Temporal regulation of extracellular signal-regulated kinase 1/2 phosphorylation, heat shock protein 70 and activating transcription factor 3 during prostaglandin F-induced luteal regression in pseudopregnant rats following heat stress.

The aim of the present study was to investigate the effects of heat stress on heat shock protein (HSP) 70 expression and mitogen-activated protein kinase (MAPK) and protein kinase (PK) B signalling during prostaglandin F (PGF)-induced luteal regression. During pseudopregnancy, rats were exposed to heat stress (HS, 40°C, 2h) for 7 days and treated with PGF or physiological saline on Day 7; serum and ovaries were collected 0, 1, 2, 8 or 24h after PGF treatment. The early inhibitory effect of PGF on progesterone was reduced in HS rats. HSP70 expression in response to PGF was significantly enhanced in HS rats. PGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was significantly greater in the HS group; however, HS rats exhibited elevated basal levels of phosphorylation of p38 MAPK, but not ERK1/2. PGF treatment increased expression of activating transcription factor (ATF) 3 at 2h, which was inhibited by heat stress. Evaluating PKB signalling revealed that phosphorylation of p-Akt (Thr308 and Ser473) was reduced at 8 and 24h after PGF treatment in both non-heat stress (NHS) and HS groups, but there were no significant differences between the HS and NHS groups at any of the time points. In conclusion, the present study provides further evidence that heat stress may enhance HSP70 and affect ERK1/2 and ATF3 expression, but not Akt activation, during PGF-induced luteal regression in pseudopregnant rats.

Spontaneous decidualization in pseudopregnant rats with vitamin E deficiency.

Successful implantation of an embryo requires adequate depth of invasion in the endometrium, which depends upon decidualization. The aim of the present study was to elucidate why humans experience spontaneous decidualization and menstruation while most other mammals do not. We established a spontaneous decidualization model in pseudopregnant rats with vitamin E deficiency (VED) to investigate mechanisms associated with spontaneous decidualization. Vaginal smears were used to monitor bleeding while vitamin E levels were analyzed with a commercial vitamin E assay kit. Trypan blue staining was used to observe the implantation site at 5.5 days post-coitum (dpc). Uterine morphology, estradiol (E2) and progesterone levels, and the anti-oxidation system were evaluated at 5.5, 7.5, and 9.5 dpc. The proportion of rats in the VED group exhibiting endometrial bleeding gradually increased (5.9%, 32.3%, and 50%) over three consecutive cycles of pseudopregnancy. Vitamin E levels in the VED group were markedly lower compared to the control group in both the plasma and uterus, while the level of vitamin E in the liver did not differ between the control and VED groups. Spontaneous decidualization in the VED group was validated by histological examination and immunohistochemistry. At 5.5 dpc, the mean serum E2 level in the VED group was more than twice that of the control group. The mean total anti-oxidizing capability, catalase level, and glutathione peroxidase activity were significantly reduced in the decidualized portion of the VED group compared to controls, while the malondialdehyde level was also significantly higher in the decidualized portion of the VED group. We hypothesize that the E2 surge at 5.5 dpc and increasing levels of reactive oxygen species are responsible for spontaneous decidualization in VED rats.

Progesterone withdrawal I: pro-convulsant effects.

