Porfirio Gómora-Arrati

FACILITATION OF ESTROUS BEHAVIOR BY VAGINAL CERVICAL STIMULATION IN FEMALE RATS INVOLVES α1-ADRENERGIC RECEPTOR ACTIVATION OF THE NITRIC OXIDE PATHWAY

In estrogen-primed female rats, vaginal cervical stimulation (VCS) provided by male intromissions or by an experimenter enhances estrous behaviors exhibited by females during subsequent mating with a male. We tested the hypothesis that alpha(1)-adrenergic receptors, acting via the nitric oxide-cGMP-protein kinase G pathway, mediate VCS-induced facilitation of female reproductive behaviors. Ovariectomized, estradiol-primed rats received intracerebroventricular (icv) infusions of vehicle or pharmacological antagonists 15 or 60min before VCS. Estrous behaviors (lordosis and proceptivity) in the presence of a male were recorded immediately (0min), and 120min following VCS. First we verified that VCS, but not manual flank stimulation alone, enhanced estrous behaviors when females received icv infusion of the vehicles used to administer drugs. Increased estrous behavior was apparent immediately following VCS and persisted for 120min. We then infused prazosin, phenoxybenzamine (alpha(1)-adrenergic receptor antagonists), yohimbine, idaxozan (alpha(2)-adrenergic receptor antagonists), or propranolol (beta-adrenergic receptor antagonist) 15min prior to the application of VCS in females primed with 5mug estradiol benzoate. Only alpha(1)-adrenergic antagonists inhibited VCS facilitation of estrous behavior, apparent 120min after VCS. Finally, we administered specific inhibitors of soluble guanylyl cyclase, nitric oxide synthase or protein kinase G icv 15 or 60min before VCS. All three agents significantly attenuated VCS facilitation of estrous behavior. These data support the hypothesis that endogenously released norepinephrine, acting via alpha(1)-adrenergic receptors, mediates the facilitation of lordosis by VCS, and are consistent with a mechanism involving alpha(1)-adrenergic activation of the nitric oxide/cGMP/protein kinase G pathway.

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.

Role of progesterone receptors during postpartum estrus in rats.

We studied the role of progesterone receptor (PR) in the display of female sexual behavior during postpartum estrus in rats. Adult female rats were treated with the PR antagonist, RU486 (1.25 and 5 mg), 3 h after parturition and sexual behavior was evaluated throughout the first postpartum day. Estradiol and progesterone serum levels changed during the first 24 h postpartum. The highest estradiol and progesterone levels were found at 9 and 12 h postpartum, respectively. The predominant PR isoform in the hypothalamus and the preoptic area was PR-A during postpartum day. The content of PR-A increased at 6 h postpartum in the hypothalamus and the preoptic area, and decreased in both regions at 9 h. PR-B content only increased in the preoptic area at 12 h postpartum. The highest display of lordotic and proceptive behaviors were found at 12 h postpartum. The treatment with 1.25 and 5 mg of RU486 respectively reduced lordosis by 61% and 92% at 12 h postpartum. These results suggest that PR is essential in the display of postpartum estrus in 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.

Leptin facilitates lordosis behavior through GnRH-1 and progestin receptors in estrogen-primed rats

Dose response curves for leptin facilitation of estrous behavior (lordosis and proceptivity) were made by infusing the peptide into the lateral ventricle (icv) of ovariectomized (ovx), ad libitum-fed rats injected 40h previously with 5μg of estradiol benzoate. Leptin doses of 1 and 3μg produced significant lordosis quotient at 60min post-injection, with maximal lordosis being displayed at 120min. Yet the intensity of lordosis was weak, and a high incidence of rejection behaviors was found. Moreover, leptin did not induce significant proceptive behaviors at any dose. The leptin doses of 1 and 3μg were selected for determining whether antide, a GnRH-1 receptor antagonist, or the progestin receptor antagonist RU486 could modify the lordosis response to leptin. Icv injection of either antide or RU486 1h before leptin significantly depressed leptin facilitation of lordosis. The results suggest that leptin stimulates lordosis by releasing GnRH, which in turn activates GnRH-1 and progestin receptors. The physiological role of leptin in the control of estrous behavior remains to be determined.

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.

The nitric oxide pathway participates in lordosis behavior induced by central administration of leptin

Intracerebroventricular (icv) administration of leptin facilitates lordosis behavior in ad libitum-fed, estrogen-primed rats. The cellular mechanism involved in this response is unknown. The present study tested the hypothesis that the nitric oxide-guanylyl cyclase, cGMP-dependent protein kinase (PKG) pathway is involved in the facilitation of lordosis behavior induced by the central administration of leptin. We tested the importance of the nitric oxide/cGMP pathway for lordosis stimulation by either icv infusion of a nitric oxide synthase inhibitor (L-NAME) or a nitric oxide-dependent, soluble guanylyl cyclase inhibitor (ODQ) 30 min before leptin administration (1 μg). This dose of leptin reliably induced lordosis behavior in ovariectomized estradiol benzoate treated rats. The lordosis induced by leptin at 1 and 2h after infusion was significantly reduced by the previous injection of either L-NAME or by ODQ. Intracerebroventricular infusion of the PKG inhibitor (KT5823) 30 min before leptin infusion, also significantly inhibited the lordosis behavior induced by leptin at 1 and 2h after hormone administration. These data support the hypothesis that the nitric oxide/cGMP/PKG pathway is involved in the facilitation of lordosis by leptin in estrogen-primed female rats.

GnRH mediates estrous behavior induced by ring A reduced progestins and vaginocervical stimulation.

The present study was designed to assess the participation of gonadotropin-releasing hormone (GnRH) in the display of estrous behavior induced by application of vaginal-cervical stimulation (VCS) and by the intracerebroventricular (icv) administration of progesterone and its ring A-reduced metabolites to ovariectomized (ovx), estradiol benzoate (E2B) primed rats. Icv injection of Antide, a GnRH-1 receptor antagonist, significantly depressed lordosis behavior in ovx, E2B-primed rats treated with icv GnRH. Application of VCS to ovx, E2B-primed rats facilitated both lordosis and proceptivity. These behavioral responses were significantly depressed by the icv administration of Antide. Similarly, icv Antide blocked the stimulatory effect on both lordosis and proceptive behaviors elicited by progesterone and its ring A-reduced metabolites: 5alpha-pregnandione (5alpha-DHP), 5alpha-pregnan-3alpha-ol-20-one (5alpha,3alpha-Pgl) and 5beta-pregnan-3beta-hydroxy-20-one (5beta,3beta-Pgl) in ovx, E2B-primed rats. By contrast, icv injection of Antide failed to interfere with the facilitatory effect of the synthetic progestin megestrol acetate on lordosis and proceptive behaviors. This progestin is not reduced in ring A. The results suggest that GnRH release is an important process in the chain of events leading to the display of estrous behavior in response to progesterone, its ring A-reduced metabolites, and VCS in female rats.