Ricardo P. Deis

Progesterone Promotes Survival of the Rat Corpus Luteum in the Absence of Cognate Receptors

Abstract Progesterone production by the corpus luteum (CL) is essential for preparation of the endometrium for implantation and for the maintenance of gestation. Progesterone modulates its own production and opposes functional luteal regression induced by exogenous agents, such as prostaglandin F2α. In the present study, we evaluated whether progesterone is also capable of interfering with the process of structural luteal regression, which is characterized by a decrease in weight and size of the gland because of programmed cell death (i.e., apoptosis). We have found that a low number of luteal cells undergo apoptosis throughout gestation. On the day of parturition, but following the initial decline in endogenous progesterone production, a small increase in the number of luteal cells undergoing cell death was observed. This increase in apoptotic cells continued postpartum, reaching dramatic levels by Day 4 postpartum, and was accompanied by a marked decrease in average luteal weight. We have established that the exogenous administration of progesterone significantly reduces the decline in luteal weight observed during structural luteal regression postpartum. This effect was associated with a decrease in the number of cells undergoing apoptosis and with enhanced circulating levels of androstenedione. Furthermore, in vivo administration of progesterone delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that neither the CL of gestation nor the newly formed CL after postpartum ovulation express the classic progesterone-receptor mRNA. In summary, the present results support a protective action of progesterone on the function and survival of the CL through inhibition of apoptosis and stimulation of androstenedione production. Furthermore, this effect is carried out in the absence of classic progesterone receptors.

Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy.

In this study, we investigated whether progesterone exerts a local action regulating the function of the corpus luteum of pregnancy in rats. The luteal activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD), involved in progesterone biosynthesis, and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), that catabolizes progesterone and reduces progesterone secretion by the corpus luteum, were evaluated after intrabursal ovarian administration of progesterone in pregnant rats that had received a luteolytic dose of prostaglandin F2alpha (PGF2alpha). Luteal 3beta-HSD activity decreased and 20alpha-HSD activity increased after PGF2alpha treatment (100 microg x 2 intraperitoneally on Day 19 of pregnancy at 12:00 p.m. and 4:00 p.m.) when compared with controls sacrificed at 8:00 p.m. on Day 20 of pregnancy. This effect of PGF2alpha on the luteal 3beta-HSD and 20alpha-HSD activities was abolished in animals that also received an intraovarian dose of progesterone (3 microg/ovary on Day 19 of pregnancy at 8:00-9:00 a.m.). In a second functional study, luteal cells obtained from 19-day pregnant rats responded to the synthetic progestin promegestone (R5020) in a dose-dependent manner, with an increase in the progesterone output. In addition, the glucocorticoid agent hydrocortisone did not affect progesterone accumulation in the same luteal cell culture. We also examined by immunocytochemistry the expression of progesterone receptors (PR) in the corpora lutea during pregnancy and demonstrated the absence of PR in this endocrine gland in all the days of pregnancy studied. In the same pregnant rats, positive staining for PR was observed in cells within the uteroplacental unit, such as cells of the decidua basalis and trophoblast giant cells of the junctional zone. In addition, positive PR staining was observed in the ovarian granulosa and theca cells of growing follicles, but not in corpora lutea of ovaries obtained from cycling rats at proestrus. In summary, this report provides further evidence of a local action of progesterone regulating luteal function in the rat despite the absence of a classic PR.

Apoptosis Induced by Antigestagen RU486 in Rat Corpus Luteum of Pregnancy

Administration of RU486 to late pregnant rats results in preterm delivery 24 h after treatment and the induction of a luteolytic process after labor. We investigated whether functional changes occurring within the corpora lutea after RU486 treatment were associated with morphologic features of apoptotic cell death. Rats on d 18 of pregnancy were treated with RU486 (5 mg/kg) at 10:00 am and killed 72 h after. We studied the number of apoptotic cells in paraffin sections of the corpora lutea by routine hematoxylin and eosin (H&E) staining, and by in situ 3′ end labeling (TdT-mediated dUTP nick-end labeling [TUNEL]). The corpora lutea were also processed for electron microscopy to study ultrastructural changes after RU486 treatment. The number of cells showing apoptotic nuclei in H&E-stained sections was higher in RU486-treated animals than in controls (vehicle-treated rats). The quantification of the number of apoptotic nuclei within the corpora lutea performed by TUNEL confirmed the higher number of apoptotic nuclei in animals receiving the antigestagen compared with controls. Ultrastructurally, the luteal cells undergoing a poptosis presented a highly deteriorated cytoplasmic organization The nuclei, in an initial step of regression, displayed condensation of the chromatin, a prominent nucleolus, and a perinuclear space. In an advanced step of degeneration, the nuclei showed evidence of large irregular aggregates of condensed chromatin. Prostaglandin F2α(PGF2α), which mediates the luteolytic action of RU486, mimicked the effect of the antigestagen on the induction of apoptosis when administered to rats on d 18 of pregnancy (100 μg at 9:00 am and 1:00 pm), which were killed 72 after the last injection. In conclusion, the present results indicate that functional luteolysis in rats is associated with structural luteal regression with the morphologic features of apoptotic cell death, as demonstrated by studying the luteolytic process induced by the administration of the antigestagen RU486.

