Two thalidomide analogs induce persistent estrous behavior and inhibit uterus contractility in rats: The central role of cAMP.

The effects of 4NO2PDPMe and 4APDPMe, which are thalidomide (Tha) analogs that act as selective phosphodiesterase (PDE-4) inhibitors, on estrous behavior (lordosis and proceptive behaviors) and on uterine contraction were studied in ovariectomized (OVX) estrogen-primed Sprague Dawley (SD) and in intact non-pregnant Wistar rats, respectively. We found that intracerebroventricular (ICV) infusion of either 4NO2PDPMe or 4APDPMe (20 to 80 µg) stimulated intense lordosis and proceptive behavior in response to mounts from a sexually active male, within the first 4 h after infusion, and persisting for up to 24 h. Inhibitors of the progesterone receptor (RU486, administered subcutaneously), the estrogen receptor (tamoxifen, ICV), the adenylate cyclase (AC)/ cyclic AMP (cAMP)/protein kinase A (PKA) pathway (administered ICV), and the mitogen activated protein kinase (MAPK) pathway (administered ICV) significantly decreased lordosis and proceptive behavior induced by Tha analogs. Uterine contractility studies showed that Tha analogs inhibited both the K+- and the Ca2+-induced tonic contractions in rat uterus. Tha analogs were equally effective, but 4APDPMe was more potent than 4NO2PDPMe. These results strongly suggest the central role of cAMP in both processes, sexual behavior, and uterine relaxation, and suggest that Tha analogs may also act as Ca2+-channel blockers.

Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats.

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17ß-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

Estrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2.

The role of classical estrogen receptors (ERs) in priming female reproductive behavior has been studied previously; however, the participation of this receptor during activation of estrous behavior has not been extensively studied. The purpose of this work was to test the possibility that the facilitation of lordosis behavior in estrogen-primed rats by progesterone (P) and its 5α- and 5ß-reduced metabolites, gonadotropin-releasing hormone (GnRH), leptin, prostaglandin E2 (PGE2) and vagino-cervical stimulation (VCS) involves interactions with classical ERs by using the selective ER modulator, tamoxifen. To further assess the role of ERs, we also explored the effects of the pure ER antagonist, ICI182780 (ICI), on estrous behavior induced by P and GnRH. Ovariectomized, estrogen-primed rats (5µg estradiol benzoate 40h earlier) were injected intraventricularly with the above-mentioned compounds, or they received VCS. All compounds and VCS effectively facilitated estrous behavior when tested at 60, 120 or 240min after infusion or application of VCS. Intraventricular infusion of tamoxifen (5µg), 30min before, significantly attenuated estrous behaviors induced in estradiol-primed rats by P, most of its 5α- and 5ß-reduced metabolites, GnRH, and PGE2, but not by VCS. Although there was a trend for reduction, tamoxifen did not significantly decrease lordosis in females treated with 5ß-pregnan-3,20-dione. ICI also inhibited lordosis behavior induced by P and GnRH at some testing intervals. These results suggest that activation of classical ERs participates in the triggering effects on estrous behavior induced by agents with different chemical structures that do not bind directly to ERs.

Glycinamide, a glycine pro-drug, induces antinociception by intraperitoneal or oral ingestion in ovariectomized rats.

AIMS: The effect of i.p. injection or oral ingestion of glycinamide, a glycine pro-drug, on two tests for nociception was assessed in ovariectomized Sprague Dawley rats. MAIN METHODS: To explore the potential analgesic effect of glycinamide the vocalization threshold to tail shock (VT) and the tail flick latency (TFL) were used. Glycinamide was administered both through the intraperitoneal route (doses 0, 25, 100, 400, 800 mg/kg) and through ad libitum oral ingestion of glycinamide solution (40 mg/ml) following a 24h period of water deprivation. KEY FINDINGS: Glycinamide exerted a significant analgesic effect on VT when injected i.p. at doses of 400 or 800 mg/kg. Analgesia occurred 10-20 min post-injection and persisted approx 45 min. At the high dose level, glycinamide exerted a weaker and more delayed effect on TFL than on the VT test. I.p. injection of 800 mg/kg glycinamide inhibited vocalizations induced by the application of suprathreshold tail shocks (30% above threshold) with a latency of approx 3 min and duration of approx 1h. The volume of a glycinamide solution (40 mg/ml) ingested by rats deprived of water for 24h was positively correlated with the degree of analgesia in the VT test. Values between 100 and 200mg glycinamide exerted clear analgesic responses. SIGNIFICANCE: Thus, glycinamide, either by systemic or oral routes, exerts a clear analgesic effect in the VT test of nociception and a much weaker action in the TFL test. This effect is probably due to the conversion of glycinamide to glycine in the brain.

