Mechanisms responsible for progesterone's protection against lordosis-inhibiting effects of restraint I. Role of progesterone receptors

Progestins and antiprogestins are widely used therapeutic agents in humans. In many cases, these are indicated for the treatment of reproductive activities. However, progesterone has widespread physiological effects including a reduction of the response to stress. We have reported that 5 min of restraint reduced lordosis behavior of ovariectomized rats hormonally primed with estradiol benzoate. When ovariectomized rats received both estradiol benzoate and progesterone priming, restraint had minimal effects on lordosis. Progesterone influences behavior through classical intracellular progesterone receptor-mediated nuclear events as well as extranuclear events. How these multiple events contribute to the response to stress is unclear. The current project was designed to initiate examination of the mechanisms responsible for progesterone's ability to protect against the effects of the restraint. In the first experiment, ovariectomized rats, primed with 10 μg estradiol benzoate, received 500 μg progesterone 4 h, 1 h, or 30 min before restraint. When progesterone was injected 4 h before restraint, progesterone eliminated the effects of restraint. In contrast, progesterone 30 min before restraint offered no protection. Effects of progesterone 1 h before restraint were equivocal allowing the suggestion that less than 4 h of progesterone priming might be sufficient. In the second experiment, the synthetic progestin, medroxyprogesterone, was shown to mimic effects of progesterone in preventing effects of restraint. Finally, the progesterone receptor antagonist, RU486, attenuated progesterone's protection against restraint. These findings offer evidence that ligand-activated progesterone receptor mechanisms contribute to the maintenance of lordosis behavior in the presence of mild stress.     

Progesterone reduces the effect of the serotonin 1B/1D receptor antagonist, GR 127935, on lordosis behavior

Ovariectomized rats were hormonally primed with 10 μg estradiol benzoate or with estradiol benzoate plus 500 μg progesterone. Rats received a bilateral infusion with 200 ng of the 5-HT_1B/1D receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1-1′-biphenyl-4-carboxamide hydrochloride (GR 127935), into the ventromedial nucleus of the hypothalamus (VMN), followed by a 5 min restraint or home cage experience. In estrogen-primed females that had experienced minimal handling between ovariectomy and use in the experiment, infusion with the water vehicle transiently inhibited lordosis behavior, and the 5-HT_1B/1D receptor antagonist amplified this inhibition. There were no effects in rats hormonally primed with estrogen and progesterone. Handling for two days before the experiment reduced the effects of the infusions in estrogen-primed rats. However, when a 5 min restraint experience followed infusion with GR 127935, there was a significant decline in lordosis behavior that persisted for 10 to 15 min after the experience. Regardless of the prior experience or type of infusion, the addition of progesterone to the hormonal priming completely prevented the lordosis inhibition. These findings are consistent with prior evidence that progesterone protects against the inhibitory effects of a 5 min restraint experience on lordosis behavior. Moreover, these are the first experiments to demonstrate an inhibitory effect of a selective 5-HT_1B/1D receptor antagonist in the VMN on lordosis behavior of estrogen primed, but not estrogen and progesterone primed, ovariectomized rats.     

Estrogen and progesterone dose-dependently reduce disruptive effects of restraint on lordosis behavior

Ovariectomized rats were hormonally primed with various doses of estradiol benzoate (EB; 0.5-10 microg) in combination with various doses of progesterone (2.5-500 microg) to induce sexual receptivity. Females were then subjected to 5 min restraint and the effect on lordosis behavior was monitored for the next 30 min. Such mild stress has been previously shown to transiently reduce lordosis behavior of ovariectomized females hormonally primed only with 10 microg EB. In the current study, doses of progesterone of 25 microg or more in combination with 10 microg EB reduced the effects of restraint. Also priming doses of EB from 4.0 to 10 microg in combination with 250 microg progesterone prevented the lordosis-inhibiting effects of restraint. These findings reinforce prior observations of the dose-dependency of both estrogen and progesterone in the facilitation of lordosis behavior and introduce the female's lordosis response to mild restraint as a potentially useful index of the female's response to stress.     

