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Technological advances during the past decade have yielded new knowledge of luteal function and its regulation. A number of new substances with luteotrophic and lutcolytic properties have been identified in the corpus luteum, and the picture which emerges of the mechanisms responsible for physiological luteolysis is becoming increasingly complex. As luteal function/regulation during the menstrual cycle in the absence of pregnancy differs both from that in normal pregnancy, and from that in pathological pregnancy, these advances in our knowledge should prove clinically useful, particularly with regard to the early diagnosis and management of pathological pregnancy.

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Human corpora lutea (CL) of the menstrual cycle and early pregnancy were excised at operation, cut into pieces, and incubated or superfused in the presence of hCG or prostaglandin (PG) E2. After incubation, the tissue levels of cAMP and the medium concentrations of progesterone (P) were determined, while the concentration of P was analyzed after superfusion. PGE2 stimulated cAMP formation in CL from all phases of the menstrual cycle as well as from early pregnancy and caused an increase in P formation in CL from the early and midluteal phases of the menstrual cycle as well as from early pregnancy. A difference was found in the latency, the lag phase until maximal response, and the duration of response between the effects of PGE2 and hCG on both cAMP and P formation. Thus, the effect of PGE2 started more rapidly and was of shorter duration than that of hCG. The stimulatory effect of PGE2 on CL from early pregnancy was of the same magnitude as that of CL from the menstrual cycle. On the other hand, hCG had less stimulatory effect on cAMP and P formation in CL from early pregnancy compared to CL from the menstrual cycle. We conclude that PGE2 stimulates P and cAMP formation in isolated human CL from all phases of the menstrual cycle as well as in early pregnancy, indicating a luteotropic effect of this PG.

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Experiments were carried out to elucidate whether the previously reported inhibitory effect of prostaglandin (PG) E2 on 3H-proline incorporation into total protein of specimens from the isolated preovulatory human follicular wall could be exerted also by other prostanoids. Moreover, investigations were designed to explore if the previously documented inhibitory effect of steroids on 3H-proline incorporation could be mediated by endogenously formed PGs. Tissue specimens from the apical wall of follicles were incubated in the presence of PGF2 alpha, 6-keto-PGF1 alpha, progesterone (P) or 17 beta-estradiol (E2). The steroids were added alone or in combination with the PG synthetase blockers, indomethacin or 5, 8, 11, 14-eicosatetraynoic acid. Following incubation with 3H-proline for 2-4 h, the incorporated radioactivity was determined. PGF2 alpha and 6-keto-PGF1 alpha had no effect on 3H-proline incorporation. Both P and E2 induced a significant decrease of 3H-proline incorporation in preovulatory follicles, whereas similar effects in unripe follicles were statistically significant only for P. The steroid effects were not influenced when blockers of PG synthesis were added concomitantly. It is concluded that among the "classical" PGs PGE2 has a unique effect on collagen metabolism in the human follicular wall. E2 and especially P have a similar inhibitory effect on collagen formation as reflected by measurements of 3H-proline incorporation, an effect which does not appear to be primarily mediated by endogenous PGs.

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From each of 43 women who had gynecologic laparotomy at different and well-classified stages of the luteal phase of the cycle, the corpus luteum (CL) was excised, cut into pieces, and incubated for short time periods (30 to 240 minutes). Incubations were performed in the absence and presence of human chorionic gonadotropin (hCG) and different concentrations of oxytocin ([OT], 0.1 to 1000 mIU/ml). After incubation, the tissue levels of cyclic adenosine 3',5'-monophosphate (cAMP) and the media concentrations of progesterone (P) were determined. Although hCG stimulated both cAMP and P formation in CL of all ages, OT alone had no effect on the basal production of these parameters. However, under certain experimental conditions, OT counteracted the stimulatory effect of hCG on both cAMP and P formation. Because the maximal antigonadotropic effect was found for concentrations of OT in the magnitude of the physiologic intraluteal concentration, these in vitro data suggest a role for OT in the luteolytic process of humans.