Antagonists of the protein kinase A and mitogen-activated protein kinase systems and of the progestin receptor block the ability of vaginocervical/flank-perineal stimulation to induce female rat sexual behaviour.

Brief vaginocervical stimulation using a glass rod (VCS) combined with manual flank-perineal stimulation (FS) rapidly (within 5 min) induced both receptive and proceptive behavioural responses to males in ovariectomised, oestrogen-primed rats. This receptive-proceptive response to males, resulting from a single brief (5-s duration) instance of manual VCS + FS, declined markedly within 4 h. However, the decline was prevented if the females were mounted by males immediately after the manual VCS + FS and 2 h later. We tested the participation of the cAMP-dependent protein kinase A system and the mitogen-activated protein kinase (MAPK) system in the response to VCS + FS by infusing either 100 ng of Rp-adenosine 3',5'-cyclic monophosphorothiate triethylamonium salt (a protein kinase A blocker) or 3.3 microg of PD98059 (a MAPK blocker) i.c.v. 15 min prior to VCS + FS. Both inhibitors blocked the ability of VCS + FS to induce the proceptive-receptive responses to males at all testing intervals. In experiment 2, systemic administration of 5 mg of RU486 1 h before VCS + FS also blocked the ability of VCS + FS to induce the proceptive-receptive responses to males. The present findings suggest that both VCS + FS and mating stimuli provided by males release neurotransmitters and neuromodulators that trigger the protein kinase A and the MAPK signalling systems, which interact with the progestin receptor to rapidly (within 5 min) induce proceptive-receptive behaviour in females.

Non-ligand activation of estrous behavior in rodents: cross-talk at the progesterone receptor.

Estrous behavior in rodents is triggered by the binding of progesterone (P) to its intracellular receptor (PR). Non-steroidal agents (i.e., gonadotropin-releasing hormone, noradrenaline, dopamine and others), acting at the membrane, can facilitate estrous behavior in estrogen-primed rats. This action is mediated through the generation of second messengers (cyclic AMP, cyclic GMP, calcium) which, in turn, phosphorylate through diverse kinase systems (protein kinases A, G or C) either the PR or associated effector proteins linking the PR to the trans-activation machinery. P or its metabolites also activate cyclic AMP-signaling pathways by acting directly on the membrane or by modulating neurotransmitter release. Molecular processes resulting from second messenger signaling pathways and those from the progesterone-RP interaction synergize to elicit a full behavioral response.

Indomethacin inhibits lordosis induced by ring A-reduced progestins: possible role of 3alpha-oxoreduction in progestin-facilitated lordosis.

Progestins with a delta-4-3-keto configuration bind to the progestin receptor (PR) and facilitate estrous behavior in estrogen-primed rats. Some ring A-reduced progestins [5alpha-dihydroprogesterone (alphaDHP), allopregnanolone, and epipregnanolone] are more potent estrus-inducing agents than progesterone when iv injected despite their lower affinity for the PR. Yet the estrus-inducing action of such progestins is reduced by the antiprogestin RU486, suggesting that binding to the PR is required for this effect. Because allo- and epi-pregnanolone are oxidized to alpha- and betaDHP, respectively, by 3alpha-hydroxysteroid oxo-reductase (3alphaHSOR), part of their estrus-inducing action may occur through the binding of such DHPs to the PR. Conversely, because 3alphaHSOR reduces alpha- and betaDHP to allo- or epi-pregnanolone, both of which exert membrane effects, the estrus-inducing effect of DHPs may involve actions independent of the PR. To test these possibilities we assessed the effect of indomethacin, a blocker of 3alphaHSOR, on the estrus-inducing action of such progestins. Because indomethacin also inhibits cyclooxygenases, we selected a dose and treatment schedule that does not interfere with prostaglandin-mediated brain processes (e.g., LHRH release). Indomethacin did not significantly modify the effect of progesterone or megestrol acetate on lordosis. Yet, it significantly reduced the action of all ring A-reduced progestins. Results suggest that: (a) oxidation is essential for lordosis facilitation by 3alpha-pregnanolones and (b) reduction of 3-keto progestins generates 3alpha-hydroxy metabolites which synergize with processes triggered by occupation of the PR by 3-keto progestins. The possible participation in this response of other events influenced by indomethacin (e.g., prostaglandin or melatonin synthesis) is discussed.