Different doses of estradiol benzoate induce conditioned place preference after paced mating

The ovarian hormones estrogen and progesterone are required for the complete display of sexual behavior in female rats. Paced mating produces a reward state in intact cycling and ovariectomized (OVX), hormonally primed females as evaluated by the conditioned place preference (CPP) paradigm. Most of the studies that have evaluated CPP induced by paced mating in OVX females have used relatively high doses of estradiol benzoate (EB). In the present study we determined if different doses of EB, combined with progesterone (P), could induce CPP after paced mating. For this purpose OVX female rats were divided in five groups that received one of different doses of estradiol benzoate (5, 2.5, 1.25 or 0.625 μg estradiol + 0.5 mg of progesterone) before being allowed to pace the sexual interaction and conditioned in a CPP paradigm. We found that the lowest dose of EB used (0.625 μg) significantly reduced the lordosis quotient and the lordosis coefficient. Even though these females paced the sexual interaction, they didn't change its original preference, suggesting that sexual interaction did not induce a positive affective, reward state. Females allowed to pace the sexual interaction with higher doses of EB developed CPP after paced mating. These results indicate that a threshold of estradiol is required for paced mating to induce CPP.     

Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows I: Basis for development of protocols

Five experiments were conducted on commercial farms in Brazil aiming to develop a fixed-time artificial insemination (TAI) protocol that achieved pregnancy rates between 40% and 55% in Bos indicus cows. These studies resulted in the development of the following protocol: insertion of an intravaginal device containing 1.9 g of progesterone (CIDR) plus 2.0 mg im estradiol benzoate on Day 0; 12.5 mg im dinoprost tromethamine on Day 7 in cycling cows or on Day 9 in anestrous cows; CIDR withdrawal plus 0.5 mg im estradiol cypionate plus temporary calf removal on Day 9; TAI (48 h after CIDR withdrawal) plus reuniting of calves with their dams on Day 11. Reduced dose of prostaglandin F_2α (PGF_2α; 12.5 mg im dinoprost tromethamine) effectively caused luteolysis. In cycling cows, fertility was greater when the treatment with PGF_2α was administered on Day 7 than on Day 9, but in anestrous cows, no effects of time of the PGF_2α treatment were found. Estradiol cypionate effectively replaced estradiol benzoate or gonadotropin-releasing hormone as the ovulatory stimulus, reducing labor and cost. In this protocol, CIDR inserts were successfully used four times (9 d each use) with no detrimental effects on fertility.     

Reduced Efficacy of 8-OH-DPAT's Inhibition of Lordosis Behavior by Prior Estrogen Treatment

The effects of bilateral VMN infusion with the 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 200, 1000, or 2000 ng), on lordosis behavior were examined in hormonally primed ovariectomized rats. When rats were given a single injection with 25 microg estradiol benzoate followed 48 h later with 500 microg progesterone, inhibition of lordosis behavior was evident at all doses of 8-OH-DPAT. However, when rats were treated with 25 microg estradiol benzoate followed 7 days later with a second injection of 25 microg estradiol benzoate and then progesterone, none of the doses of 8-OH-DPAT effectively inhibited lordosis behavior. In some rats, cannulae were located near the most rostral portion of the VMN. In these rats, there was no effect of the second estrogen treatment on the response to 8-OH-DPAT. Therefore, a second experiment was performed to specifically evaluate the effects of two estradiol benzoate treatments on the response to bilateral 8-OH-DPAT infusion in the rostral VMN. In contrast to the reduced effectiveness of the 8-OH-DPAT infusion in the mid to caudal VMN in rats given two injections with estradiol benzoate, 2000 ng 8-OH-DPAT continued to effectively inhibit lordosis behavior following the 5-HT1A receptor agonist's infusion into the more rostral areas. These findings are discussed in relation to earlier studies in which the potency, but not the efficacy, of 8-OH-DPAT was reduced following systemic treatment with the 5-HT1A receptor agonist.     

