Prolactin inhibits the steroidogenesis in midfollicular phase human granulosa cells cultured in a chemically defined medium*

FERTILITY AND STERILITY Copyright~ 1988 The American Fertility Society

Vol. 49, No.4, Aprill988 Printed in U.S.A.

Prolactin inhibits the steroidogenesis in midfollicular phase human granulosa cells cultured in a chemically defined medium*

Eduardo Cutie R., M.D.t Nieves A. Andino, M.D. Female Reproductive Laboratory, National Institute of Endocrinology, World Health Organization Collaborating Center in Human Reproduction, Habana, Cuba

In vitro studies were conducted on prolactin (PRL) effects on human granulosa cell steroidogenesis. Cells derived from healthy midfollicular phase follicles were cultured in a chemically defined medium supplemented with androstenedione (~4 A) 10- 7 M. Cultures treated with follicle-stimulating hormone (FSH) showed a dose-dependent increase of estradiol (E 2 ) and progesterone (P) secretion. The authors demonstrated that PRL (~10 ng/ml) inhibits basal as well as FSH (10 ng/ml)-stimulated E 2 and P secretion. This PRL effect was overcome only by FSH maximal stimulating doses (100 ng/ml). These results suggest a direct inhibitory effect of PRL on granulosa cell steroidogenesis acting as a negative modulator of FSH action. These effects might be related to the ovarian dysfunction observed in hyperprolactinemia. Fertil Steril 49:632, 1988

The effect(s) of prolactin (PRL) on the human ovary remains controversial. Some clinical findings point out to a possible direct deleterious action of PRL on ovarian function. 1·2 The presence of PRL receptors on human ovarian tissues 3 might indicate a possible role of the hormone on the female gonadal function. Several in vitro studies seem to demonstrate that PRL is able to modify the steroid output of human ovarian cells. 4- 7 However, other authors have not found any effect of the hormone on this tissue. 8 •9 It has been suggested that the ovarian dysfunction observed in hyperprolactinemic states might be explained through decreased steroid biosynthesis during folliculogenesis. 10•11 It is well known that, during the midfollicular

phase of the cycle, the ovarian follicle destined to ovulate goes through a developing process, depending mainly on its capacity to synthesize steroids. Human granulosa cells are the main source of follicular estradiol (E 2) production, and this steroid represents the cornerstone of follicular development.12 Bearing this in mind, we considered it important to evaluate the in vitro effect(s) of PRL on E 2 and progesterone (P) secretion by human granulosa cells derived from healthy midfollicular phase follicles.

MATERIALS AND METHODS Hormones and Reagents

Received September 15, 1987; revised and accepted December 30, 1987. * Supported in part by the Human Reproduction Programme from the World Health Organization, Geneva, Switzerland. t Reprint requests: Eduardo Cutie R., M.D., Female Reproductive Laboratory, National Institute of Endocrinology, Hospital "Fajardo," Habana 4, Cuba.

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Cutie R. and Andino PRL effects on human granulosa cells

Human follicle-stimulating hormone (FSH) (National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases [NIADDK] hFSH-3, AFP-4822B, bioactivity 3100 IU/mg) and human PRL (NIADDK hPRL-1-7, AFP-9900, bioactivity 35 IU /mg) were resuspended in sterile phosphateFertility and Sterility

buffered saline (PBS) 10 mM, pH 7.2, dispensed in 1 ~-tg/10 ~-tl aliquots and stored at -20°C until used. Androstenedione (Ll4 A), E 2 , P, amphotericin B, gentamicin, and HEPES were purchased from Sigma Chemical Company, (St. Louis, MO). McCoy's 5a medium (modified, without serum), sodium bicarbonate, and trypsine were obtained from GIBCO (Grand Island, NY). Organic solvents were obtained from Merck (Darmstadt, FRG), and reagents for scintillation cocktail from British Drug Houses Chemicals Ltd. (Poole, England). Patients

From each of 19 women (aged 23 to 41 years) undergoing legal sterilization via minilaparotomy under general anesthesia, a small wedge resection of the ovary containing the largest follicle(s) was performed. Forty-five follicles were obtained. All patients had regular menstrual cycles and had not been under hormonal treatment for at least 3 months prior to the sterilization. In all cases, surgery was scheduled to be performed on the fourth to eighth day of the cycle after the patients gave their informed consent to participate in the investigation. Granulosa Cell Cultures

Ovarian wedges were rinsed in sterile saline HEPES 20 mM and gentamicin 50 ~-tglml. Each follicle was freed from interstitial tissue and its diameter measured. Follicular fluid was aspirated with a 20-gauge needle. The fluid was centrifuged at 2000 X g for 10 minutes and the supernatant frozen for later steroid determinations. Collapsed follicles were placed in culture medium (McCoy's 5a modified, without serum, sodium bicarbonate 2.2 gm/1, HEPES 20 mM, gentamicin 50 ~-tg/ml, amphotericin B 2.5 ~-tg/ml, pH 7.4). Each follicle was slit open and granulosa cells were obtained through gentle scraping of the inner follicular wall

