Participation of ovarian 20 alpha-hydroxysteroid dehydrogenase in luteotrophic and luteolytic processes during rat pseudopregnancy

In normal rats, before Day 12 of pseudopregnancy, minimal levels of 20 alpha-HSD activity were detected in functional CL whereas those in the residue were 3-5 times higher. When ovulation was blocked for more than 2 weeks by placing rats in a continuously lit environment before the induction of pseudopregnancy, only minimal levels of 20 alpha-HSD activity were detectable in the functional CL and residue before Day 12. In normal pseudopregnant rats, there was a linear increase in 20 alpha-HSD activity from Day 12 to 15 in the functional CL and residue, but the rate of elevation was much higher in functional CL. This tendency was much more clear-cut in rats in the continuous lighting. In immature rats in which pseudopregnancy was induced by PMSG and hCG treatment, 20 alpha-HSD activity peaked twice. The first small peak was attributed to the early regression of some of the large number of corpora lutea, and the changes in 20 alpha-HSD activity in most of the corpora lutea paralleled those in rats in continuous lighting. Bromocriptine abolished the prolactin surges, and in normal pseudopregnant rats an increase in 20 alpha-HSD activity in functional CL started from 12 h and the rate of the increase was accelerated from 36 h afterwards, while a relatively small increase was observed in the residue at 18 h and later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of LH/hCG receptors and physical state of ovarian membranes in rat pseudopregnancy

Previous investigations have demonstrated that increased ovarian function during pseudopregnancy in the rat may be associated with alterations of the physical state of membranes. Changes in rigidity of membrane lipids were observed during the formation as well as regression of corpora lutea. The effects of cyclooxygenase inhibitors (indomethacin and acetylsalicylic acid (ASA)) and of selected steroids (estradiol, testosterone and dihydrotestosterone) on the functional state of luteinized ovaries were studied. The compounds were administered to the animals in silastic capsules on different days after hCG injection. ASA and indomethacin administration on days 10 and 11 after hCG injection resulted in an increase in the LH/hCG receptor binding activity and rigidity of ovarian membrane lipids, as determined by fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene (DPH) probe. This effect was apparent within 7 days after indomethacin and ASA treatment. Both estradiol and testosterone significantly increased the ovarian LH/hCG binding activity, however estradiol did not affect the membrane lipid rigidity. Unlike testosterone, the administration of dihydrotestosterone induced a decrease in membrane lipid rigidity and reduced the accessibility of the LH/hCG receptor. Inhibitors of prostaglandin F2alpha (PGF2alpha) synthesis, as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats.     

Beta-adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.     

Plasma gonadotropins and prolactin in pseudopregnancy in the rat

Radioimmunoassay presented a method of measuring plasma levels of FSH,LH and prolactin in pseudopregnant rats. Plasma prolactin levels doubled 15 minutes following cervical probing (p .01) on the day of estrus. Plasma LH levels were not significantly elevated. Due to the use of ether anesthesia at blood collecting 3 hr before and 15 minutes after stimulation, only 1 of 16 rats developed pseudopregnancy. On Day 4 of pseudopregnancy in rats mated with vasectomized males; plasma LH was lower (p .05) than in normal rats on the first day of diestrus, perhaps due to the suppressive action of ovarian progesterone and some estrogen. FSH was higher than in normal rats (p .05) perhaps due to the lesser sensitivity of FSH to the inhibitory effect of progesterone. Large decidoumata developed by Day 9 in uterine horns traumatized on Day 4 (153 plus or minus 8 mg uterus weight compared to 1510 plus or minus 204 mg). Thus, the corpora remain functional after LH and prolactin are at normal and subnormal levels. On Day 9 plasma prolactin was lower than at Day 1 of diestrus (p .001). Plasma FSH was elevated (p .01). Plasma LH was unchanged. The degree of rise of LH levels 5 days following ovariectomy on Day 4 of psuedopregnancy or on the first day of diestrus was greater in the former group (p .02), perhaps due to rebound of LH from suppression by ovarian steroids. FSH rose equally in both groups. Prolactin remain about the same. Increased prolactin release by the adenohypophysis was briefer than expected.     