Neonatal exposure to estradiol decreases hypothalamic allopregnanolone concentrations and alters agonistic and sexual but not affective behavior in adult female rats

Exposure of developing female rats to estradiol during the perinatal period induced long-lasting dysregulation of gonadal axis and decreased cerebrocortical and plasma concentrations of allopregnanolone. We have now examined the effects of neonatal estradiol administration in female rats on hypothalamic allopregnanolone concentrations and on exploratory, affective, agonistic and sexual behaviors as well as social learning. A single administration of β-estradiol 3-benzoate (EB, 10 μg) on the day of birth resulted in a delay of vaginal opening, acyclicity and ovarian failure. These alterations were associated with a significant decrease in the concentrations of allopregnanolone in the hypothalamus at 21 and 60 days, but not at 7 days, after birth. Neonatal administration of EB also increased agonistic behaviors in adult rats, such as dominant behaviors and following of an ovariectomized intruder, while living attacks unaffected. EB-treated rats showed also an increase in anogenital investigation, associated with a drastic reduction in spontaneous and induced female sexual behaviors (receptivity and proceptivity). In contrast, neonatal administration of EB did not affect locomotor activity, anxiety- and mood-related behaviors, the social transmission of flavor preferences, and seizures sensitivity. These effects of estradiol suggest that it plays a major role in regulation of both the abundance of allopregnanolone and the expression of agonistic and sexual behaviors, while failing to influence affective behaviors and social learning. Thus, the pronounced and persistent decrease in hypothalamic allopregnanolone concentration may be related to the manifestation of agonistic and sexual behaviors.     

Progesterone concentrations, exogenous equine chorionic gonadotropin, and timing of prostaglandin F2α treatment affect fertility in postpuberal Nelore heifers

Two experiments were performed to test the hypothesis that elevated progesterone concentrations impair pregnancy rate to timed artificial insemination (TAI) in postpuberal Nelore heifers. In Experiment 1, postpuberal Nelore heifers (n = 398) received 2 mg estradiol benzoate (EB) and either a new progesterone-releasing intravaginal device containing 1.9 g of progesterone (CIDR) (first use) or a CIDR previously used for 9 d (second use) or for 18 d (third use) on Day 0, 12.5 mg prostaglandin F_2α (PGF_2α) on Day 7, 0.5 mg estradiol cypionate (ECP) and CIDR withdrawal on Day 9, and TAI on Day 11. Largest ovarian follicle diameter was determined on Day 11. The third-use CIDR treatment increased largest ovarian follicle diameter and pregnancy rate. Conception to TAI was reduced in heifers with smaller follicles in the first- and second-use CIDR treatments, but not in the third-use CIDR treatment. In Experiment 2, postpuberal Nelore heifers received the synchronization treatment described in Experiment 1 or received 12.5 mg PGF_2α on Day 9 rather than Day 7. In addition, 50% of heifers received 300 IU equine chorionic gonadotropin (eCG) on Day 9. Heifers were either TAI (Experiment 2a; n = 199) or AI after detection of estrus (Experiment 2b; n = 125 of 202). In Experiment 2a, treatment with eCG increased pregnancy rate to TAI in heifers that received PGF_2α on Day 9 but not on Day 7 and in heifers that received a first-use CIDR but not in heifers that received a third-use CIDR. Treatments did not influence reproductive performance in Experiment 2b. In summary, pregnancy rate to TAI in postpuberal Nelore heifers was optimized when lower concentrations of exogenous progesterone were administered, and eCG treatment was beneficial in heifers expected to have greater progesterone concentrations.     

The effect of estrogen treatment on scopolamine inhibition of lordosis.