Ring A reductions of progestins are not essential for estrous behavior facilitation in estrogen-primed rats.

In Experiment 1 six dose levels (range 0.66-2000 microg) of progesterone (P) and two synthetic progestins with a double bond at C6: megestrol acetate (MA) and chlormadinone acetate (CA), which cannot be reduced at C5, were injected to estrogen-primed (2 microg estradiol benzoate 42 h earlier) ovariectomized (ovx) rats. The three progestins elicited significant lordosis and proceptive behaviors. Potency analysis showed that MA was the most potent progestin for stimulating estrous behavior, followed by P and CA. These results suggest that ring A reduction of progestins to 5alpha/5beta metabolites is not essential for the facilitation of estrous behavior in ovx estrogen-primed rats. Progestins with the 3-ketone group and a double bond at C4 can also be reduced at C3 to yield 3alpha-hydroxysteroid metabolites potentially capable of stimulating estrous behavior. In Experiment 2, the relevance of the formation of 3alpha-hydroxysteroid metabolites for estrous behavior facilitation was tested by concurrently injecting indomethacin (1.5 mg), a blocker of 3alpha-hydroxysteroid oxidoreductase, with 400 microg of P, MA, or CA to ovx estrogen-primed rats. Indomethacin failed to block the stimulatory effect of these progestins on estrous behavior. These results suggest that 3-ketosteroid reduction is also not essential for estrous behavior facilitation by progestins.

Progesterone receptor participates in the stimulatory effect of LHRH, prostaglandin E2, and cyclic AMP on lordosis and proceptive behaviours in rats.

We explored the effect of the antiprogestin RU486 on the estrous behaviour (lordosis and proceptivity) induced in sexually experienced ovariectomized oestrogen primed rats by: 5 microg luteinizing hormone-releasing hormone (LHRH), 100 microg prostaglandin E2 (PGE2), or 2 mg dibutyryl cyclic AMP (db cAMP). Pretreatment with 5 mg RU 486 (but not with vehicle) 60 min before the injection of the above-mentioned agents significantly decreased both lordosis and proceptive behaviours normally induced by such agents. Results suggest that the estrus-inducing action of LHRH, PGE2 and db cAMP occurs through the activation of the progesterone receptor.

Ring A reduced progestins potently stimulate estrous behavior in rats: paradoxical effect through the progesterone receptor.

The effect of ring A reductions at C5 and C3 on the capacity of the progesterone (P) molecule to stimulate estrous behavior was studied in ovariectomized estrogen primed rats (5 micrograms estradiol benzoate, EB, 40 h before progestin administration). Dose-response curves (dose range: 0.75-200 micrograms) for the lordosis quotient (LQ), lordosis score (LS), and proceptivity were constructed for P and all its ring A reduced metabolites: 5 alpha-pregnanedione (alpha DHP), 5 beta-pregnanedione (beta DHP), 3 alpha,5 alpha-pregnanolone (3 alpha,5 alpha-Pgl), 3 alpha,5 beta-pregnanolone (3 alpha,5 beta-Pgl), 3 beta,5 alpha-pregnanolone (3 beta,5 alpha-Pgl), and 3 beta,5 beta-pregnanolone (3 beta,5 beta-Pgl). Progestins were dissolved in propylene glycol and IV injected through an indwelling jugular catheter. Tests for lordosis and proceptivity were made at 5, 30, and 120 min after progestin injection. Weak, though significant lordosis behavior was observed at 5 min following the injection of some of the progestins, particularly the pregnanolones. Maximal responses were obtained at 120 min postinjection for all progestins. Dose response curves of the LQ, LS, and proceptivity were dualistic for alpha DHP and both 3 alpha pregnanolones, smaller responses being observed with high doses. Relative potency analysis revealed that alpha DHP, 3 alpha,5 beta-Pgl, 3 beta,5 alpha-Pgl, and 3 alpha,5 alpha-Pgl were considerably more potent for eliciting lordosis than P (14, 13.7, 9, and 4-fold, respectively). The same order of relative potencies was found for both LS and proceptivity. 3 beta,5 beta-Pgl and beta DHP were only slightly more potent than P (2 and 1.5-fold, respectively). In a second study, the antiprogestin RU486 (5 mg, SC), injected 60 min before one of four selected progestins (alpha DHP, 3 alpha,5 alpha-Pgl, 3 alpha,5 beta-Pgl, and 3 beta,5 beta-Pgl), significantly inhibited their action on estrous behavior (lordosis and proceptivity) when tested at 60 and 120 min postinjection. On the other hand, RU486 failed to inhibit early lordotic responses obtained at 5 and 30 min following 3 alpha,5 alpha-Pgl and 3 alpha,5 beta-Pgl. Similarly RU486 was ineffective in inhibiting lordosis in ovariectomized rats treated only with estradiol (3 micrograms of EB/day for 7 days). Data suggest that: (i) ring A reduction of the P molecule plays an important role in the normal facilitation of estrous behavior in the rat; and (ii) ring A reduced progestins provoke this effect by acting, at least partially, through the progesterone receptor.