Table 1

with a platinum loop. Cells were dispersed by repeating pipetting through a Pasteur pipette and counted in a hemocytometer to determine total number of follicular cells. The suspension obtained was centrifuged in 100 X 16 mm tubes (Sterilin Ltd., Feltham, England) at 200 X g for 10 minutes. The resulting pellet was resuspended in warm culture medium. An aliquot of the suspension was counted using a hemocytometer. Cell viability was determined with the trypan blue exclusion test (viability> 60%). The remaining cell suspension was diluted with culture medium to a concentration of 15,000 to 20,000 viable cells/200 ~-tl and cultured at this cellular density on 96 wells with 0.32 cm 2 of culture area (Costar Europe Ltd., Sloterweg, The Netherlands). Cultures were grown at 37°C in a humidified 95% air:5% C0 2 incubator for 12 to 18 hours. Media then was discarded and hormonal treatments started, as described subsequently. We considered valid only those experiments conducted with cells derived from healthy follicles following previously described criteria. 12 •13 Briefly, healthy follicles had more than 50% of the ideal granulosa cell number corresponding to its follicular diameter and a rate of Ll 4 A/E 2 < 5 in follicular fluid (Table 1). Hormonal Treatments

All cultures received Ll 4 A 10- 7 M in ethanolic solution because this dose produced a maximal E 2 secretion (data not shown). Final concentration of ethanol in culture medium was 0.01 %. To validate our culture system, FSH doses of 0, 10, 100, and 1000 ng/ml were used, and the capacity of the cells to respond to the gonadotropin with an enhanced steroid output was evaluated. Having proved that cells were able to respond to FSH, we studied the effect(s) of different doses of PRL (0, 10, 100, 1000 ng/ml) on granulosa cell steroidogenesis in basal and FSH-stimulated conditions.

Characteristics of the Healthy Midfollicular Phase Follicles Used for the Experiments (n

Mean+ SEM Range a

Y

Cell number

19)

Follicular fluid steroids

Follicles Follicular diameter

=

ya

ll 4A

E2

mm

Xl(f

%

ng/ml

ng/ml

6.1 + 0.3 4-10

2.4 + 0.3 1.1-6.6

72 + 3.6 53-100

408 +50 196-1075

476 + 149 54-2778

ll4 A/E 2 1.6 + 0.2 0.3-3.8

Number of cells recovered from a follicle of a certain diameter Maximum number of recoverable cells from a full developed follicle of that same diameter ·

= 100 X ~----;-----;---;;------;-:--;:--;;-------=-:;:-:--;------;--;;-;-;~----;::-;--------:;,---­

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Cutie R. and Andino PRL effects on human granulosa cells

633

Hormonal treatments lasted 48 hours in all experiments. Conditioned media were collected and stored at -20°C until assayed for E 2 and P by radioimmunoassay (RIA). The plated cells were trypsinized at 37°C for 10 minutes with 25 ~l of a PBS solution containing 0.02% trypsine, 0.05% ethylenediaminetetraacetic acid. The reaction was stopped, adding 25 ~l of 0.02% trypsine soya bean inhibitor solution, and cells were counted in a hemocytometer. Granulosa cell numbers were not significantly different among treatment groups (data not shown). Granulosa cells obtained from each of the follicles were processed separately and each experiment contained cells from individual follicles. Results are expressed in picograms (pg) of secreted steroids per 48 hours per 1000 cells present in the well. Analytical Methods

E 2and P levels in conditioned culture media were measured by RIAs previously described, 14 and were set up and validated in our laboratory. In the present experiments, samples were assayed directly since the values corresponded closely to those obtained in lipid extracts (r = 0.987). The follicular fluid samples were assayed for E 2 and !l4A after extraction with organic solvents. Recoveries were always higher than 85%. E 2 was measured employing an antiserum with a CrOSS reaction <6% for Other C18 steroids, <2% for C19 steroids, and <3% for C21 . The intra-assay coefficient of variation (CV) was <10% and the interassay CV was <13% for E 2 RIA (sensitivity, 4.25 pg/tube). P was assayed using a well-characterized antiserum 15 being the intra- and interassay CV were <10% and <12%, respectively (sensitivity, 6.25 pg/tube). The !l4A antiserum cross-reacted <0.8% with other C19 steroids. Intra- and interassay CVs for !l4A RIA were <11% and <12%, respectively (sensitivity, 12.5 pg/tube).