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Oxytocin immunoreactivity was determined in follicular fluid from human follicles at different stages of development. The concentration of oxytocin was highest in pre-ovulatory follicles. The measured oxytocin was found to co-elute with synthetic oxytocin in an h.p.l.c. system. The influence of oxytocin on the incorporation of [3H]proline into isolated human follicular wall was studied in vitro. Oxytocin induced a decrease of radiolabelling in both unripe and pre-ovulatory follicles, indicating an inhibitory effect on collagen synthesis. This effect of oxytocin was not affected by indomethacin. An oxytocin analogue, 1-deamino-2-D-Tyr(OEt)-4-Thr-8-Orn-vasotocin, did not inhibit the incorporation of [3H]proline. The pre-ovulatory augmentation of oxytocin concentration in follicular fluid might reflect a physiological role for oxytocin in the ovulatory process. This assumption is strengthened by the observation that oxytocin may influence follicular collagen metabolism in vitro and by this means participate in the regulation of follicular rupture in the human.

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In order to investigate the influence of danazol on steroidogenesis and gonadotropic responsiveness of human follicular cells, granulosa and thecal cells of preovulatory follicles were isolated and separately incubated for short term periods. Human chorionic gonadotropin (hCG) (100 IU/ml), FSH (0.5 IU/ml) and danazol (10 micrograms/ml) alone or in combination were added to the incubation medium. Following incubation the cellular cyclic adenosine 3'5' monophosphate (cAMP) levels and the medium content of progesterone (P), androstenedione (A) and 17 beta-estradiol (E2) were determined. All follicles included in the study were classified as nonatretic and well developed, i.e. less than 3 days before ovulation. Human chorionic gonadotropin caused an increase in cAMP formation in both cell-types and this effect was significantly counteracted by danazol in vitro. In granulosa cells danazol tended to counteract a stimulatory effect of FSH on cAMP formation. No significant influence of danazol was found on the basal steroid formation of both cell types during short term incubation. On the other hand, danazol significantly counteracted the FSH stimulated P formation of the granulosa cells and the hCG stimulated A and E2 formation of the thecal cells. It is concluded that danazol inhibits gonadotropin-stimulated steroidogenesis locally in the human follicular cells and that this effect of danazol is mediated via the cyclic AMP system.

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Corpora lutea (CL) were excised from each of 24 women undergoing laparotomy at different and well-classified stages of the luteal phase of the menstrual cycle. Pieces were incubated for intervals between 30 min and 4 h, the incubations being carried out in the presence or absence of human chorionic gonadotrophin (HCG) and oestradiol-17beta (E2) along and in combination. Following incubation, the tissue levels of cAMP and the media concentrations of progesterone and, in certain cases, pregnenolone, were determined. HCG stimulated the production of cAMP and progesterone in CL of all ages. This effect on the formation of progesterone was significantly counteracted by E2 in CL of the early and mid-luteal phases. The addition of indomethacin to the incubation media did not alter this anti-gonadotrophic effect of E2 in-vitro. The formation of cAMP was not influenced by E2 in-vitro. These results suggest that the well-known luteolytic effect of E2 in-vivo is caused by inhibition of the gonadotrophin-stimulated synthesis of progesterone by corpora lutea, an effect mediated neither by prostaglandins nor cAMP.

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Granulosa and thecal cells from human preovulatory follicles, extirpated from the ovaries of women undergoing sterilization by minilaparotomy, were incubated for 2 hours in the presence or in the absence of human gonadotropin (hCG). The follicles were obtained from 16 women, 8 of whom had previously been treated with clomiphene citrate (CC) to induce follicular maturation. The tissue contents of cyclic AMP (cAMP) and the content of progesterone (P), testosterone (T) and estradiol-17 beta (E2) in the incubation medium were measured. Granulosa and thecal cells from control and CC-induced follicles all responded to hCG in vitro with increased formation of cAMP. Both granulosa and thecal cells from CC-induced follicles produced larger amounts of P in vitro than cells from spontaneously matured follicles. There were no other differences in the patterns of steroid formation or reactivity to hCG between the cells from the two types of follicle. It is concluded that CC treatment in vivo induces the growth and maturation of preovulatory follicles with apparently normal biochemical characteristics, as far as the content of and capacity to form cyclic AMP, progesterone, testosterone and estradiol-17 beta are concerned.

