Anti-progesterone Serum Research Articles

Luteinizing Hormone (LH) and Corticosterone in Proestrous Afternoon Restore the Follicle-Stimulating Hormone Secretion at Early Estrus in Adrenalectomized LH-Releasing Hormone Antagonist-Treated Rats1

Administration of the antiprogestagen RU486 in the morning of proestrus abolishes the secondary surge of FSH during early estrus in the rat without preventing the drop in serum inhibin. In addition, the injection of an ovulatory dose of LH to RU486-injected rats does not restore the secondary surge of FSH. Since RU486 is a potent antiprogesterone with antiglucocorticoid activity, in the first experiment we compared the effects of RU486 and a specific antiprogesterone serum (APS), administered on proestrus, on the secretion of FSH during early estrus. While RU486 and APS reduced the primary surge of LH and FSH, only RU486 abolished the secondary secretion of FSH. In the second experiment, rats injected with LHRH antagonist (LHRHa) and ovine LH (oLH) were adrenalectomized (ADX) or sham-ADX in the morning and injected with corticosterone (B) or oil in the afternoon of proestrus. LHRHa completely eliminated, and oLH restored, the secretion of FSH at 0200 h in estrus. ADX reduced FSH serum concentration at 0200 h in estrus, and B reversed this effect in rats injected with LHRHa and oLH. The results of these experiments indicate that, in the rat, the LHRH-independent secretion of FSH during early estrus is evoked by the combined effects of the preovulatory surge of LH and the rise in serum B on proestrous afternoon.

Fractionation of an antiserum to progesterone by affinity chromatography: effect of pH, solvents and biospecific adsorbents.

Several progesterone-AH Sepharose 4B matrices were prepared as biospecific adsorbents suitable for affinity chromatography to fractionate antibodies of different affinity and specificity from a polyclonal antiserum to progesterone-11 alpha-hemisuccinate-BSA. From an affinity column of progesterone-11 alpha-hemisuccinate-AH Sepharose 4B no antibodies can be eluted, even with glycine buffer (pH 2.6) and 30% of 2-methoxyethanol. The use of biospecific adsorbents, prepared by coupling with AH Sepharose 4B progesterone derivatives [5-pregnene-3,20-dione di(ethyleneacetal)-11 alpha-ol-11 alpha-hemisuccinate; 4-pregnene-11,20 beta-diol-3-one-11 alpha-hemisuccinate 20 beta-benzoate; progesterone-3-carboxymethyloxime] having a low cross-reactivity with the antiserum, makes the elution of various antibody fractions of variable affinity and specificity possible. 2-Methoxyethanol or N,N-dimethylformamide gradients, in acetate or TRIS buffer, were equally efficient for fractionating the antiprogesterone serum, while a decreasing pH gradient was less effective and eluted antibody fractions that were further separated into various binding components by a solvent gradient. Antibodies eluted from the affinity columns by an eluent containing a high solvent concentration have affinities higher than antibodies eluted at lower solvent concentration.

Progesterone production and clearance in cyclic ewes passively imminized against progesterone

The effects of passive immunization of ewes against progesterone on plasma progesterone concentrations and on the metabolic clearance rate (MCR) and production rate (PR) of progesterone were investigated. Three treatment groups were studied: 1) nonimmunized controls, 2) ewes passively immunized with antiprogesterone serum, and 3) immunized progestagen-treated ewes, treated concomitantly with anti-serum and with a synthetic progestagen that is not bound by the antiserum. Progesterone levels in the immunized ewes reached a maximum of 27.7±4.8 nmol/l and were significantly higher (P<0.05) than in the nonimmunized controls (9.2±1.1 mol/l) or the immunized progestagen-treated ewes (15.6±1.6 nmol/l). Mean progesterone MCR in the immunized ewes was 1.6±0.5 and 2.1±0.3 liter/min on Days 7 and 13 of the estrous cycle, respectively, compared with 0.8±0.2 and 1.4±0.3 liter/min, respectively, in nonimmunized controls. The progesterone production rate in the immunized ewes was significantly higher than in nonimmunized controls, and reached 12.0±2.2 and 19.7±1.6 nmol/min on Days 7 and 13 of the estrous cycle, respectively, compared with 4.6±0.6 and 10.0±2.5 nmol/min in nonimmunized controls (P<0.03 for both comparisons). Treatment with progestagen had no significant effect on progesterone MCR or PR of immunized ewes. The LH pulse frequency on Days 10 to 11 of the cycle was 0.7±0.3, 1.8±0.3 and 0.0±0.0 pulses/6 h in the control, immunized and immunized progestagen-treated groups, respectively (P<0.05). It is concluded that the increased plasma progesterone levels in the immunized ewes are the result of an increased progesterone production rate, which may have been induced by an increase in gonadotrophin secretion or by a direct effect of the anti-progesterone serum on the ovary.

