Marcos García-Juárez

Differential effect of kinase A and C blockers on lordosis facilitation by progesterone and its metabolites in ovariectomized estrogen-primed rats

Dose response curves for lordosis behavior was obtained for progesterone (P) and its two ring A-reduced metabolites: 5alpha-pregnanedione (alpha-DHP) and 5alpha,3alpha-pregnanolone (5alpha,3alpha-Pgl) by infusing these progestins in the right lateral ventricle (rlv) of ovariectomized (ovx) estradiol-treated rats (2 microg estradiol benzoate; EB), 40 h before intracerebro-ventricular (icv) injection. Effective doses 50 (ED50) revealed that ring A-reduced progestins were more potent than P itself to induce lordosis behavior. Two dose levels, one producing the maximal effect and the other one producing a submaximal response (ED50-ED60), were selected for testing the capacity of RpAMPS, a kinase A blocker, and H7, a kinase C blocker, to modify the response to the three progestins. rlv injection of RpAMPS significantly depressed the lordosis response to the two dose levels of P and alpha-DHP but failed to significantly inhibit that of 5alpha,3alpha-Pgl. The administration of H7 prevented the effect of both 5alpha-reduced progestins without affecting the response to P. The results suggest that P and its ring A-reduced metabolites stimulate lordosis behavior through different cellular mechanisms: P acting mainly through the cAMP-kinase system; alpha-DHP through both kinase A and kinase C signaling pathways and 5alpha,3alpha-Pgl through the kinase C system.

A role for Src kinase in progestin facilitation of estrous behavior in estradiol-primed female rats

This study tested the hypothesis that the Src/Raf/MAPK signaling pathway is involved in the facilitation of the lordosis and proceptive behaviors induced by progesterone (P) and its ring A-reduced metabolites in ovariectomized, estradiol-primed rats. Intraventricular (icv) infusion of PP2 (7.5, 15 and 30 microg), a Src kinase inhibitor, significantly depressed P-dependent estrous behavior (lordosis and proceptivity) in estradiol-primed rats. Icv infusion of 30 microg of PP2 also significantly attenuated estrous behavior induced by the ring A-reduced P metabolites 5 alpha-dihydroprogesterone (5 alpha-DHP) and 5 alpha-pregnan-3alpha-ol-20-one (allopregnanolone). PP2 did not inhibit estrous behavior induced by administration of high doses of estradiol alone to ovariectomized rats. We also assessed if the ventromedial hypothalamus (VMH) is one of the neural sites at which progestins activate Src signaling to facilitate estrous behavior. Bilateral administration of 15 microg of PP2 into the VMH inhibited the stimulation of both lordosis and proceptive behaviors elicited by subcutaneous P administration to estradiol-primed rats. These results suggest that progestins act through Src/Raf/MAPK signaling to initiate estrous behaviors in estrogen-primed rats. This event is one component of the cellular pathways leading to the display of estrous behaviors induced by P and its ring A-reduced metabolites in female rats.

Nitric oxide and ERK/MAPK mediation of estrous behavior induced by GnRH, PGE2 and db-cAMP in rats

We tested the hypothesis that GnRH, PGE2 and db-cAMP act via the nitric oxide (NO)-cGMP and MAPK pathways to facilitate estrous behavior (lordosis and proceptivity) in estradiol-primed female rats. Estradiol-primed rats received intracerebroventricular (icv) infusions of pharmacological antagonists of NO synthase (L-NAME), NO-dependent soluble guanylyl cyclase (ODQ), protein kinase G (KT5823), or the ERK1/2 inhibitor PD98059 15 min before icv administration of 50 ng of GnRH, 1 microg of PGE2 or 1 microg of db-cAMP. Icv infusions of GnRH, PGE2 and db-cAMP enhanced estrous behavior at 1 and 2 h after drug administration. Both L-NAME and ODQ blocked the estrous behavior induced by GnRH, PGE2 and db-cAMP at some of the times tested. The protein kinase G inhibitor KT5823 reduced PGE2 and db-cAMP facilitation of estrous behavior but did not affect the behavioral response to GnRH. In contrast, PD98059 blocked the estrous behavior induced by all three compounds. These data support the hypothesis that the NO-cGMP and ERK/MAPK pathways are involved in the lordosis and proceptive behaviors induced by GnRH, PGE2 and db-cAMP. However, cGMP mediation of GnRH-facilitated estrous behavior is independent of protein kinase G.

