISO,
All reagents (grade A) and essential fatty acidfree BSA" were purchased from Sigma Chemical Company, St. Louis, MO. hCG, 10,000 IV/mg, was used in concentrations varying between 1 and 100 IV/ml of incubation medium. The higher concentration was used in most experiments.
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CALCULATIONS AND STATISTICS
Two types of follicles were collected: normal preovulatory follicles and preovulatory follicles from patients who had been treated with hCG in vivo. Within each experiment, the different cell samples of the follicle were, for each cell type, divided into two subgroups according to the presence or absence ofhCG in the incubation medium: experimental samples and control samples. Each subgroup consisted of four to eight individual observations. The means from each subgroup of the individual follicles were then pooled to form a group representing all follicles of the same type.
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Figure 2 Concentration-response relationship between hCG and cAMP formation in granulosa and thecal cells derived from preovulatory follicles exposed to hCG in vivo. Incubation time, 2 hours. The concentrations of hCG are expressed as IU/ml. Each bar represents the mean of 7 to 8 observations. Vertical lines indicate SEM. In granulosa cells the stimulatory effects of 100 IUlml hCG and 10 IUlml hCG versus control are statistically significant (P < 0.01). Fertility and Sterility
58
Dennefors et aI.
heG and steroids in human follicular cells
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Figure 3 Release of steroids into the incubation medium after a 2-hour incubation period of granulosa cells in the absence and presence of 100 IU/ml hCG. Each bar represents the mean of 11 to 25 observations. Vertical lines indicate SEM. A, Cells from preovulatory follicles. B, Cells from preovulatory follicles exposed to hCG in vivo. No comparisons with control values are significant.
cAMP formation in the granulosa cells from both types offollicles and in the thecal cells from spontaneously matured follicles. However, no significant increase in cAMP formation was observed in thecal cells from follicles exposed to heG in vivo. In two experiments on cells from follicles exposed to heG in vivo, the effects of various concentrations of heG in vitro on cAMP formation were studied; the results are shown in Figure 2. In granulosa cells heG in vitro caused a concentration-dependent increase in cAMP formation, whereas in thecal cells no stimulatory effects were found.
The thecal cells from follicles exposed to heG in vivo formed predominantly P in vitro, while A was the major steroid formed by thecal cells from follicles of the natural cycles. Thus, the amounts of P formed in vitro were much higher in thecal cells from follicles exposed to heG in vivo than from follicles that developed in the natural cycle. E2 was formed in similar and considerable amounts by the thecal cells from both types of follicles. The addition of heG in vitro elicited a statistically significant increase in A formation and a tendency toward an increase of P formation in the thecal cells from follicles not exposed to heG in vivo. However, in vitro heG had no effect on the formation of any of the three steroids measured in the thecal cells from follicles exposed to heG in vivo (Fig. 4). DISCUSSION
All follicles of the present study contained more than 50% of the optimum number of granulosa cells for their respective diameter, according to McNatty et al.,9 and the oocytes recovered from these follicles appeared healthy and mature. These findings indicate that all of the follicles used in the study were healthy and well developed according to the classification scheme of McNatty et a1. 9
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The basal steroid formation and the influence of heG (100 IU/ml) in vitro after 2 hours of incubation are shown in Figures 3 and 4. The major steroid formed by the granulosa cells from both types of follicles was P. Granulosa cells from follicles exposed to heG in vivo formed very high amounts of P in vitro, while the P formation by the granulosa cells from follicles of the natural cycles was much lower. There was no difference between the two types of follicles in the amounts of A and E2 formed by the granulosa cells. heG in vitro tended to increase the mean value ofP ofthe granulosa cells from both types of follicles, while the production of A and E2 was not altered (Fig. 3). Vol. 39, No.1, January 1983
THECRL·CELLS
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Figure 4 Release of steroids into the incubation medium after a 2-hour incubation period ofthecal cells in the absence and presence of 100 IU/ml hCG. Each bar represents the mean of 11 to 25 observations. Vertical lines indicate SEM. A, Cells from normal preovulatory follicles. B, Cells from preovulatory follicles exposed to hCG in vivo. Note the different scale on the y-axis as compared with Figure 3. The stimulatory effect of hCG on A formation by thecal cells from normal follicles is significant (P < 0.05). No other comparisons with control values are significant.