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A young woman with typical polycystic ovary syndrome (PCO) underwent laparotomy for moderately differentiated endometrial cancer. Specimens from the hyperplastic thecal and stromal tissue of the ovaries were incubated for 2 hours in the presence or absence of hCG, 100 IU/ml. Following incubation the tissue content of cyclic AMP and the amounts of progesterone (P), androstenedione (A), testosterone (T) and estradiol-17 beta (E2) in the incubation medium were analysed. For comparison, thecal cells from normal ovaries of regularly menstruating women were incubated under identical conditions. In vivo, the PCO ovaries secreted several-fold greater amounts of T than normal ovaries. In vitro, the thecal cells were much more active, steroidogenically, than the stromal cells of the PCO ovary. Furthermore, the hyperplastic thecal cells of the PCO ovary produced several-fold greater amounts of androgens, and appeared more sensitive to stimulation with hCG, as compared with thecal cells from normal ovaries. The results indicate that in women with PCO associated with endometrial cancer the hyperplastic thecal cells are a significant site of abnormal androgen production and abnormal sensitivity to gonadotropin.

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In order to characterize the patterns of steroid production and gonadotropin responsiveness in growing human follicles, follicular thecal and granulosa cells were incubated for two hours in the presence or absence of human chorionic gonadotropin (hCG). After incubation, tissue cyclic AMP (cAMP) levels and medium content of progesterone (P), androstenedione (A) and estradiol-17 beta (E) were determined. A was the dominant steroid formed by the thecal cells, regardless if these were derived from small (diameter: 4-7.5 mm) or from large (diameter: 8-15 mm) follicles. Granulosa cells from small follicles formed minimal amounts of all steroids measured, while granulosa cells from large follicles produced considerable amounts of E in vitro. Thecal cells from both small and large follicles increased their production of cAMP in the presence of hCG. Steroid formation was significantly increased by hCG in thecal cells from large follicles only. Granulosa cells from large follicles responded to hCG in vitro with increased cAMP and steroid formation, while granulosa cells from small follicles appeared insensitive to hCG in vitro.

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From each of 14 women undergoing laparotomy at different stages of the luteal phase of the cycle, the corpus luteum (CL) was excised, cut into pieces and incubated for 120 min. Incubations were performed in the absence and presence of hCG and prostaglandin F2 alpha (PGF2 alpha). In addition, noradrenaline (NA) was added to the incubation medium of newly formed CL, and the adrenergic blocker propranolol to incubated specimens of mid-luteal phase CL. After incubation the tissue levels of protein and cyclic AMP (cAMP) and the media concentrations of progesterone (P) were determined. In young CL, PGF2 alpha did not interfere with the stimulatory effect of hCG on neither cAMP nor P formation, while in CL of the mid-luteal phase PGF2 alpha significantly counteracted the stimulatory effect of hCG on these two parameters. In the presence of propranolol, however, this antigonadotrophic effect of PGF2 alpha in vitro was abolished. Furthermore, when NA was added to specimens of young CL, PGF2 alpha significantly counteracted the stimulatory effect of hCG on both cAMP and P formation. These in vitro data show that endogenous catecholamines may regulate the antigonadotrophic, 'luteolytic' effect of PGF2 alpha in human CL.

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The incorporation of proline, a precursor for collagen specific hydroxyproline, is regarded to reflect the metabolism of collagen. In vitro experiments were carried out to investigate the influence of sex steroids on the incorporation of [3H]proline into the human follicular wall. Tissue pieces from the apical wall of follicles at different stages of development were incubated in the presence of the steroids and [3H]proline, and the tissue bound radioactivity was determined. Progesterone induced a decrease of radiolabelling in both unripe and pre-ovulatory follicles. Androstenedione caused a similar effect but only in pre-ovulatory follicles. The influence of oestradiol-17 beta on the incorporation of [3H]proline was less pronounced with a tendency towards a decrease of radiolabelling in both types of follicles. It is suggested that the observed biochemical changes induced by sex steroids are of importance for connective tissue remodelling preceding human follicular rupture.

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In order to show whether or not danazol exerts direct effects on the cells of the human ovarian follicle, follicular granulosa and thecal cells of follicles in different stages of development are currently being investigated in short-term incubation experiments and during more prolonged cell culture. Cells are isolated by free-hand dissection from follicles of 8 to 25 mm diameter, corresponding approximately to cycle day 7 to 14. Granulosa and thecal cells have so far been incubated for 2 to 4 hours in the presence or absence of hCG and/or danazol. A small number of cell cultures of granulosa cells have been performed for up to 6 days in the presence or absence of danazol, hFSH, and hLH, in various combinations. The progesterone (P), estradiol-17 beta (E) and androstenedione (A) content of the incubation or culture medium and the tissue content of cyclic AMP (cAMP) were measured. Danazol (10 micrograms/ml) inhibited thecal cAMP production in most short-term experiments but only the hCG-stimulated cAMP production of granulosa cells. Short-term production of P and E by granulosa and of A and E by thecal cells was also inhibited by danazol in certain circumstances. In tissue cultures of granulosa cells lasting up to 4 days, danazol (1 and 10 micrograms/ml) inhibited both the FSH and LH-stimulated P production. Provided that any unspecific effects of the concentrations of danazol used can be ruled out, the results seem to indicate that this substance may act directly upon steroidogenesis at the ovarian follicular level.

