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 apoptosis 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 F2,alpha(PGF2alpha), 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 microg 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.

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.

Expression of heat shock protein 25,000 in rat uterus during pregnancy and pseudopregnancy.

In previous studies, we found that the human estrogen-regulated heat shock protein (hsp) 27 (human homologue of rat hsp25) is modulated in the endometrium during the different phases of the menstrual cycle and that it is present in endometrial predecidual cells and in decidual cells attached to the placenta. In the present report, we describe the cell type-specific pattern of hsp25 expression in the rat uterus during the periimplantation period as well as during early and late decidualization and placentation. The hsp25 expression pattern was also analyzed in pseudopregnant rats with deciduomas. Immunocytochemistry was performed with an antibody generated against a chimeric hybrid protein containing the N-terminal of the murine hsp25 and the C-terminal of the human hsp27. During pregnancy at the time of implantation, hsp25 was expressed in the endothelial cells of the endometrial vessels and in the luminal epithelium of the antimesometrial region. As pregnancy advanced, hsp25 appeared in predecidual/decidual cells close to the implantation region and then expanded to the mesometrial region. This expression pattern was very similar during pseudopregnancy. Hsp25 was strongly expressed in trophoblastic giant cells beginning on Day 11 of gestation; less expression was noted in the junctional and labyrinth zones of the chorioallantoic placenta (in some cells lining the vascular spaces). In all the disparate cell types that expressed hsp25, the presence of the protein did not correlate with cell proliferation or with apoptosis but with the state of differentiation. Some placental PRL-family members with molecular weights similar to that of hsp25 are also present in antimesometrial decidua and in differentiated trophoblast giant cells; therefore, in this study we eliminated the possibility that our antibody was recognizing prolactin. We also determined that the hybrid hsp25/27 protein did not bind prolactin receptors, and noted that the hsp25 immunostaining pattern was not identical to that of decidual prolactin. In conclusion, the striking cell type-specific timing of hsp25 expression points to hsp25 as a molecule that is important during the implantation, decidualization, and placentation processes.

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.

Steroid receptors and heat-shock proteins in patients with primary biliary cirrhosis.

Primary biliary cirrhosis has a definite female preponderance. Increased estrogen levels have been found in patients with this disease; however no studies indicate the status of sex hormone steroid receptors in primary biliary cirrhosis patients. In this study the occurrence and distribution of estrogen receptors, progesterone receptors and androgen receptors in liver biopsy specimens from patients with primary biliary cirrhosis were examined and compared with these receptors in the normal liver. In addition, three heat-shock proteins associated with steroid receptors (90 kD, 70 kD and 27 kD) were examined. All of the receptor proteins were detected on immunocytochemical study using specific receptor antibodies; monoclonal and polyclonal antibodies were also used to detect the heat-shock proteins. Normal bile duct epithelial cells displayed low-to-moderate amount of estrogen receptors and abundant 90- kD, 70- kD and 27-kD heat-shock protein expression, whereas normal hepatocytes showed moderate estrogen receptor and 90-kD heat-shock protein and high 70-kD heat-shock protein expression. Expression of 70-kD heat-shock protein was due mainly to the constitutive form of this protein (hsc72). In patients with primary biliary cirrhosis, significant increases in estrogen receptor and 90-kD heat-shock protein content were seen in bile duct cells and in hepatocytes. Levels of 27-kD heat-shock protein were also increased in some of the primary biliary cirrhosis biopsy specimens. The expression of progesterone receptor and androgen receptor was very low in normal and primary biliary cirrhosis bile duct cells and hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Colocalization of estrogen and progesterone receptors with an estrogen-regulated heat shock protein in paraffin sections of human breast and endometrial cancer tissue.