Effects of dihydrotestosterone on progesterone secretion in pseudopregnant rats

To determine if the administration of dihydrotestosterone (DHT) suppresses serum progesterone concentrations in pseudopregnant rats and if so what role the decidua play in mediating this effect, two pellets (8 mg) of DHT were inserted under each ovarian bursa on Day 9 of pseudopregnancy in hysterectomized or intact rats with or without the decidua. The treatment induced within 24 hr a 50% decline in serum progesterone concentrations in decidua-bearing rats only; a further reduction was observed on Days 11 and 12. To further determine if the effect of DHT on serum progesterone concentrations is due to its effect on luteal luteinizing hormone (LH)-receptor content, rats were similarly treated on Day 9 and the capacity of corpora lutea to bind 125I-labeled human chorionic gonadotropin (hCG) on Day 12 in all three groups of rats was examined. DHT treatment had no effect on luteal LH-receptor content in any group. These results suggest that DHT is luteolytic in pseudopregnant rats only in the presence of the decidua and it is speculated that this effect is mediated by suppressing the decidual luteotropin.     

Effect of indomethacin on the progesterone secretion of hysterectomized pseudopregnant rats

The length of pseudopregnancy and the progesterone levels in rats pseudopregnant and hysterectomized on their 2nd day and injected daily with indomethacin (500 g/0.2 ml) from the 2nd to 10th day do not differ from those exhibited by oil treated rats. On the contrary, indomethacin treatment from 11th day on, results in both a lengthening of the diestrous phase and a delayed luteolysis. These findings point out to an extrauterine prostaglandin synthesis mechanism participating in the functional luteolysis in histerectomized pseudopregnant rats.     

The effects of 5-bromo-2-thienyl-ethyl-ketone thiosenicarbazone on ovarian cyclicity and ovulation in the rat.

Pseudopregnant and cyclic rats were injected for 5 to 26 days with daily doses of 5 and/or 3 mg of 5-bromo-2-thienyl-ethyl-ketone thiosemicarbazone (70026) starting on Day 0 (the day of oestrus). The vaginal smear cytology, record of ovulation and ability to breed and conceive were compared with the results for corn oil-injected controls. Both doses of 70026 were found to cause a reappearance of pro-oestrous and/or oestrous vaginal smears within 4 to 6 days in the pseudopregnant rats, but ovulations did not occur. The 5-mg dose of 70026 inhibited ovulation and interrupted the oestrous cycle in cyclic rats, even though the daily 3-mg dose seemed to have little effect on ovulation, ovarian cyclicity, breeding or conception. In spite of the absence of an ovulation accompanying the induced pro-oestrous and/or oestrous vaginal smears in the pseudopregnant rats, the pattern of the vaginal smears suggested the occurrence of a 'delayed pseudopregnancy' in most of the pseudopregnant rats treated daily with 3 mg, but in few of those treated with 5 mg, 70026.     

Effects of hysterectomy and uterine decidualization on in vivo levels of prostaglandins in the corpus luteum of adult pseudopregnant rats