In Vivo Hormonal Environment Leads to Differential Susceptibility of the Corpus Luteum to Apoptosis In Vitro

Abstract We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.

Dual regulation of luteal progesterone production by androstenedione during spontaneous and RU486-induced luteolysis in pregnant rats

The effect of androstenedione on luteal progesterone production was studied during luteolysis preceding parturition as well as that induced by the antiprogestin RU486 in late pregnant rats. Luteal cells from animals on days 19, 20 or 21 of pregnancy and incubated with 10 microM androstenedione increased progesterone production by 99, 136, and 277%, respectively. The animals receiving androstenedione (10 mg/rat s.c.) on day 19 of pregnancy showed an increase in serum progesterone levels, a decline in luteal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity and an increase in corpus luteum weight without modifying 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity on day 21 of pregnancy. Androstenedione and testosterone but not dihydrotestosterone were able to prevent the decrease in serum progesterone concentration and corpus luteum weight observed 58 h after treatment with RU486 (2 mg/kg) on day 18 of pregnancy. However, the three androgens studied inhibited the luteal 3 beta-HSD activity but 20 alpha-HSD activity was not affected, when compared with animals receiving RU486 alone. The co-administration of androstenedione with the aromatase inhibitor 4-hydroxyandrostenedione or with the specific antioestrogen ICI 164,384 did not modify the effects induced by androstenedione in RU486-treated rats, indicating that the action of androstenedione on progesterone production and secretion at the time of luteolysis seems to occur through an androgenic mechanism and is not mediated by previous conversion of the androgens to oestrogens. In all experiments the high luteal 20 alpha-HSD activity, that characterizes a luteolytic process, was not modified by androgens. Androstenedione administered to adrenalectomized rats was also able to prevent the decrease in serum progesterone concentration observed in spontaneous or RU486-induced luteolysis. The administration of androstenedione to RU486-treated rats induced a decrease in luteal progesterone content concomitant with an increase in serum progesterone levels. These studies demonstrate that androgens during luteolysis, are able to stimulate luteal progesterone secretion, prevent the loss in corpora lutea weight and enhance the decrease in 3 beta-HSD activity, without affecting the increase in 20 alpha-HSD activity.

Levonorgestrel inhibits luteinizing hormone-stimulated progesterone production in rat luteal cells

The effects of the synthetic progestin levonorgestrel (LNG) on basal and LH-stimulated progesterone production were studied in collagenase-dispersed luteal cells obtained from 9-day pregnant rats. Luteal cells responded to ovine LH (oLH) with an increase in progesterone output which was maximal at a dose of 100 ng/ml. No effect of LNG was observed at 0.1-10 microM, but at 100 microM, it inhibited basal progesterone production. On the other hand, a dose of 10 microM LNG suppressed the stimulation of progesterone secretion induced by oLH, dibutyryl-cAMP and pregnenolone. It is suggested that the possible mechanism of action of the progestin involves a post-cAMP site and, in some way, may lead to an interference with 3 beta-hydroxysteroid dehydrogenase activity, which catalyzes the formation of progesterone from pregnenolone, the last step of progesterone biosynthesis. This study provides a different point of view supporting an autocrine control mechanism by which progesterone, the principal steroidogenic product of luteal cells, may exert a negative ultra-short loop regulation of its own biosynthesis.

Hormonal profile and reproductive performance in lactation deficient (OFA hr/hr) and normal (Sprague-Dawley) female rats.