treatments was evaluated by Duncan's test. A P value of 0.05 or less was considered significant. RESULTS Effect of Follicle-Stimulating Hormone on Estradiol and Progesterone Secretion

As shown in Figure 1A, medium levels of E 2 were low in control cultures (no FSH added), whereas treatment with 10 ng/ml FSH significantly increased E 2 secretion (P ~ 0.05). Peak E 2 secretion was induced at an FSH dose of 100 ng/ml. Figure 1B shows a significant increase of P secretion concomitant to the increment of FSH concentration (P ~ 0.05). It was also the '100 ng/ml FSH dose that produced a peak stimulation. Effect of Prolactin on Estradiol and Progesterone Secretion in Basal Conditions

Figures 2A and 2B show, respectively, the effect of PRL on E 2 and P secretion by human granulosa cells. As shown in Figure 2A, there is a statistically significant (P ~ 0.05) decline of E 2 levels at PRL concentrations of 10 ng/ml or higher. PRL treatment did not result in a dose-related decrease in steroid secretion compared with the different PRL doses (10 to 1000 ng/ml) and did not reflect a statistically significant difference. The effect of PRL on P secretion (Fig. 2B) was similar to that found for E 2, with an inhibition of the secretion of the steroid with PRL doses ~10 ng/ml and no dose-related effect. Effect of Prolactin on Estradiol and Progesterone Secretion in Follicle-Stimulating Hormone-Stimulated Conditions

Figures 3A and 3B show the effect of increasing doses of PRL on E 2 and P secretion, respectively, ....

8

Data Analysis

RIA data were analyzed with a program making use of a weighted logit-log regression analysis. Experimental data are presented as the mean± standard error of the mean (SEM) of measurements of triplicate cultures. Similar results were obtained in at least three separate experiments. Analysis of variance was used to assess overall statistical differences of the observed effects. The significance of individual 634

FSHing/mll

FSH(ng/mll

Figure 1 Dose-dependent effect of FSH on E 2 (A) and P (B) secretion by human granulosa cells cultured in the presence of A4A 10-7 M. Each bar represents the mean (±SEM) of triplicate cultures (*P ..;; 0.05 versus control; **P ..;; 0.05, 10 versus 100 ng/ml FSH).

Cutie R. and Andino PRL effects on human granulosa cells

Fertility and Sterility

B

A

PRLing/m/1

PRLtng/ml)

Figure 2 . Effects of increasing doses of PRL on E 2 (A) and p (B) secretion of human granulosa cells cultured in the presence of !l4 A 10-7 M. Each bar represents the mean (±SEM) of triplicate cultures (*P,.,; 0.05 versus control).

when cells were treated with FSH 10 ng/ml (plain bars). PRL doses ~10 ng/ml produced a statistically significant decline (P :o;;;; 0.05) in E 2 and P secretion as compared with controls (no PRL added). Although there was a tendency to lower steroid output with PRL dose increments, the differences were not statistically significant. The effects of varying doses of PRL on the secretion of both steroids when cells were stimulated with FSH 100 ng/ml are shown in Figures 3A and 3B (stippled bars). As can be observed, this FSH dose was able to overcome the inhibitory effects of PRL on E2 and P secretion. Treatment groups were not statistically different. Treatment with the different PRL doses did not modify plating efficiency or cell viability (trypan blue exclusion test) either in the presence or absence of FSH, indicating that PRL effects are not related to a decrease in granulosa cell number (data not shown). The previously mentioned observations, together with the recovery in steroid output found when using a maximal stimulating FSH dose, excludes any possibility of cell damage by the PRL preparation.

A

DISCUSSION

We have studied the effects of PRL on E 2 and P secretion by human granulosa cells obtained from midfollicular phase healthy follicles. To this purpose, we used a chemically defined culture system able to increase cellular steroid output when supplemented with FSH. To validate our culture system, we considered of the utmost importance the evaluation of the ability of the cells to respond to the main tropic hormone of this follicular developmental stage, i.e., FSH. Our results confirm previous reports that human Vol. 49, No.4, April1988