A possible role of the uterus in regulating content of luteal prostaglandins (PGs) was investigated. Pseudopregnancy was induced in adult virgin female rats by mating them with vasectomized male rats. On Day 5 of pseudopregnancy, decidualization of the uterus was induced or hysterectomy was performed. As controls, intact pseudopregnant animals with a luteal phase of 13 +/- 1 days were used. Measurements of in vivo tissue levels of PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were performed by RIA after homogenization and extraction procedures in CL of pseudopregnancy and remainder of ovaries on Days 5, 13, and 19. Serum levels of progesterone and 20 alpha-dihydroprogesterone were determined by RIA. In hysterectomized animals, PGF2 alpha levels increased 2.5-fold in corpora lutea on Day 13 compared with levels on Day 5 of pseudopregnancy, but were still lower than in control rats undergoing functional luteolysis on Day 13. Decidual-tissue-bearing rats exhibited low levels of PGF2 alpha on Day 13 of pseudopregnancy. On Day 19, when luteolysis had occurred in decidual-tissue-bearing and hysterectomized rats, as judged by plasma levels of progestins, luteal content of PGF2 alpha was elevated to a similar level as that in control animals undergoing functional luteolysis on Day 13. When data pooled from control, decidual-tissue-bearing and hysterectomized rats were analyzed, a highly significant inverse correlation (r = -0.72, n = 46, p less than 0.001) between luteal PGF2 alpha content and ratio of plasma progestins was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

The lack of a physiologic effect of the stress-induced decrease of the proestrous prolactin surge in the rat

Experiments were performed to determine whether the restraint stress-induced decrease in the proestrous prolactin (PRL) surge blocked luteolysis of the corpora lutea (CL), affected ovulation, or prevented the induction of pregnancy/pseudopregnancy in the next cycle. In all experiments rats were either stressed on proestrus and estrus, administered daily sc injections of 1 mg/day of 2-Br-alpha-ergocryptine (CB-154) for 4 days starting on diestrus II or not treated. In one experiment animals were sampled on the afternoon of proestrus to determine the effect of restraint stress on plasma PRL values and sacrificed on the morning of proestrus in the next cycle. Ovaries were removed, weighed, fixed and examined for number of CL. Restraint stress resulted in a significant increase in ovarian weight when compared to controls; CB-154 resulted in significant increases in ovarian weight when compared to stress and control animals. However, only CB-154-treated animals had a significant increase in the number of CL when compared to controls. In another experiment, animals were sacrificed on estrus of the next cycle and the oviducts examined for the number of ova. There were no differences among groups. In the final experiment, animals were placed with males of proven fertility on proestrus of the next cycle and examined for evidence of sperm in the vaginal lavage and/or vaginal plugs. CB-154 prevented the induction of pregnancy or pseudopregnancy due to a carry over effect of the drug on PRL surges. Restraint stress had no significant effect on the induction of pregnancy or pseudopregnancy. We conclude that there is no physiological significance to the stress-induced decrease of the proestrous PRL surge with respect to ovarian function or fertility.     

Possible role of cyclooxygenase II in the acquisition of ovarian luteal function in rodents

The development of the corpus luteum (CL), which involves angiogenesis, is essential for the establishment of early pregnancy. We investigated the roles of the prostaglandin synthases cyclooxygenase (COX) I and COX-II in angiogenesis and progesterone production in the newly formed CL, using inhibitors of the COX enzymes and the gonadotropin-induced pseudopregnant rat as a model. Injection of indomethacin, a nonselective COX inhibitor, on the day of ovulation and the following day decreased serum levels of progesterone, as did injection of the selective COX-II inhibitor NS-398. In contrast, a selective COX-I inhibitor, SC-560, had no effect on serum progesterone concentrations. None of the inhibitors had any effect on the weight of the superovulated ovaries or on the synthesis of progesterone by cultured luteal cells. To determine whether changes in angiogenesis are responsible for the decrease in progesterone synthesis, we measured hemoglobin and CD34 levels in luteinized ovaries following injection of COX inhibitors and measured the relative frequency of cells positive for platelet-endothelial cell adhesion molecule as a specific marker for endothelial cells. All of these parameters were reduced by the COX-II inhibitors, suggesting that changes in the vasculature are responsible for the decrease in serum progesterone. Histological examination of ovarian corrosion casts indicated that NS-398 inhibited the establishment of luteal capillary vessels following the injection of hCG. The results are consistent with the hypothesis that the activity of COX-II is associated with the formation of functional CL via its stimulation of angiogenesis.     