Lactation deficiency may have important consequences on infant health, particularly in populations of low socioeconomic status. The OFA hr/hr (OFA) strain of rats, derived from Sprague-Dawley (SD) rats, has deficient lactation and is a good model of lactation failure. We examined the reproductive performance and hormonal profiles in OFA and SD strains to determine the cause(s) of the lactation failure of the OFA strain. We measured hormonal (PRL, GH, gonadotropins, oxytocin, and progesterone) levels by RIA in cycling, pregnant, and lactating rats and in response to suckling. Dopaminergic metabolism was assessed by determination of mediobasal hypothalamic dopamine and dihydroxyphenylacetic acid (DOPAC) concentrations by HPLC and tyrosine hydroxylase expression by immunocytochemistry and western blot. OFA rats have normal fertility but 50% of the litters die of malnutrition on early lactation; only 6% of the mothers show normal lactation. The OFA rats showed lower circulating PRL during lactation, increased hypothalamic dopamine and DOPAC, and impaired milk ejection with decreased PRL and oxytocin response to suckling. Before parturition, PRL release and lactogenesis were normal, but dopaminergic metabolism was altered, suggesting activation of the dopaminergic system in OFA but not in SD rats. The number of arcuate and periventricular neurons expressing tyrosine hydroxylase was higher in SD rats, but hypothalamic expression of TH was higher in OFA rats at the end of pregnancy and early lactation. These results suggest that the OFA rats have impaired PRL release linked with an augmented dopaminergic tone which could be partially responsible for the lactational failure.

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

Abstract The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.

Participation of Opioid and Serotoninergic Systems in Prolactin Secretion Induced by Hypothalamic Action of Estradiol

The aim of the present study was to determine the central effect of estradiol (E2) on the pattern of secretion of prolactin (PRL) in virgin rats and the participation of opioid and serotoninergic systems in the regulation of this secretion. Bilateral cannulae containing E2 (group E) or cholesterol (group C) were implanted in the arcuate nucleus on the day of estrus (day 0). Blood samples were obtained at 09.00, 14.00, or 18.00 h on days 1, 3, 6, or 9. All rats were blood sampled once. In group E, the PRL levels at 09.00 h on days 1 and 3 were similar to those from group C. However, higher values were obtained at 14.00 and 18.00 h, thus showing a diurnal rhythm with low levels in the morning and high values during the afternoon. No rhythm in PRL secretion was observed on days 6 and 9 in group E in which serum PRL was similarly increased with respect to group C at all times. The progesterone (P) levels paralleled PRL concentration, being significantly higher in group E at 18.00 h on day 1, at 14.00 and 18.00 h on day 3, and at all three times on days 6 and 9; the P measurements were consistent with luteotropic actions of PRL. Naloxone (NAL; 2 mg/kg i.p.) was injected at 17.30 h on days 3, 6, or 17, and 30 min later the animals were blood sampled. p-Chlorophenylalanine (pCPA; 200 mg/kg s.c.) was administered at 07.00 h on days 2, 5, or 16, and blood samples were taken 35 h later. Control E rats were injected with vehicle and blood 18.00 h on days 3 and 6 was not modified by pretreatment with NAL or pCPA. Serum P was significantly reduced after pCPA administration on days 3 and 6 and after NAL only on day 6. The increase in PRL at 18.00 h on day 17 induced by E2 was dramatically enhanced by NAL or pCPA, while these treatments did not significantly modify serum P levels. Our results indicate an inhibitory influence from both opioid and serotoninergic systems on PRL secretion induced by the long-term application of E2 in the arcuate nucleus.

Dopaminergic Mechanisms Involved in Prolactin Release after Mifepristone and Naloxone Treatment during Late Pregnancy in the Rat

Background/Aims: During late pregnancy, the antiprogesterone mifepristone facilitates prolactin release. This effect is enhanced by administration of the opioid antagonist naloxone, suggesting an inhibitory-neuromodulatory role of the opioid system. Since hypothalamic dopamine (DA) is the main regulator of prolactin release, in this study we explored the role of DA on prolactin release induced by mifepristone and naloxone treatment. Methods/Results: Rats on day 19 of pregnancy were used. Naloxone treatment did not modify the 3,4-dihydroxyphenylacetic acid/DA (DOPAC/DA) ratio or serum prolactin concentration in control rats. After mifepristone treatment, DA activity diminished significantly without modifying serum prolactin levels. Naloxone administration to antiprogesterone-treated rats did not change the DOPAC/DA ratio but increased serum prolactin. Tyrosine hydroxylase (TH) expression in medial basal hypothalamus (MBH) protein extracts was lowered by pretreatment with mifepristone, with no additional effect of naloxone. While mifepristone decreased the intensity of TH immunoreactivity in the arcuate and periventricular nuclei and in fibers of the median eminence, naloxone treatment had no further effect. Conclusions: (1) A reduction of tuberoinfundibular dopaminergic (TIDA) neuron activity is suggested by the fall of the DOPAC/DA ratio and the low expression of MBH TH; (2) this reduction facilitates prolactin secretion by naloxone, indicating that progesterone stimulates DA neurons to maintain low serum prolactin; (3) naloxone action seems to depend on a previous decrease of DA tone induced by mifepristone, without involve a direct effect on neuronal DA activity, and (4) endogenous opioids may inhibit prolactin secretion through a non-dopaminergic neuronal system that regulates prolactin secretion in which as yet undetermined prolactin-releasing factors may participate.