granulosa cells increase their steroid output when supplemented with FSH. 16 •17 However, we have demonstrated (to our knowledge, for the first time) that human cells from the midfollicular phase, with an in vivo induction of the aromatase system, respond to the in vitro FSH stimulus with a dose-dependent increase of E 2 and P secretion. In this culture system in basal conditions, PRL inhibited directly the E 2 secretion of human granulosa cells from the midfollicular phase. Lee et al. 6 reported such an effect of PRL on granulosa-lutein cells from preovulatory follicles, and Demura et al. 5 also demonstrated an inhibitory effect of the hormone on steroid release using a human ovarian perifusion system. Our finding that PRL inhibits P secretion in basal conditions confirms previous reports using human granulosa cells from different stages of follicular development4 and granulosa-lutein cells derived from patients hormonally treated for an in vitro fertilization program. 7 On the contrary, Wang et al. 18 found a stimulatory action of PRL on P secretion by rat granulosa cells, which might be an indication of a species-specificity of the hormonal action on the ovary. Treating cells simultaneously with an FSH concentration of 10 ng/ml and increasing doses of PRL result in an inhibition of the E 2 and P secretion at all PRL doses. This is in accordance with previous reports on a PRL inhibitory action on steroid release by follicles stimulated with human chorionic gonadotropin (hCG). 5 Some authors have found inhibitory actions of PRL on human granulosa cell steroidogenesis, but using higher doses of the lactogenic hormone 4- 7 and not doses as low as those used in our experiments (10 ng/ml). The differences encountered might be related to the different experimental approaches 0 FSH /0 ng/rnl BI"SH /OOng/m/

r

r

PRLtng/ml)

r

0

B

FSH /0 ng/m/ FSH /00 ng/m/

rrr PRL lng/miJ

Figure 3 Secretion of E 2 (A) and P (B) by human granulosa cells cultured in the presence of t:.4A 10-7 M, FSH 10 ng/ml (plain bars), or FSH 100 ng/ml (stippled bars), and increasing doses of PRL. Each bar represents the mean (+SEM) of triplicate cultures.

Cutie R. and Andino PRL effects on human granulosa cells

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and/or to the different sensitivity of the human cells derived from the midfollicular phase to inhibitory factors, such as PRL. Our finding that PRL may act as a negative modulator of human granulosa cell steroidogenesis might shed some light on the possible mechanisms through which hyperprolactinemia alters ovarian function. Particularly important is the partial inhibition of E 2 secretion exerted by PRL in our experiments. Estrogens are potent mitogenic factors for granulosa cells/9 stimulate their own synthesis,20 and act as obligatory mediators of FSH actions. 21 ·22 As a consequence, adequate intrafollicular levels of E 2 are necessary for normal follicular development. 12 In fact, it has been reported a negative correlation between intrafollicular levels of PRL and those of E2 and p .10.23 The interference of the follicular steroid biosynthesis produced by PRL might explain the disturbances observed in ovarian function in hyperprolactinemia. This direct deleterious action of PRL on ovarian steroidogenesis and its implications in folliculogenesis also have been suggested by other authors. 10·11 Our observation that an FSH peak stimulatory dose (100 ng/ml) is able to overcome PRL inhibition of human granulosa cell steroidogenesis might explain the restoration of ovarian cyclicity observed in hyperprolactinemic patients treated with human menopausal gonadotropins and hCG, 24 and is in accordance with the suggestion that the inhibitory effects of PRL depend on a balance between its concentration and that of gonadotropins. 25 In conclusion, our results demonstrate that PRL has a direct inhibitory effect on the steroidogenesis of human granulosa cells derived from healthy follicles of the midfollicular phase of the cycle cultured in a chemically defined system where cells are able to respond to an FSH stimulus. We might speculate that PRL is a negative modulator of follicular steroidogenesis and that such an action might be associated with the ovarian dysfunction observed in hyperprolactinemic states. Acknowledgments. We are grateful to the National Pituitary and Hormone Distribution Program, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (NIADDK) for FSH and PRL. We wish to thank Vivian H. T. James, D.Sc., for !::.4A antiserum; Fortune Kohen, M.D., for P antiserum; Focko F. Rommerts, Ph.D., for E 2 antiserum; Eduardo Cutie, D.Sc. and Delso Aquino, M.D., for human material; Ada J. Machado, M.D., for statistical analysis of results; Mrs. Lucia Paret, Mrs. Norma Banos, and Mrs. Maria A. Carbonell, for excellent tech-

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Cutie R. and Andino PRL effects on human granulosa cells

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22. Knecht M, Brodie AMH, Catt KJ: Aromatase inhibitors prevent granulosa cell differentiation: an obligatory role for estrogens in luteinizing hormone receptor expression. Endocrinology 117:1156, 1985 23. Bohnet HG, Baukloh V: Prolactin concentrations in follicular fluid following ovarian hyperstimulation for in vitro fertilization. Horm Res 22:189, 1985 24. Farine D, Mashiach S, Ben-Rafael Z, Oelsner G, Blankstein J, Lunenfeld B, Serr DM: Retrospective evaluation of human menopausal gonadotropin and human chorionic gonadotropin induction of ovulation in galactorrheic and hyperprolactinemic women. Fertil Steril 38:187, 1982 25. McNeilly AS, Glasier A, Jonasson J, Howie PW: Evidence for direct inhibition of ovarian function by prolactin. J Reprod Fertil 65:559, 1982

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