Previous evidence indicates that the cholinergic muscarinic antagonist, scopolamine, inhibits lordosis in female rats. In the experiments reported here, the effects of various doses and repeated administrations of estrogen on the scopolamine inhibition of lordosis were examined. In the first experiment, intraperitoneal injections of scopolamine (1 mg/rat) completely inhibited lordosis in ovariectomized rats primed with low doses of estradiol benzoate (0.25 or 0.5 micrograms for 3 days) and progesterone (500 micrograms). However, scopolamine was significantly less effective in inhibiting lordosis in females primed with a higher dose of estradiol benzoate (25 micrograms for 3 days) and progesterone (500 micrograms). When hormone priming was repeated on subsequent weeks, scopolamine continued to inhibit lordosis in females that received 0.25 micrograms estradiol benzoate but was less effective in females primed with 0.5 micrograms. Scopolamine failed to inhibit lordosis in females treated with 25 micrograms estradiol benzoate on these later tests. In the second experiment, various doses of scopolamine (1, 2, or 4 mg/rat) were administered intraperitoneally to females primed with the highest dose of estradiol benzoate (25 micrograms) and progesterone (500 micrograms). Lordosis was inhibited equally by all scopolamine doses during the first week. As in the first experiment, scopolamine failed to inhibit lordosis at all doses on subsequent weeks of testing. These results indicate that the ability of scopolamine to inhibit lordosis is reduced by increasing the dose or the number of estrogen exposures. Because higher doses of scopolamine failed to restore its inhibitory effect on lordosis an upregulation of muscarinic receptors by estrogen cannot account for the reduced effectiveness of scopolamine.     

Effects of low versus physiologic plasma progesterone concentrations on ovarian follicular development and fertility in beef cattle

The objective of this study was to determine the effects of low versus physiologic plasma progesterone concentrations during the ovulatory wave on fertility in cattle. Suckled beef cows (Bos taurus; n = 129) and pubertal heifers (Bos taurus; n = 150) at random stages of the estrous cycle were given a luteolytic dose of prostaglandin F_2α (500 μg cloprostenol; PGF) twice, 11 d apart. Ten days after the second PGF treatment, cattle were given estradiol benzoate im (1.5 and 1.0 mg for cows and heifers, respectively) and a progesterone-releasing intravaginal device (Cue-Mate) with a single pod containing 0.78 g progesterone (Day 0). Cattle in the low-progesterone group (n = 148) received a luteolytic dose of PGF on Day 0, whereas those in the high-progesterone (i.e., physiologic plasma concentrations) group (n = 131) were allowed to retain their corpora lutea. On Day 8, the Cue-Mate was removed, and PGF was given to both groups. Fifty-four hours to 56 h later, cattle received 12.5 mg of porcine LH (pLH) im and were concurrently artificially inseminated. The dominant follicle in the low-progesterone group was larger (P < 0.001) than that in the high-progesterone group on the day of insemination (14.9 ± 0.3 mm vs. 12.7 ± 0.3 mm, mean ± SEM). At 7 d after ovulation, the low-progesterone group had a larger corpus luteum (24.5 ± 0.54 mm vs. 21.9 ± 0.64 mm, P < 0.01) and higher plasma progesterone concentration (4.0 ± 0.3 vs. 3.1 ± 0.2, P < 0.01) than that of the high-progesterone group. However, pregnancy rates did not differ (79 of 148, 53.4%, and 70 of 131, 53.4%) for low- and high-progesterone groups, respectively). In summary, low circulating progesterone concentrations during the growing phase of the ovulatory follicle resulted in a larger dominant follicle and a larger CL that produced more progesterone, with no significant effect on pregnancy rate.     