Dennefors et aI. heG and steroids in human follicular cells
59
The pattern of steroidogenesis of the isolated cells from naturally developed follicles found in the present study is in accordance with earlier reports 5, 10 and demonstrates that steroidogenesis is not strictly compartmentalized between the two cell types in the normal human preovulatory follicle. In follicles exposed to hCG in vivo, the amounts of P formed by the isolated granulosa cells were more than 15 times higher than those of A or E2 (Fig. 3). This finding is compatible with the observed histologic appearance of marked luteinization of the granulosa cells. Other studies l l have shown that the P formation of cultured human granulosa cells from follicles exposed to hCG in vivo is several times higher than the formation of any other steroid measured. From studies in the rat in our laboratory it has been shown that a prolonged influence of LH in vivo induces a block in the formation of A and estrogens in the granulosa cells of preovulatory follicles. 12 The results of the present study support the existence of a similar block by hCG in vivo on certain steps in steroidogenesis in isolated human preovulatory granulosa cells. In this context it should be emphasized that the total steroidogenic capacity of the granulosa cells of this study was not completely assessed, since no androgen substrate was added to the incubation medium. Consequently, the results obtained may not be fully representative for the intact follicle. Also, thecal cells from human follicles exposed in vivo to hCG predominantly formed P in vitro, whereas the amounts of A and E2 were lower. The thecal cells from both types of follicles in the present study formed E2 in similar amounts, indicating their preserved aromatizing capacity after hCG treatment in vivo. These patterns of in vitro steroid formation by the two cell types from hCG-treated follicles are thus different from one another and, furthermore, are different from those of isolated cells from human preovulatory follicles of the natural cycle. In this latter type of follicle, both cell types produced approximately one-tenth of the amount of P as those in follicles exposed to hCG in vivo. It must be emphasized, however, that human preovulatory follicles of the natural cycle may have been exposed to varying amounts of LH in vivo. For such reasons, comparisons of the in vitro steroid formation between these two types of follicles must be interpreted with care. Nevertheless, it 60
seems evident from this study that exogenous hCG in "ovulatory" doses creates a higher degree of luteinization of the follicular cells than the endogenous gonadotropin surge. Since all follicles of the present study contained oocytes that were morphologically normal and mature, it may be assumed that this "hyperluteinization" does not interfere with the viability of the oocyte, although the fertilizability has not been tested. The granulosa cells from both types of follicles in this study responded to hCG in vitro in terms of increased cAMP formation. This is in agreement with the finding that follicular maturation in the human is associated with a dynamic increase in the number of LHIhCG receptors on the granulosa cells. 13 It thus seems that an "ovulatory" dose of hCG in vivo does not substantially interfere with the responsiveness to hCG in vitro, as far as the granulosa cells are concerned. On the other hand, thecal cells exposed to hCG in vivo did not respond to hCG in vitro, in terms of cAMP or steroid formation, and this is in contrast to the thecal cells from preovulatory follicles derived from the natural cycle, which were highly sensitive to the in vitro hCG (100 IUfml). It has been reported in earlier studies on rats that after an injection of hCG in vivo the intact preovulatory follicle becomes completely desensitized to hCG or LH in vitro for several days.14 Furthermore, experiments from our laboratory on the same species indicate that the presence of follicular fluid is a prerequisite for the development of LH refractoriness in isolated granulosa cells. 15 The reason for the nonrefractoriness of the human granulosa cells observed in the present study may be the fact that follicular fluid was removed by washing the cells prior to incubation. Experiments are in progress to investigate the importance of human follicular fluid in the development of refractoriness in this type of granulosa cell. No animal or human data have been reported as yet concerning development of gonadotropic refractoriness in isolated thecal cells. In conclusion, the results of the present study demonstrate that a single injection of hCG, in doses often used in clinical practice to induce ovulation, causes a marked luteinization of both follicular cell types with a high production of P in vitro. Furthermore, the isolated thecal cells, but not the granulosa cells, develop complete refractoriness to further stimulation with hCG in vitro for at least 24 hours.