Inhibition of lymphocyte aggregation by progesterone

Investigations were carried out on the influence of progesterone and murine trophoblast culture supernatants on the capacity of lymphocytes to form aggregates (clusters), an important feature in immunologic recognition and response. Both progesterone and trophoblast supernatants inhibited this cluster formation in a dose-dependent way. The effect of trophoblast supernatants appeared to be mediated mainly by progesterone since they lost their inhibitory effect on the cluster formation after treatment with anti-progesterone serum (APS). Preparations with Il-2 activity of rat and mouse origin could either prevent or restore the suppressive effect of both progesterone and trophoblast supernatants on lymphocyte aggregation. The interference with lymphocyte interaction by trophoblast may represent one of the mechanisms by which the fetal allograft is protected against maternal recognition.

Changes in plasma progesterone, estradiol, follicle-stimulating hormone and luteinizing hormone during diestrus and ovulation in rats with 5-day estrous cycles: effect of antibody against progesterone.

Progesterone secretion remained significantly higher during diestrus in the 5-day cyclic rat than in the 4-day cyclic animal. Injection of a sufficient amount of antiprogesterone serum (APS) at 2300 h on metestrus in a 5-day cycle advances ovulation and completion of the cycle by 1 day in the majority of animals (75 and 80%, respectively). Progesterone (250 micrograms) administered with APS eliminated the effect of the antiserum. Within 2 h after administration of APS, levels of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) elevated significantly, while a significant elevation of plasma estradiol above the control value followed as late as 36 h after the treatment. None of the 5-day cyclic rats treated with APS showed ovulatory increases of FSH and LH at 1700 h on the second day of diestrus, although 3 of the 4 animals receiving the same treatment ovulated by 1100 h on the following day. The onset of ovulatory release of gonadotropins might have been delayed for several hours in these animals. These results indicate that recurrence of the 5-day cycle is due to an elevated progesterone secretion on the morning of diestrus, and suggest that a prolongation of luteal progesterone secretion in an estrous cycle suppresses gonadotropin secretion. Rather than directly blocking the estrogen triggering of ovulatory LH surge, the prolonged secretion of luteal progesterone may delay the estrogen secretion itself, which decreases the threshold of the neural and/or hypophyseal structures for ovulatory LH release.

SUPPRESSION OF CELLULAR IMMUNE REACTIONS BY RAT OVARIAN CELLS

The influence on the immunologic response by rat ovarian cells of follicle and stromal tissue origin was investigated using the mixed lymphocyte reaction (MLR), the phytohemagglutinin (PHA) assay, the cytotoxic T lymphocyte (CTL) assay, and a cytotoxic T-cell-mediated microcytotoxicity test. The MLR between BN/Mai Pfd (RT-In) stimulator splenocytes (mitomycin-C treated) and R/A Pfd (RT-1u) responder lymph node cells was markedly suppressed by ovarian cells of follicle and/or stromal tissue origin. A similar in vitro inhibition was observed with cell-free supernatants from ovarian cells, not only in the rat MLR but also in the mouse MLR between Balb/c stimulator splenocytes and C57B1 responder lymph node cells. Moreover, the reactivity of rat lymphocytes was suppressed when they were cocultured with ovarian cells in the PHA and CTL assays. Preincubation of the supernatants with serial diluted antiprogesterone serum (APS) and antiestradiol serum (AES) revealed that the suppressive effect of these supernatants could be completely abolished with APS--and, to a lesser extent, by AES. As shown by the cell-mediated microcytoxicity assay both the ovarian cells and the steroids secreted by these cells were, however, ineffective in suppressing the effector phase. It is suggested that the steroid secretion by the ovary may play an important role in the prolonged survival of ovarian grafts.

Immunosuppressive properties of murine trophoblast

The modification of the immunological response by murine trophoblast cells of different sources was investigated using the mixed lymphocyte reaction (MLR) and the cell mediated lympholysis (CML) test. MLR between C57BL (H-2 b) stimulator splenocytes (mitomycin C treated in the unidirectional MLR) and BALB/c (H-2 d) responder lymph node cells were markedly suppressed by trophoblast of ectoplacental cone (EPC) and placental origin. The same in vitro effect was observed with supernatants (SN) of trophoblast cells and with supernatants of blastocysts. Addition of anti-progesterone serum (APS), anti-testosterone serum (RAT), and anti-immunoglobulin serum (RAHIg) in serial dilutions to the trophoblast-MLR system revealed that the immunosuppressive effect of trophoblast giant cells and trophoblast giant cell culture supernatants can be abolished with APS. Identical results were obtained with APS added to immunosuppressive doses of progesterone. CML between C57BL responder lymph node cells and mitomycin C-treated BALB/c stimulator spleen cells was also markedly suppressed when trophoblast of EPC origin was added. A similar suppression of cytotoxic T-cell induction was seen when progesterone was added to the system. The immunosuppressive action of trophoblast as detected in vitro is likely to play an important role in the maintainance of pregnancy by protecting the semiallogeneic conceptus against immune aggression by the maternal immune system.