Prediction of estrus cyclicity in Asian elephants (Elephas maximus) through estimation of fecal progesterone metabolite: development of an enzyme-linked immuno-sorbent assay

Asian elephants (Elephas maximus), prominent “flagship species”, are listed under the category of endangered species (EN - A2c, ver. 3.1; IUCN Red List 2009) and there is a need for their conservation. This requires understanding demographic and reproductive dynamics of the species. Monitoring reproductive status of any species is traditionally being carried out through invasive blood sampling and this is restrictive for large animals such as wild or semi-captive elephants due to legal, ethical, and practical reasons. Hence, there is a need for a non-invasive technique to assess reproductive cyclicity profiles of elephants, which will help in the species’ conservation strategies. In this study, we developed an indirect competitive enzyme linked immuno-sorbent assay (ELISA) to estimate the concentration of one of the progesterone-metabolites i.e., allopregnanolone (5α-P-3OH) in fecal samples of Asian elephants. We validated the assay which had a sensitivity of 0.25 μM at 90% binding with an EC₅₀ value of 1.37 μM. Using female elephants, kept under semi-captive conditions in the forest camps of Mudumalai Wildlife Sanctuary, Tamil Nadu and Bandipur National Park, Karnataka, India, we measured fecal progesterone-metabolite (5α-P-3OH) concentrations in six animals and showed their clear correlation with those of serum progesterone, measured by a standard radio-immuno assay. Statistical analyses using a Linear Mixed Effect model showed a positive correlation (P < 0.1) between the profiles of fecal 5α-P-3OH (range: 0.5-10 μg/g) and serum progesterone (range: 0.1-1.8 ng/mL). Therefore, our studies show, for the first time, that the fecal progesterone-metabolite assay could be exploited to predict estrus cyclicity and to potentially assess the reproductive status of captive and free-ranging female Asian elephants, thereby helping to plan their breeding strategy.     

Glutamate release in the ventromedial hypothalamus of the female rat during copulation: Modulation by estradiol

Binding of glutamate or its ionotropic receptor agonists in the ventromedial hypothalamus (VMH) of female rats inhibits both appetitive and consummatory aspects of sexual behavior. Because vaginocervical stimulation activates glutamate neurons in the VMH, and administration of estradiol benzoate (EB) and progesterone (P) delays this effect, the present study examined the effects of hormonal priming on glutamate release within the VMH of female rats paired with sexually vigorous males. Ovariectomized, sexually experienced rats were implanted with guide cannula aimed at the ventrolateral VMH, through which microdialysis probes were inserted prior to testing. Females were assigned randomly to one of three hormone treatment conditions: EB + P, EB alone, or the oil vehicle. Testing was conducted over 5 h, including a 120-min period of habituation to the testing chamber, a 60-min period of baseline sample collection, and a 120-min period during which a sexually vigorous male was introduced into the testing chamber. Dialysates were collected every 20 min during the test and were analyzed for glutamate using HPLC. Females primed with oil had large and significant increases in glutamate release from baseline once the male was introduced to the chamber. Treatment with EB alone decreased glutamate release in response to male cues. Although treatment with EB + P did not differ significantly from EB alone, the degree of reduced glutamate release was less than with EB alone. These results indicate that priming with EB reduces glutamate transmission in the VMH in response to male cues. Taken together with our previous findings, estradiol blunts the activation of glutamate neurons in the VMH thus allowing female rats to copulate.     