Evidence for dopaminergic regulation of prolactin and a luteotropic complex in the ferret

The role of dopaminergic agents in prolactin (Prl) release and the luteotrophic role of Prl and luteinizing hormone (LH) were investigated in pseudopregnant female ferrets. A single injection of the dopamine antagonist pimozide (0.63 mg/kg) resulted in a tenfold elevation of plasma Prl in anestrous females. Subcutaneous injection of pimozide on alternate days from Day 2 through Day 16 of pseudopregnancy elevated both Prl and progesterone levels. Daily treatment with the dopamine agonist 2 alpha-bromoergocryptine (bromocriptine, 4 mg/kg), from Day 2 through Day 16 of pseudopregnancy lowered levels of both plasma Prl and progesterone. Neither pimozide nor bromocriptine had a direct effect on progesterone secretion by luteal cells in vitro. Daily intraperitoneal administration of a monoclonal antibody against gonadotropin-releasing hormone from Day 2 through Day 10 of pseudopregnancy lowered both plasma LH and progesterone, but had no effect on plasma Prl concentrations. Daily administration of equine antisera against bovine LH or 100 IU of human chorionic gonadotrophin to pseudopregnant ferrets lowered progesterone levels. It is concluded that Prl release is influenced by dopaminergic compounds, and both Prl and LH are required for luteal maintenance in the ferret.     

Functional and morphological changes in the hypothalamo-pituitary-gonadal axis of aged female rats.

Age-related functional and morphological alterations in the hypothalamo-pituitary-gonadal axis were investigated in old recurrently pseudopregnant (RPP) female rats, and these alterations were compared with those in young diestrous rats. LHRH in the median eminence (ME) and mediobasal hypothalamus (MBH) as well as plasma FSH, LH, and progesterone were measured by RIA. LHRH in the lateral ME (LME) and pituitary FSH and LH were evaluated by morphometry and densitometrical immunocytochemistry. Furthermore, by light microscopy, we classified and counted the number of ovarian follicles and corpora lutea. LHRH concentrations in the ME and MBH were similar in old and young rats, whereas in old rats, plasma FSH was markedly increased, LH was moderately increased, and plasma progesterone was unchanged. The number and the total area and immunoreactivity of LHRH-labeled axon cross sections in the LME were reduced in old rats. The number of nucleated FSH-labeled cells and total FSH area and immunoreactivity were almost twice in old compared with young animals. The measurements of LH-labeled cells were not different between the two groups. In old rats, the numbers of ovarian follicles and corpora lutea were reduced and that of atretic follicles increased. In conclusion, age-related morphological impairments of LHRH axons associated with an increased number of FSH gonadotropes and higher plasma FSH in our old RPP rats suggest hypothalamic and pituitary disturbances, which may largely contribute to the complex hormonal disarrangement responsible for the decline of reproductive functions in old female rats.     

Relative roles of oestradiol and of the uterus in the maintenance of the corpus luteum in the pseudopregnant brown hare (Lepus europaeus)

Brown hares were made pseudopregnant by sterile matings or PMSG-hCG treatment (day of mating or hCG injection = Day 0 of pseudopregnancy). Progesterone secretion by the CL began 3-4 days after the ovulatory stimuli, reached maximum on Days 8 to 11 and decreased thereafter to reach low levels from Day 9 to 18, depending on the female. Cauterization of all large ovarian follicles on Day 7 resulted in an immediate luteolysis in young females, but had no effect in older ones. Oestradiol capsules implanted from Day 7 to Day 46 were able to maintain progesterone secretion until at least Day 30, in intact females as well as in females with all large follicles cauterized. Hysterectomy on Day 7 or 8 was followed by an immediate drop in progesterone concentrations; oestradiol capsules implanted at the time of hysterectomy prevented the drop in progesterone values, which remained elevated until Day 38. The induction of ovulation in females hysterectomized 2 months before resulted in CL of slightly shortened life-span. The injection of PGF-2 alpha on Day 7 of pseudopregnancy was followed by an immediate luteolysis. These results suggest that oestradiol secreted by the large ovarian follicles is the main luteotrophic factor in the brown hare. In old hares, the large amount of interstitial tissue could secrete oestrogens, and thus maintain pseudopregnancy. On Day 7 of pseudopregnancy, the uterus secretes a luteotrophic substance acting either directly on the ovary, or via the pituitary, to maintain oestradiol secretion by the follicles. In long-term hysterectomized females, the CL would be able to develop independently of any trophic substance, but for a reduced duration.     