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The present review briefly summarizes the current view of the mechanisms whereby prostaglandins (PGs) may act to modulate ovarian function. Particular concern is devoted to the aspects of ovarian function which are under current investigation by the authors, namely follicular maturation and rupture, granulosa cell and oocyte maturation, and the formation and maintenance as well as the regression of the corpus luteum (CL). PGs are formed in the granulosa cells of the preovulatory follicle in response to gonadotropin action and are at their highest levels around the time of ovulation. PGs are believed to be obligatory for follicular rupture, but their mechanism of action is still unknown. In the process of oocyte maturation PGs can mimic the stimulatory effects induced by LH or hCG, but are probably not obligate mediators of these effects. The importance of PGs for CL formation and maintenance is so far unsettled, and PGs may be regarded more as modulators than as inducers for these processes. The most well-documented effect of PGs is the induction of CL regression. Evidence is presented that PGs may have this effect also in the human.

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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 (hCG). To six of these women, an ovulatory dose of hCG (9000 IU) 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 17 beta-estradiol (E2) were determined. All follicles appeared healthy and well developed, and the oocytes recovered were morphologically normal and mature. Exposure to hCG 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 hCG in vitro.

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The effect of prostaglandin (PG) E2 on the incorporation of 3H-glucosamine in the human follicular wall was investigated in vitro. Follicles were obtained from women undergoing sterilization via minilaparotomy. Tissue pieces of the follicular wall were incubated in the presence of PGE2 and 3H-glucosamine and the tissue-bound radioactivity was determined. PGE2 induced a significant increase of radiolabelling in the apex of the mature follicle wall but did not significantly affect the incorporation in other parts of the wall, nor in the wall of less developed follicles. The incorporation of glucosamine, a precursor of glucosaminoglycans, is regarded to reflect the synthesis of proteoglycans. The demonstrated increase in the incorporation of 3H-glucosamine in the apical wall of the mature follicle in response to PGE2 indicates that PG-mediated stimulation of proteoglycan synthesis is of importance for follicular rupture.

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Follicular wall specimens were taken from various parts of human preovulatory follicles as well as from less developed follicles. The tunica albuginea was isolated and incubated for 2 h in Krebs Ringer bicarbonate buffer containing 3H-proline in the presence or absence of PGE2 (0.1 microgram/ml). Following incubation the incorporated radioactivity was determined and related to the protein content of each strip. PGE2 had a specific effect on the incorporation of 3H-proline into protein in the follicular wall. In the apical part of the preovulatory follicle a significantly decreased incorporation was registered, indicating a reduced net synthesis of collagen. However, PGE2 was without effect on specimens derived from the non-apical parts of the preovulatory follicles as well as from the less developed follicles. It is suggested that the registered biochemical changes induced by PGE2 are of importance for the process of human ovulation.

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From each of 24 women undergoing minilaparotomy at various stages of the luteal phase of the cycle, the corpus luteum (CL) was excised in toto, decapsulated, cut into pieces, and incubated for short time periods (5--120 min). Incubations were carried out in the absence and presence of hCG, prostaglandin F2 alpha (PGF2 alpha) and PGE2, both alone and in combination. Dating of the CL was done meticulously using several parameters. After incubation, the tissue levels of cAMP and the media concentrations of progesterone (P) were determined. The basal P production in vitro was highest in CL of the midluteal phase. hCG stimulated cAMP formation in CL of all ages, with the highest levels of cAMP being produced in CL of the midluteal phase. PGE2 was found to increase cAMP formation and potentiate the hCG effect in young CL, but not in CL of the midluteal phase. PGF2 alpha, alone or in combination with hCG, had no effect on cAMP or P formation in either young or old CL, while in CL of the midluteal phase, PGF2 alpha significantly counteracted the stimulatory effect of hCG on both cAMP and P formation. These in vitro data show that PGF2 alpha is capable of inducing functional luteolysis in humans.

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