Changes in concentration of hypothalamic cytosolic progestin receptors at the initiation of pseudopregnancy in rats.

Pseudopregnancy (psp) can be induced by a single injection of progesterone in cyclic rats and the administration of anti-progesterone serum can block the establishment of psp in cervically stimulated rats. To further investigate neuroendocrine mechanisms for the initiation of either psp or prolactin (PRL) surges in connection with the neurotropic action of progesterone, cytosolic progestin receptors (PRc) were identified and measured in preoptic (POA) and basal hypothalamic (BH) areas. Chronological determinations of PRc concentrations in both areas revealed large fluctuations in the morning of the estrous day but not in the morning of the diestrous day. The differences between the maximal and the minimal PRc concentrations observed were more than 100-fold in POA and 3-fold in BH. Cervical stimulation applied in the afternoon of the proestrous day significantly altered the changing pattern of PRc concentrations in POA but not in BH. One of the two peaks of PRc concentrations in POA was magnified and advanced earlier to 0300 h, and then the 1st PRL surge peaking at 0700 h occurred. This PRL release seemed to stimulate progesterone secretion and an elevation of peripheral progesterone levels coincided with the 2nd peak of PRc concentrations in POA at 0900 h. This coincidence may be a prerequisite for the further continuation of PRL surges. These results strongly suggest that the spontaneous oscillation of the PRc concentration in hypothalamic neurons is involved in the regulation of PRL secretion and that cervical stimulation shifts the phase and changes the amplitude.

Blockade of pseudopregnancy in the rat by treatment with antiprogesterone serum.

The role of peripheral progesterone in the initiation of pseudopregnancy (psp) was investigated. Antiprogesterone serum was generated in a rabbit immunized with progesterone-i lo-hemisuccinate coupled with ovine serum albumin and injected i.v. once or twice into cervically stimulated female rats. The uterine cervical stimulation was performed on proestrus (Day 0) at 1900 h. The antiserum was injected at various times between Days 0—2. Serum prolactin (PRL) and progesterone concen trations were determined by respective radioimmunoassays from Days 1 to 5. The amount of “?�free” progesterone was calculated by subtracting the amount of progesteronebound to the previ ously injected progesterone antibody from the total amount of progesterone. Injections of antiprogesterone serum (1 ml) at 0700 h on Days 1 and 2 prevented the initiation of psp in 9 of 10 rats, but the other combination of 2 injections was ineffective. In psp blocked rats, nocturnal surges of PRL decreased gradually in magnitude and were absent on Day 5, while diurnal surges were absent from Day 1. Free progesterone stayed at low levels between Days 3—5. Another group of rats received a single injection of antiprogesterone serum. Only the treatment at 0700 or 0800 h on Day 1 was effective in preventing the initiation of psp in 5 of 15 rats. Only those rats which underwent psp in spite of this treatment were used to measure changes in levels of free progesterone. Free progesterone concentrations were lowered on Days 2 and 3 and started to rise on Day 4 in contrast to normal psp rats whose first significant rise was observed on Day 3. The nocturnal surge was detectable throughout the period examined, but the magnitude on Day 2, 1 day after the antiprogesterone treatment, was suppressed. These results indicate that the magnitude of the nocturnal surge of PRL is attenuated if circu lating progesterone is curtailed at 0700 h of the previous morning and that the occurrence of the diurnal surge of PRL is highly susceptible to the level of peripheral progesterone. Antiprogesterone treatment at 0700 h on Day 1 or 2 will trigger the following sequential events: attenuation of the nocturnal surge, preclusion of the diurnal surge, impairment of progesterone secretion and, finally, interruption of psp.

Prevention of the abortifacient action of antiprogesterone serum by progesterone

Earlier studies1−4 showed that when rats of 10 days' gestation are passively immunized with antiprogesterone (A-P) globulins the biologically available progesterone (P) unbound by A-P (Pu) not only falls precipitously but also remains low for days, despite the rapid clearance of A-P. This finding suggested that the effective reduction of Pu affects P synthesis, probably through action on the fetoplacental unit. If so, pregnancy should be protected from the effect of A-P by P treatment to prevent the reduction of Pu. The present studies demonstrated that if P treatment was delayed for six hours after the administration of A-P, Pu did not return to physiologic levels, and pregnancy did not continue. However, if P was given three hours after A-P, at the same time, or three hours before A-P, the reduction of Pu was short-lived or prevented, and thus pregnancy was protected.