Effect of decidual tissue on the uterine production of prostaglandins in pseudopregnant rats.

The concentration of prostaglandin F (PGF) in uterine vein plasma of non-traumatized pseudopregnant rats and pseudopregnant rats with deciduomata were not significantly different from each other at any of the times of pseudopregnancy studied (Days 7, 10 and 12). There was a significant increase in PGF levels on Day 10 in both groups of pseudopregnant animals (P less than 0-05) compared to the Day 4 values, and PGE values were significantly greater on Day 10 in the decidual tissue-bearing rats (P less than 0-01). A slight but not significant elevation in PGE concentration was observed on Days 7 and 12 in rats with deciduomata, but there was no significant difference in the control rats on Days 4, 7, 10 or 12. The results indicate that the prolongation of pseudopregnancy in rats with deciduomata is not due to a decreased production of uterine PGs and lend support to the recent suggestion of a luteotrophic effect of decidual tissue in the rat.     

Effects of PGE1 or PGE2 on luteal function in pseudopregnant rats

Effects of PGE1 or PGE2 on luteal function were studied in 163 pseudopregnant rats. PGE1 (10, 100, or 300 micrograms) given intrauterine every 6 hr did not shorten pseudopregnancy (P greater than 0.05), however, the same doses of PGE2 given intrauterine every 6 hr advanced luteolysis (P less than 0.05). PGE1 (100 or 300 micrograms) given every 4 hr intramuscular maintained levels of progesterone in peripheral blood above controls (P less than 0.05) while 100 or 300 micrograms of PGE2 hastened the decline in progesterone (P less than 0.05). The antiluteolytic effect of PGE1 was not via an inhibition of PGF secretion (P greater than 0.05) by the uterus or by induction of ovulation in treated animals. Moreover, PGE1 (100, 200, or 500 micrograms) given intramuscular every 4 hr from day 4 of pseudopregnancy until the next proestrus delayed luteal regression around 3 days (P less than 0.05). PGE2 at doses of 100, 200, or 500 micrograms every 4 hr given intramuscular consistently shortened pseudopregnancy (P less than 0.05). Lower doses were without effect (P greater than 0.05). Based on the above data it is concluded that PGE2 is consistently luteolytic whereas PGE1 is not luteolytic in pseudopregnant rats and that PGE1 may be an antiluteolysin.     

Prostaglandins F in uterine and ovarian compartments and in plasma from the uterine vein, ovarian artery and vein, and abdominal aorta of pseudopregnant rats with and without deciduomata

Prostaglandins F were measured in uterine and ovarian compartments and in uterine venous, ovarian arterial and venous and abdominal aorta plasma and the uptake of 3H-PGF2 alpha by ovarian compartments of 240 pseudopregnant rats with or without bilateral deciduomata in five experiments. Concentrations of PGF in deciduomal tissue, uterine venous plasma, ovarian arterial and venous plasma, corpora lutea, and remainder of the ovary and 3H-PGF2 alpha in the ovary were consistently as high or higher in pseudopregnant rats with deciduomata as in the endometrium, ovarian compartments, or samples of plasma from the same blood vessels of pseudopregnant rats without deciduomata. Levels of PGF were consistently 3 to 7 fold higher in uterine venous than in plasma from the abdominal aorta. It is concluded that extended luteal maintenance by deciduomal tissue is by some mechanism other than an inhibition of PGF synthesis by the uterus, transfer of PGF locally to the ovary, or uptake of PGF by the ovarian compartments.     