Blockage of substance P-induced scratching behavior in rats by the intrathecal administration of inhibitory amino acid agonists.

Intrathecal administration of 20 micrograms of substance P induced scratching behavior in most tested rats (80%). Scratching appeared in bouts of short latency and variable duration, intensity and frequency (range 1-60, mean number of scratching bouts in one hour test: 8.93 +/- 1.86). Intrathecal administration of glycine (400 micrograms but not 66 micrograms) significantly decreased the effect of substance P on this behavior. Taurine, in dosages equimolar to glycine, abolished the response to substance P at the high dose level (700 micrograms), but did not significantly affect it at the lower level (120 micrograms). The GABAA agonist, muscimol, abolished the effect of substance P at the 3 micrograms dose level, but the 0.5 microgram dose did not produce a significant effect. Baclofen, a GABAB agonist, was highly effective in significantly reducing the action of SP at 0.9 and 0.15 microgram; only two of 8 rats receiving the low dose of baclofen (0.15 microgram) exhibited scratching. The results suggest that the spinal inhibitory amino acids modulate nociceptive impulses generated by the action of substance P in dorsal horn neurons of the spinothalamic tract.

Intrahypothalamic injection of RU486 antagonizes the lordosis induced by ring A-reduced progestins.

We explored the possibility that ring A-reduced progestins facilitate lordosis in estrogen primed rats through their interaction with an intracellular progestin receptor (PR) by using RU486. This drug binds with high affinity to the PR, thus preventing the action of progesterone (P). Ovariectomized estrogen-primed rats (2 micrograms estradiol benzoate 40 hr earlier) were bilaterally injected into the ventromedial hypothalamic nucleus (VMHN) with 1 microgram of: P, 5 alpha-pregnanedione or 3 beta,5 beta-pregnanolone in 1 microliter oil. All three progestins effectively facilitated lordosis, tested at four and eight hours after intrahypothalamic injections. The ability of RU486 to counteract progestin-induced lordosis was assessed by infusing 10 micrograms of this agent into the VMHN along with any of the progestins. RU486 antagonized the lordosis induced not only by P (67% reduction) but also that induced by 5 alpha-pregnanedione and by 3 beta,5 beta-pregnanolone (47% and 93% reductions, respectively). Results suggest that ring A-reduced progestins may act through the PR mechanism to facilitate lordosis, i.e., in a fashion similar to P.

Aberraciones cromosómicas en 334 individuos con perdidas gestacionales diversas: (incluyendo 144 parejas) / Chromosome aberrations in 334 subjects with repeated abortion (including 144 comples)

La evaluación citogenética de 334 individuos (incluyendo 144 parejas) con abortos repetidos y/o pérdidas gestacionales diversas demostró una aberración cromosómica en 15 de los individuos (4.49%) y en 14 de las parejas (9.72%). La comparación de la frecuencia de anormalidades en individuos que no habían procreado hijos normales contra aquélla de los que sí lo habían hecho (5.62% vs. 1.18%) reveló una diferencia estadísticamente significativa (p<0.02). Estos datos apoyan la conveniencia de efectuar estudios citogenéticos parentales en la investigación etiológica de pérdidas gestacionales, particularmente cuando no ha habido descendencia normal. Tanto los resultados obtenidos como el tipo de anormalidades observadas (8 translocaciones recíprocas, 2 translocaciones Robertsonianas, 2 mosaicos para el cromosoma X, 1 inversión pericéntrica, 1 sitio frágil y 1 anillo no identificado en mosaico) fueron similares a los obtenidos por otros autores