Pro-convulsant withdrawal properties have been reported for a variety of GABA-modulatory drugs, such as the benzodiazepines (BDZs, [S.E. File, The history of BDZ dependence: a review of animal studies, Neurosci. Biobehav. Rev. 14 (1990) 135-146; P.R. Finley, P. E. Nolan, Precipitation of BDZ withdrawal following sudden discontinuation of midazolam, DICP 23 (1989) 151-152]), barbiturates and ethanol [N. Kokka, D.E. Sapp, U. Witte, R.W. Olsen, Sex differences in sensitivity to pentylenetetrazol but not in GABAA receptor binding, Pharm. Biochem. Behav. 43 (1992) 441-447]. In this report, we test the hypothesis that pro-convulsant effects are produced by withdrawal from the GABA-modulatory neurosteroid 3alpha-OH-5alpha-pregnan-20-one (3alpha,5alpha-THP) after sustained exposure to elevated circulating levels of its parent compound progesterone (P). Seizure activity was precipitated by picrotoxin or with the BDZ inverse agonist n-methyl-beta-carboline-3-carboxamide (beta-CC), and a seizure rating determined 24 h after abrupt discontinuation of P following a multiple withdrawal/chronic administration paradigm. In some cases, a pseudopregnant rat model was employed to produce increased ovarian production of P prior to withdrawal (ovariectomy). Rats undergoing P withdrawal exhibited greater seizure-like activity than vehicle-treated controls, and received seizure scores in the same range as rats undergoing BDZ withdrawal. Administration of a 5alpha-reductase blocker, MK-906, along with P, prevented this pro-convulsant effect of P withdrawal, suggesting that the GABA-modulatory 3alpha,5alpha-THP is the active compound responsible for this withdrawal effect. Combined administration of P and diazepam produced synergistic effects upon withdrawal and produced a seizure score higher than observed after withdrawal from either agent alone. These results suggest that P exhibits withdrawal properties via the neuroactive steroid 3alpha, 5alpha-THP, that include exacerbation of seizure activity. These results may have clinical relevance, as increased incidence and severity of seizures has been reported in susceptible women during times of declining circulating levels of P across the menstrual cycle [T. Backstrom, B. Zetterlund, S. Blom, M. Romano, Effects of intravenous progesterone infusions on the epileptic discharge frequency in women with partial epilepsy, Acta Neurol. Scand. 69 (1984) 240-248; A.G. Herzog, Progesterone therapy in women with complex partial and secondary generalized seizures, Neurology 45 (1995) 1660-1662].

Antiproliferative effects of inducible nitric oxide synthase inhibition on decidualization in pseudopregnant rats.

The aim of this study was to assess the involvement in decidual proliferation of nitric oxide (NO), a regulator of many cellular processes, that is synthesized from L-arginine by NO synthase. The investigation was conducted on pseudopregnant (PG) rats in which the decidual cell reaction, the basis for the decidualization process, was surgically induced by uterine trauma on PG Day 4. Groups of animals (n = 5) were pretreated with either 2 doses/day of N(G)-nitro-L-arginine methyl ester (L-NAME) that inhibits NO synthase, or twice daily doses of L-NAME plus L-arginine combined. Drug application times coincided with 3 hr after lights on or 3 hr before lights off. The two treatment regimens (PG Days 1-4 or 5-8) respectively preceded or followed decidual induction. Animals were sacrificed at mid-light on PG Day 9, the day of maximal growth response to the deciduogenic stimulus. Parallel, time-dependent increases in both NO synthase activity and decidual growth occurred mainly in the endometrium. L-NAME produced reductions in endometrial and myometrial growth that were reversed by the combined L-NAME plus L-arginine treatments. These inhibitory effects by L-NAME were caused by only the pretraumal (PG Days 1-4) administration. Hormonally, circulating progesterone levels were similarly affected by this early treatment and may also contribute to the reduced decidual sensitivity. In contrast, serum estradiol, along with the zinc metalloenzymes, alkaline phosphatase and the matrix metalloproteinases--prominent decidualization biomarkers--were all unaffected by either the pre- or post-decidual induction dosings. The study demonstrates that inducible NO synthase/endogenous NO may physiologically participate in uterine metabolism during the decidual cell reaction. Moreover, by virtue of L-NAME inhibition of the decidual response, it appears that NO synthase/NO may influence decidual growth either by directly increasing uterine sensitivity to the deciduogenic stimulus or by indirectly affecting endometrial vascularity and subsequent availability of decidual metabolites.

Radiation-induced mammary tumorigenesis in pseudopregnant rats mated with vasectomized partners.