Mounting behavior in pregnant and pseudopregnant female rats

Mounting behavior by female rats with regular 4-day estrous cycles was significantly reduced after the induction of pregnancy, pseudopregnancy, or treatment with progesterone. The mounting behavior shown by pseudopregnant female rats was comparable to that shown by ovariectomized females. Pseudopregnant and unmated females showed mounts with pelvic thrusting with the same frequency after treatment with estradiol benzoate (EB). EB stimulated the display of mounts with pelvic thrusting in ovariectomized females, and this behavior was not affected by concurrent progesterone treatment. It is suggested that the reduction of mounting in pregnant and pseudopregnant female rats is due to decreased ovarian estrogen secretion.     

Immature rats show ovulatory defects similar to those in adult rats lacking prostaglandin and progesterone actions

Gonadotropin-primed immature rats (GPIR) constitute a widely used model for the study of ovulation. Although the equivalence between the ovulatory process in immature and adult rats is generally assumed, the morphological and functional characteristics of ovulation in immature rats have been scarcely considered. We describe herein the morphological aspects of the ovulatory process in GPIR and their response to classical ovulation inhibitors, such as the inhibitor of prostaglandin (PG) synthesis indomethacin (INDO) and a progesterone (P) receptor (PR) antagonist (RU486). Immature Wistar rats were primed with equine chorionic gonadotropin (eCG) at 21, 23 or 25 days of age, injected with human chorionic gonadotropin (hCG) 48 h later, and sacrificed 16 h after hCG treatment, to assess follicle rupture and ovulation. Surprisingly, GPIR showed age-related ovulatory defects close similar to those in adult rats lacking P and PG actions. Rats primed with eCG at 21 or 23 days of age showed abnormally ruptured corpora lutea in which the cumulus-oocyte complex (COC) was trapped or had been released to the ovarian interstitum, invading the ovarian stroma and blood and lymphatic vessels. Supplementation of immature rats with exogenous P and/or PG of the E series did not significantly inhibit abnormal follicle rupture. Otherwise, ovulatory defects were practically absent in rats primed with eCG at 25 days of age. GPIR treated with INDO showed the same ovulatory alterations than vehicle-treated ones, although affecting to a higher proportion of follicles. Blocking P actions with RU486 increased the number of COC trapped inside corpora lutea and decreased ovulation. The presence of ovulatory defects in GPIR, suggests that the capacity of the immature ovary to undergo the coordinate changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age.     

Changes in corpus luteum content of prostaglandin F2 alpha and E in the adult pseudopregnant rat

Conflicting reports exist regarding the source of luteolytic PGF2 alpha in the rat ovary. To assess the quantities of different PGs, measurements of PGF2 alpha, PGE and PGB were performed by radioimmunoassay in the adult pseudopregnant rat ovary throughout the luteal lifespan. Ovaries of 84 rats were separated by dissection into two compartments, corpora lutea of pseudopregnancy and remainder of ovary. Tissue samples were homogenized and prostaglandins extracted and determined by radioimmunoassay. During the mid-luteal and late-luteal phases, levels of PGs were significantly higher in the corpora lutea of pseudopregnancy than in the remainder of ovary. An increase of PGF2 alpha-content in the corpus luteum was registered with peak-levels of 53.9 +/- 8.5 (mean +/- SEM, N = 18) ng/g tissue wet weight at day 13 of pseudopregnancy. PGE-levels reached peak-values at day 11 of pseudopregnancy (271.6 +/- 28.4 ng/g w w, mean +/- SEM, N = 12). PGB-levels were below detection limits in all compartments for all ages studied. The present study demonstrates increased availability of PGF2 alpha in the corpus luteum during the luteolytic period, and points toward either increased luteal synthesis or luteal binding of PGF2 alpha during the luteolytic period.     

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.