Progesterone receptor isoforms differentially regulate the expression of tryptophan and tyrosine hydroxylase and glutamic acid decarboxylase in the rat hypothalamus.

Progesterone exerts a variety of actions in the brain through the interaction with its receptors (PR) which have two isoforms with different function and regulation: PR-A and PR-B. Progesterone may modulate neurotransmission by regulating the expression of neurotransmitters synthesizing enzymes or their receptors in several brain regions. The role of PR isoforms in this modulation is unknown. We explored the role of PR isoforms in the regulation of tryptophan (TPH) and tyrosine (TH) hydroxylase, and glutamic acid decarboxylase (GAD) expression in the hypothalamus of ovariectomized rats. Two weeks after ovariectomy, animals were subcutaneously injected with 5 μg of estradiol benzoate (EB), and 40 h later, progesterone (P) was intracerebroventricularly (ICV) injected. Each animal received two ICV injections of 1 μg/μl (4 nmol) of PR-B and total PR (PR-A+PR-B) sense or antisense (As) oligonucleotides (ODNs). First injection was made immediately before sc EB injection, and 24h later animals received the second one. Twenty-four hours after P administration, rats were euthanized and brains removed to measure the expression of PR-A and PR-B, TPH, TH and GAD by Western blot. We observed that sense ODNs modified neither PR isoforms nor enzymes expression in the hypothalamus, whereas PR A+B antisense (PR A+B As) clearly decreased the expression of both PR isoforms in this region. ICV administration of PR-B As only decreased PR-B isoform expression with no significant effects on PR-A expression. A differential protein expression of TPH, TH and GAD was observed after PR isoforms antisense administration. PR-B As administration decreased the expression of TPH (65% with respect to control). In contrast, PR A+B As and PR-B As administration increased (51.6% and 34.4%, respectively) TH expression. The administration of PR A+B As and PR-B As diminished GAD expression (33.4% and 41.6%, respectively). Our findings indicate that PR isoforms play a differential role in the regulation of the content of TPH, TH and GAD in the rat hypothalamus.

Role of progesterone receptor isoforms in female sexual behavior induced by progestins in rats.

Progesterone and its ring A reduced metabolites regulate female sexual behavior through the direct or indirect activation of progesterone receptor (PR) which has two isoforms with different function and regulation: PR-A and PR-B. The contribution of each PR isoform to the regulation of lordosis in rats is unknown. We explored the role of PR isoforms in lordosis display induced by progesterone and two of its ring A reduced metabolites: 5α-pregnan-3,20-dione (5α-DHP), and 5β,3β-pregnan-20-one (5β,3β-Pgl) in adult ovariectomized rats. Two weeks after ovariectomy, the animals were injected subcutaneously with 5 μg of estradiol benzoate (EB), and 40 h later, progestins were injected intracerebroventricularly. PR-B and total PR (PR-A + PR-B) sense or antisense oligonucleotides were administered intracerebroventricularly immediately before EB injection and 24 h later. Lordosis was evaluated 30, 120 and 240 min after progestin administration. Western blot analysis of both PR isoforms was performed in the hypothalamus and preoptic area 24 h after lordosis tests. All progestins induced maximal lordosis 120 min after administration, and antisense oligonucleotides against both PR isoforms inhibited lordosis in all animals. PR-B antisense oligonucleotides also inhibited lordosis induced by progesterone and 5α-DHP although with less efficacy than total PR antisense oligonucleotides, but the former inhibited lordosis induced by 5β,3β-Pgl in a similar manner as total PR antisense oligonucleotides. In the hypothalamus and preoptic area, the content of both PR isoforms or PR-B alone was diminished by the administration of total or PR-B antisense oligonucleotides, respectively. These results suggest that the PR-B isoform is essential for the display of the lordosis behavior in rats.

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 μg 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.

Lordosis facilitation by LHRH, PGE2 or db-cAMP requires activation of the kinase A signaling pathway in estrogen primed rats.