Pseudopregnancy was induced in Wistar-MS female rats by mating with male rats 1 month after vasectomy. Pseudopregnant rats received whole-body irradiation with 2.6 Gy gamma-rays at day 7 of pseudopregnancy and were then implanted with a DES pellet as a tumor promoter. In the control groups, virgin 2.5 month-old rats, and normal pregnant rats at day 7 of pregnancy were also exposed to the same dose of radiation. The incidence of mammary tumors in rats irradiated in pseudopregnancy (52.6%) was significantly higher than that in the irradiated virgin rats (20.8%), but was not significantly different from that observed in rats irradiated in normal pregnancy (66.7%). The appearance of the first mammary tumor in the rats irradiated in pseudopregnancy occurred 4 months earlier than that in the normal pregnancy group. The proportion of adenocarcinomas in total tumors was 16.7, 0 and 38.5% in rats irradiated in pseudopregnancy, virgin and pregnancy groups, respectively. The incidence of adenocarcinomas was not significantly different among the three groups.

Effects of alloxan-induced diabetes on corpus luteum function in the pseudopregnant rat.

The effects of alloxan-induced diabetes mellitus on rat ovarian structure and function were examined throughout pseudopregnancy (PSP). Animals received either saline (C) or alloxan (40 mg/kg) treatment on the day of proestrus (PA) preceding PSP or on day 1 (D-1A) of PSP (day 0 = ovulation). Serum samples were analyzed by radioimmunoassay for progesterone (P) and 17-beta-estradiol (E) levels and compared with the corresponding changes in ovarian and uterine weights in C, PA, and D-1A rats. In addition, the effects of daily treatment with 6 IU ovine insulin (AI) on serum P levels were assessed in D-1A-treated rats and compared with controls. Alloxan treatment effectively elevated blood glucose levels (P less than or equal to 0.01) in PA and D-1A groups as compared with controls or AI rats. Alloxan treatment reduced both ovarian and uterine weights of PA and D-1A groups as compared with C and AI rats. Serum P levels were significantly reduced in PA (P less than or equal to 0.01) and D-1A (P less than or equal to 0.05-0.01) rats as compared with control rats throughout PSP. Daily insulin treatment reversed the suppressive effects of D-1A treatment on serum P levels, but did not restore luteal function to control levels. Neither C nor D-1A groups exhibited any marked differences in serum E levels throughout PSP. The results of these studies indicate that the administration of alloxan before the onset of PSP effectively inhibits luteal function, whereas D-1A treatment induces early luteolysis as compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Decidual changes in the endometrium and morphological adaptation of the associated supplying arteries in the normal and diabetic pseudopregnant rat.

After an electrically induced pseudopregnancy in rats in which deciduomas were produced by an intraluminal oil injection, the decidual tissue was studied morphologically on days 7, 10 and 13. A constant and dynamic wave of mitotic figures was found, which started on day 7 in the antimesometrial decidua, moving to the mesometrial decidua on day 10 and finally to the mesometrial triangle area on day 13 of pseudopregnancy. This and other morphological changes were compared with those found in pseudopregnant rats with streptozotocin-induced diabetes. On days 7 and 10 the incidence and distribution of mitotic figures were practically identical in both groups and statistically no significant difference was found in the uterine weight between the two groups on these two days. On day 13, two statistically significant differences were observed in the diabetic group: a fall in the uterine weight and a fall in the incidence of mitotic figures in the mesometrial triangle decidual cells. Associated with decidualization, a series of histological changes were studied in the arteries of the mesometrial triangle. Although the difference was not statistically significant, changes seemed to occur to a lesser degree in the diabetic group. It is concluded that the diabetic state has no influence in the early decidualization period, but it does have influence late in pseudopregnancy: a fall in the mitotic activity in the decidual tissue, a fall in the uterine weight and a less marked change in the spiral arteries which during pregnancy would supply the developing placenta with maternal blood.