Nomegestrol acetate, a novel progestogen for oral contraception

Nomegestrol acetate (NOMAC) is a potent, highly selective progestogen, which is structurally similar to 19-norprogesterone and characterized as a full agonist at the progesterone receptor, with no or minimal binding to other steroid receptors, including the androgen and glucocorticoid receptors. In animal models, NOMAC demonstrated moderate antiandrogenic activity and strong antiestrogenic activity. In clinical studies, the progestogen was associated with effective suppression of gonadotropic activity and ovulation in premenopausal women, and a neutral impact on hemostasis, lipids, and carbohydrate metabolism. In normal and cancerous human breast tissue, NOMAC has shown favorable effects on estrogen metabolism, and in human breast cancer cell lines in vitro, it does not stimulate cell proliferation. The pharmacologic profile of NOMAC suggested that it would be well suited for combination with a physiologic estrogen in a combined oral contraceptive (COC), with the aim of achieving effective contraception with good cycle control and a favorable safety profile. A monophasic COC containing NOMAC 2.5 mg and 17β-estradiol (E2) 1.5 mg, administered in a 24/4-day regimen, is currently under clinical investigation. In a phase III study, NOMAC/E2 provided consistent and robust ovulation inhibition, with contraceptive effects that compared favorably with those of drospirenone 3 mg/ethinyl estradiol (EE) 30 μg. Investigators for a second phase III study reported less overall impact with NOMAC/E2 on hemostatic, lipid, inflammatory, and carbohydrate metabolism parameters than with levonorgestrel 150 μg/EE 30 μg. These clinical findings are promising; however, full publication of results from the pivotal phase III trials of NOMAC/E2 is pending.     

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 µg), a Src kinase inhibitor, significantly depressed P-dependent estrous behavior (lordosis and proceptivity) in estradiol-primed rats. Icv infusion of 30 µg of PP2 also significantly attenuated estrous behavior induced by the ring A-reduced P metabolites 5α-dihydroprogesterone (5α-DHP) and 5α-pregnan-3α-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 µg 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.     

Ovarian hormones,copulatory stimuli,and female sexual behavior in the Mongolian gerbil

The effects of repeated estradiol benzoate (1.0 or 3.3 μg) and progesterone (0.5 mg) injections and mating experiences (10 min or 4 hr) were examined in ovariec-tomized female Mongolian gerbils (Meriones unguiculatus) paired with sexually vigorous but unfamiliar males. Estradiol benzoate alone for 2 days did not elicit female sexual responses. Estradiol benzoate for 2 days followed by progesterone facilitated lordosis in the female. In females injected daily with 1.0 μg estradiol benzoate, progesterone exposure for approximately 1 day produced a significant inhibition in subsequent lordosis, although these females continued to respond to the male with lordosis quotients of approximately 60. A 4-hr mating experience produced a nearly complete elimination of sexual activity in tests observed 24 hr later with a fresh male. Footstomping was positively associated with sexual activity in both sexes, although females footstomped more often in the 1.0 rather than the 3.3-μg estradiol benzoate condition. Fighting was most frequent in tests in which lordosis quotients were low. Interactions among copulatory experiences and inhibitory effects of progesterone summed to terminate female sexual receptivity and alter other social interactions in the gerbil.     

Sensitization of sexual behavior in ovariectomized rats by chronic estradiol treatment

The ovariectomized (OVX) rat treated with estradiol benzoate (EB) is used to elucidate neuroendocrine mechanisms of sexual behavior. Chronic behavioral and pharmacological manipulations can be confounded by rising baselines, since females are behaviorally more sensitive to repeated EB injections. The literature lacks a systematic examination of chronic effects of EB administered alone to the sexually experienced OVX rat. Long–Evans rats were repeatedly treated (8 tests) with s.c. injections of 2, 5, or 10 μg EB at different time intervals (4 or 8 days). Female sexual behaviors as well as receipt of mounts, intromissions and ejaculations from the male were observed in the unilevel 4-hole pacing chamber. The effects of adrenalectomy (ADX) and strain (Long–Evans vs. Wistar) were also assessed. Long–Evans OVX rats treated with 5 μg EB every 8 days showed persistently low levels of sexual behavior. Sensitization was most robust following 10 μg EB at 4-day intervals. Very few sexual behaviors were ever induced by 2 μg EB. ADX did not affect the development of behavioral sensitization by 10 μg EB. Therefore, to achieve a low steady state of sexual behaviors in sexually experienced Long-Evans OVX rats 5 μg of EB administered every 8 days is optimal, whereas a persistently high level of sexual behaviors is induced with 10 μg EB administered every 4 days. OVX Wistar rats are behaviorally more sensitive to EB. Behavioral sensitization to EB may serve as a mechanism to optimize reproductive success.     