Dennefors et al. heG and steroids in human follicular cells
Fertility and Sterility
Acknowledgments. Human chorionic gonadotropin was a gift from Leo Ltd., Helsingborg, Sweden. The RIAs of serum hormones were performed by Prof. Goran Lindstedt, Department of Clinical Chemistry, Sahlgrens Hospital, Gothenburg, Sweden. Antisera were donated by Drs. Hans Lindner and Sara Bauminger, Department of Hormone Research, Weizman Institute, Rehovot, Israel. REFERENCES 1. Edwards RG, Steptoe PC: Induction of follicular growth, ovulation and luteinization in the human ovary. J Reprod Fertil (Suppl) 22:121, 1975 2. Black WP, Martin BT, Whyte WG: Plasma progesterone concentration as an index of ovulation and corpus luteum function in normal and gonadotrophin-stimulated menstrual cycles. J Obstet Gynaecol Br Commonw 79:363, 1972 3. Williams RF, Hodgen GD: Disparate effects of human chorionic gonadotropin during the late follicular phase in monkeys: normal ovulation, follicular atresia, ovarian acyclicity, and hypersecretion of follicle-stimulating hormone. Fertil Steril 33:64, 1980 4. Dennefors BL: Studies on the endocrine function of the human ovary. Experiments conducted on isolated ovarian compartments. Thesis ISBN 91-7222-423-1, Goteborg, 1981 5. Dennefors BL, Nilsson L, Hamberger L: Steroid and 3'5'monophosphate formation in granulosa and thecal cells from human preovulatory follicles in response to human chorionic gonadotropin. J Clin Endocrinol Metab 54:436, 1982 6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folinphenol reagent. J Bioi Chern 193:265, 1951 7. Gilman AG: A protein binding assay for adenosine 3'5'cyclic monophosphate. Proc Natl Acad Sci USA 67:305, 1970
Vol. 39, No.1, January 1983
8. Lindner HR, Bauminger S: Production and characterization of antisera to steroid hormones. In Recent Advances in Reproductive Endocrinology, Edited by PG Crosignani, VHTJames. New York, Academic Press Inc., 1974, p 193 9. McNatty KP, Smith DM, Makris A, Osathanondh R, Ryan KJ: The microenvironment of the human antral follicle: interrelationships among the steroid levels in antral fluid, the population of granulosa cells, and the status of the oocyte in vivo and in vitro. J Clin Endocrinol Metab 49:851, 1979 10. McNatty KP, Makris A, De Grazia C, Osathanondh R, Ryan KJ: The production of progesterone, androgens and estrogens by granulosa cells, thecal tissue, and stromal tissue from human ovaries in vitro. J Clin Endocrinol Metab 49:687, 1979 11. Fowler RE, Fox NL, Edwards RG, Walters DE, Steptoe PC: Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors. J Endocrinol 77:171, 1978 12. Hamberger L, Hillensjo T, Ahren K: Steroidogenesis in isolated cells of preovulatory rat follicles. Endocrinology 103:771, 1978 13. Rajaniemi HJ, Ronnberg L, Kauppila A, Ylostalo R, Jalkanen M, Saastamoinen J, Selander K, Pystynen P, Vihko R: Luteinizing hormone receptors in human ovarian follicles and corpora lutea during menstrual cycle and pregnancy. J Clin Endocrinol Metab 52:307, 1981 14. Lamprecht SA, Zor U, Tsafriri A, Lindner HL: Action of prostaglandin E2 and of luteinizing hormone on ovarian adenylate cyclase, protein kinase and ornithine decarboxylase activity during postnatal development and maturity in the rat. J Endocrinol 57:217, 1973 15. Nordenstrom K, Sjogren A, Hamberger L: Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid. Acta Endocrinol (Copenh) 95:84, 1980
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heG and steroids in human follicular cells
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