Dose-response curves for lordosis and proceptive behaviors were obtained for luteinizing hormone releasing hormone (LHRH), prostaglandin E2 (PGE2) and dibutyryl cyclic AMP (db-cAMP), by infusing them in the right lateral ventricle (i.c.v.) of ovariectomized (OVX) estradiol benzoate (E2B; 2 microg) treated rats. Two dose levels, one producing the maximal effect and the other one producing a submaximal response (approximately ED50) were selected for testing the capacity of Rp-cAMPS, a kinase A blocker, to modify the behavioral response to the three compounds. I.c.v. injections of Rp-cAMPS, significantly depressed both lordosis and proceptive responses induced by LHRH, PGE2 and db-cAMP. The results show that these agents use the cAMP-kinase A signaling pathway to elicit their stimulating effect on estrous behavior in the rat.

Src kinase signaling mediates estrous behavior induced by 5β-reduced progestins, GnRH, prostaglandin E2 and vaginocervical stimulation in estrogen-primed rats

The progesterone receptor (PR) is a dual function protein that acts in the nucleus as a transcriptional factor and at the cytoplasm as a scaffold for the Src-MAPK signaling pathway. Several agents lacking affinity for the PR, such as 5β-reduced progestins, GnRH or prostaglandin E(2) (PGE(2)) facilitate estrous behavior in ovariectomized (ovx), estrogen-primed rats yet their action is blocked by the antiprogestin RU486. We hypothesize that these agents act by using the PR-Src-mitogen activated protein kinase alternative pathway. To test this hypothesis we used PP2, a specific inhibitor of the Src kinase family. Intraventricular infusion of 30 μg of PP2, 30 min before behavioral testing, significantly attenuated estrous behaviors induced in estradiol benzoate (E(2)B)-primed rats by 5β-dihydroprogesterone (5β-DHP), 5β-pregnan-3β-ol-20-one (5β,3β-Pgl), GnRH, PGE(2) and by manual flank/vaginocervical stimulation. These results suggest that the Src signaling system, by activating mitogen-activated protein kinases, participates in the facilitation of estrous behavior in E(2)B-primed rats induced by agents lacking affinity for the PR.

Differential effects of progesterone and genital stimulation on sequential inhibition of estrous behavior and progesterone receptor expression in the rat brain

The effect of genital stimulation, either by vaginocervical stimulation (VCS) using a calibrated vaginal probe combined with manual flank stimulation (FS), or by mounts performed by the male, on the hypothalamus and preoptic area concentration of the progesterone receptors A (PR-A) and B (PR-B) was assessed in ovariectomized (ovx) estrogen-primed rats. VCS/FS or stimulation provided by male mounts, even without intromission, significantly decreased PR-B concentration in the hypoythalamus. Down regulation of PR produced by genital stimulation was quantitatively similar to that elicited by progesterone (P) administration. Bilateral or unilateral transection of the pelvic or the pudendal nerves prevented down regulation elicited by VCS/FS. Repeated VCS/FS elicited lordosis behavior in most ovx estrogen primed rats, but the lordosis intensity was lower than that observed in response to P. P administered to ovx estrogen primed rats, induced sequential inhibition, i.e., failure to display estrous behavior in response to a second P injection (24h after the initial P injection). VCS/FS failed to elicit sequential inhibition, since rats responded with normal estrous behavior to the second injection of P. This suggests that down regulation by VCS, by contrast with P, failed to inhibit the subpopulation of PR involved in the facilitation of estrous behavior by P.

Sexual receptivity facilitated by unesterified estradiol: Dependence on estrogen and progestin receptors and priming dose of estradiol benzoate.

In some conditions, female sexual behavior in ovariectomized rats can be induced by continuous exposure of estradiol (E2) alone or by a single injection of a high dose of the long-lasting, esterified estradiol benzoate (EB). However, there are inconsistencies in the literature on the role of estrogens during priming or in the facilitation on female sexual behavior in EB-primed rats, as well as the cellular mechanisms involved. Either subcutaneous (sc) or intracerebral (icv) administration of some doses of free unesterified E2, induced lordosis in EB-primed rats. Either sc or icv injection of E2, immediately prior to testing, induced high levels of sexual receptivity when the female rats were primed with an EB sc injection of 2 μg EB. The roles of progesterone receptor (PR) and estrogen receptor on lordosis induced by sc or icv administration of E2 were explored. Tamoxifen or RU486 administrated sc or icv; each reduced lordosis induced by E2. Similarly, antisense oligonucleotides directed at PR-B or total PR (PR-A + PR-B) administrated icv immediately before EB injection inhibited lordosis induced by daily injections of EB. These results suggest that lordosis facilitated by free E2 is dependent on priming dose of EB. Furthermore both ERs and PRs are involved in this action of E2.