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 24 h 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.     

Pregnancy rates and corpus luteum-related factors affecting pregnancy establishment in bovine recipients synchronized for fixed-time embryo transfer

The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus × Bos taurus indicus) nonlactating cows and heifers (n = 259) were treated with the following protocol: 2 mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9 g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F_2α (PGF_2α) and CIDR withdrawal (Day 8); and 1 mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo-derived (n = 90) or in vitro-produced (n = 87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo-derived embryos than for in vitro-produced embryos (58.8% vs. 31.0%, respectively; P < 0.001). Mean (±SD) plasma progesterone (P₄) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2 ± 5.0 vs. 3.8 ± 2.4 ng/mL; P = 0.02). Mean pixel values (71.8 ± 1.3 vs. 71.2 ± 1.1) and pixel heterogeneity (14.8 ± 0.3 vs. 14.5 ± 0.5) were similar between pregnant and nonpregnant recipients (P > 0.10). No significant relationship was detected between pregnancy outcome and plasma P₄, corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum-related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome.     

Effects of estradiol and progestins on follicular regression before, during, and after follicular deviation in postpartum beef cows

The objective was to evaluate the effect of estradiol benzoate (EB), in association with three progestin protocols, on ovarian follicular regression of suckled beef cows treated at three stages of follicular development (pre-deviation, deviation, or post-deviation). Thirty-six suckled beef cows (60-90 d postpartum, given 125 μg cloprostenol on two occassions, 12 h apart). Forty-eight hours after the first cloprostenol treatment, all follicles >5 mm were ablated and transrectal ultrasound scanning (8 MHz) was performed every 24 h until Day 7 (Day 0 = treatment). When the largest follicle reached a designated diameter of 5-7, 8-10 or >10 mm, cows were randomly allocated to receive 2 mg of EB im in association with an intravaginal device containing 250 mg of medroxyprogesterone acetate (MPA) with or without 100 mg of progesterone (P₄) given im, or an intravaginal device containing P₄ (3 × 3 factorial design). Treatments induced follicular regression in all cows, independent of follicular stage or treatment. There was no interaction between progestin treatment and follicular stage, nor was there any difference in the time of follicular regression or new wave emergence among follicular stages. Treatment with MPA plus P₄ delayed follicular regression. In conclusion, EB in association with various progestins induced regression of growing follicles and emergence of a new follicular wave in postpartum beef cows, regardless of the stage of follicular development.     

Estrogen receptor ligands counteract cognitive deficits caused by androgen deprivation in male rats

Androgen deprivation causes impairment of cognitive tasks in rodents and humans, and this deficit can be reverted by androgen replacement therapy. Part of the effects of androgens in the male may be mediated by their local metabolism to estradiol or 3-alpha androstanediol within the brain and the consequent activation of estrogen receptors. In this study we have assessed whether the administration of estradiol benzoate, the estrogen receptor β selective agonist diarylpropionitrile or the estrogen receptor α selective agonist propyl pyrazole triol affect performance of androgen-deprived male Wistar rats in the cross-maze test. In addition, we tested the effect of raloxifene and tamoxifen, two selective estrogen receptor modulators used in clinical practice. The behavior of the rats was assessed 2 weeks after orchidectomy or sham surgery. Orchidectomy impaired acquisition in the cross-maze test. Estradiol benzoate and the selective estrogen receptor β agonist significantly improved acquisition in the cross-maze test compared to orchidectomized animals injected with vehicle. Raloxifene and tamoxifen at a dose of 1 mg/kg, but not at doses of 0.5 or 2 mg/kg, also improved acquisition of orchidectomized animals. Our findings suggest that estrogenic compounds with affinity for estrogen receptor β and selective estrogen receptor modulators, such as raloxifene and tamoxifen, may represent good candidates to promote cognitive performance in androgen-deprived males.