We have studied by immunocytochemistry and monoclonal antibodies the presence and localization of estrogen receptors, progesterone receptors, and a 24-kD estrogen-regulated heat shock protein in biopsies from breast and endometrial cancer patients. Three different tissue processing protocols were used to colocalize the antigens in the same tissue sections: a) frozen sections, b) formalin fixation with routine paraffin embedding, and c) picric acid-formaldehyde (PAF) fixation with a rapid embedding in paraffin. Frozen sections showed good receptor staining but poor 24-kD protein immunoreactivity, while routine paraffin sections (with or without DNase pretreatment) were inadequate to reveal the nuclear receptor proteins at the same level seen in frozen sections. On the other hand, all three proteins could be detected satisfactorily in PAF-fixed paraffin-embedded tissue. Using this procedure we were able to visualize 24-kD protein and estrogen receptor or progesterone receptor in individual cells in paraffin sections. The study revealed that in all of the estrogen receptor positive breast and endometrial tumor samples, almost 90% of the cells expressing the cytoplasmic 24-kD protein contained estrogen receptor in the cell nucleus. In contrast, 24-kD immunoreactive cells did not express progesterone receptors in almost 40% of the progesterone receptor positive tumor samples.

Intensification of the immunocytochemical reaction by staining both sides of tissue sections.

We describe how the efficiency of immunostaining may be increased by staining paraffin sections on both sides. This modification exposes more antigenic binding sites per unit tissue area, as shown by the peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex methods. Using antigen-rich tissue samples, the modified procedure made it possible to use more dilute primary antiserum or to reduce the incubation time of the tissue with the primary antibody. Alternatively, in tissue samples with sparse antigenic sites, the procedure made it possible to visualize and document very weak immunoreactivities.

Immunocytochemical evidence for the ability of the human pharyngeal hypophysis to respond to change in endocrine feedback.

Two pharyngeal hypophyses from patients with endocrine disorder were examined light microscopically and immunocytochemically. The pharyngeal hypophysis from a patient with primary hypothyroidism was hypertrophic, with TSH cell hyperplasia; while that from a patient treated with metoclopramide, a dopamine-receptor-blocking drug, showed PRL cell hyperplasia. These findings strongly suggest that under certain circumstances the pharyngeal hypophysis is able to respond with specific changes to variations in the endocrine feedback.

Identification of seven hormone-producing cell types in the human pharyngeal hypophysis.

Eight adult human pharyngeal pituitary glands taken at autopsy were studied by immunocytochemistry to reveal the presence of ACTH-, lipotropin-, FSH-, LH-, TSH-, PRL-, and GH-immunoreactive cells. All of these cell types were found and quantitated in pharyngeal hypophyses from patients with no evidence of endocrine disorder. The percentage of the seven hormone-producing cell types varied from gland to gland from 1-30%; there were no marked histological differences between sexes or attributable to age. The cellular composition of the pharyngeal hypophysis shows that this gland has the capacity to produce at least seven hormones.

Constitution and behavior of follicular structures in the human anterior pituitary gland.

The follicular structures present in the human pituitary gland were studied, at the light-microscopic level, using histochemical and immunocytochemical techniques. The antisera applied in the peroxidase-antiperoxidase procedure were anti-hFSH beta, anti-hLH beta, anti-hPRL, anti-hGH, anti-hTSH beta, anti-hLPH beta, anti-pACTH, and anti-hACTH. In the 10 normal pituitaries examined, follicles were always found in the three areas of the adenohypophysis. The wall of the pars distalis follicles showed the seven immunoreactive cell types studied, while follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were the only ones present in the wall of the pars tuberalis follicles. Most of the cell types studied were also present in the wall of the intermediate area follicles, but these follicles had characteristics not found in the other two areas. They were very large, with frequent interconnections forming a three-dimensional network of anastomotic cavities, and the colloid had different histochemical affinity. None of the hormones studied could be detected by immunocytochemistry within the follicular colloid. Three of the ten pituitary adenomas examined showed numerous follicular structures. Some of the follicles in the adenomatous pituitaries were similar to those found in the normal adenohypophysis, but there were also follicles filled with only traces of colloid and numerous blood cells in the cavity, and follicles filled with neoformed connective tissue. In one of these cases, FSH/LH immunoreactive adenoma cells were seen in the wall of the follicles. The results obtained suggest that the finding of pituitary adenomas with follicular structures is not uncommon and that the follicles originate from the tumor cells. In addition, the follicles seem to have several functional stages, explaining the finding of different types of follicular formation.