Mary S. Erskine

Behavioral effects of 3α-androstanediol I: modulation of sexual receptivity and promotion of GABA-stimulated chloride flux

Pregnane neurosteroids may initiate sexual receptivity not only via actions at intracellular receptors, but by affecting gamma-aminobutyric acid (GABA) receptor complexes (GBRs). To investigate whether GBR-mediated actions of an androgenic neurosteroid 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol; 3 alpha-Diol) may influence the expression of sexual behavior, ovariectomized (ovx) rats received daily injections of 3 alpha-Diol (0.6, 3.0, 6.0 and 7.5 mg/kg) or vehicle (10% (v/v) ethanol in propylene glycol) at 10.00 h, and s.c. injections of estradiol-17 beta (E2: 1 microgram/0.2 ml in 10% ethanol) at 13.00 h and 19.00 h. Progesterone (P: 0.5, 1.0, 2.0 and 4.0 mg/kg) or sesame-oil vehicle was given at 12.30 h on the day following two days of 3 alpha-Diol and E2 treatment. In Expt. 1, levels of sexual receptivity were measured at 18.00-19.00 h, 56-57 h after the first injection of 3 alpha-Diol and 4 h after P or vehicle injection. 3 alpha-Androstanediol (6.0 mg/kg) attenuated sexual behavior (lordosis quotient, lordosis rating) and facilitated aggressive/rejection behaviors following 0.0, 1.0, 2.0 and 4.0 mg/kg P. The highest dosage of 3 alpha-Diol (7.5 mg/kg) facilitated sexual behavior and inhibited aggression behaviors following 0.0, 1.0, 2.0 and 4.0 mg/kg P. In Expt. 2, GABA-stimulated chloride flux was greater in cortical synaptoneurosomes of animals that received hormone treatments associated with inhibited receptivity (E2 + P + 3 alpha-Diol 3.0 mg/kg) than following treatments that facilitated receptivity (E2 + P and E2 + P + 3 alpha-Diol 7.5 mg/kg) or unreceptive ovx animals. In Expt. 3, circulating concentrations of 3 alpha-Diol resulting from the 0.0, 3.0 and 7.5 mg/kg s.c. doses administered to E2- and P-primed animals was measured by radioimmunoassay. Circulating levels of 3 alpha-Diol at the completion of behavioral testing were comparable to those previously ascertained across the estrous cycle. These data indicate that 3 alpha-Diol influences the expression of E2 and P-induced receptivity, and suggest that 3 alpha-Diol, like other neurosteroids, may exert its effects on sexual behavior by actions at GBRs.

Mating-induced FOS-like immunoreactivity in the rat forebrain: a sex comparison and a dimorphic effect of pelvic nerve transection

Previous research has shown that mating induces the expression of the immediate‐early gene, c‐fos, as detected by the increased presence of nuclear FOS‐like immunoreactivity (FOS‐IR), in specific forebrain regions of both male and female rats. In the male both olfactory/vomeronasal (O/V) and genital/somatosensory (G/S) inputs appear to contribute to the neural FOS response to mating whereas in the female G/S input carried by the pelvic nerves appears to mediate the forebrain FOS response. To date, however, no direct sex comparison of the mating‐induced forebrain FOS response has been made in rats maintained under the same steroidal conditions nor has the contribution of afferent sensory input from the pelvic nerves been assessed in males. We first compared the level of FOS‐IR in brain regions of mated and unpaired gonadectomized male and female rats given 5μg/kg estradiol benzoate (EB) for 7 days and 500 μg progesterone (P) 4 h prior to testing. One h after experiencing 1 ejaculation, both sexes showed increased FOS‐IR in the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), medial amygdala, the ventro‐lateral portion of the ventromedial nucleus of the hypothalamus (VMN), and the midbrain central tegmental field (CTF). This increase was significantly greater in the MPOA and medial amygdala of mated females than of males. Bilateral transection of the pelvic nerves significantly attenuated the increase in FOS‐IR after mating in the CTF of male rats and in the MPOA, BNST, VMN, medial amygdala and CTF of females. Thus, following mating there is no sex difference in the brain regions which express c‐fos, but there is a dimorphism in the contribution of afferent information conveyed by the pelvic nerves to the mating‐induced FOS response. The neural FOS response of the female to mating is heavily dependent upon the G/S afferent inputs carried by the pelvic nerves whereas the males neural c‐fos response may depend on O/V input plus G/S input conveyed via other afferent pathways such as the pudendal nerves.

Patterns of Induction of the Immediate-Early Genes c-fos and egr-1 in the Female Rat Brain following Differential Amounts of Mating Stimulation

Vaginocervical stimulation received either during mating or by artificial mechanical means has been shown to induce FOS expression in medial amygdala, preoptic area, hypothalamus, and midbrain of female rats. While mating-induced increases in FOS-like immunoreactivity (FOS-IR) have been shown to require intromissive stimulation from males, the pattern of FOS-IR in animals receiving numbers of intromissions across a range relevant to the induction of the prolactin surges of early pregnancy has not been explored. Experiment 1 examined brain FOS-IR following 15 mounts without intromission or 5, 10, or 15 intromissions in ovariectomized females treated with estrogen and progesterone; these treatments are known to be less than or more than sufficient to trigger prolactin surges in cycling females. FOS was expressed in a graded fashion in the medial amygdala with respect to the numbers of intromissions received and in an all-or-nothing manner in preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus of the hypothalamus. In experiment 2, 15 intromissions induced expression of another immediate-early gene, egr-1, in each of these same areas as well as in a second division of the bed nucleus of the stria terminalis and in the paraventricular nucleus of the hypothalamus. These studies demonstrate that mating is differentially effective in inducing FOS expression in responsive brain areas and point to the medial amygdala as a site in which summation of intromissive stimulation may occur. Furthermore, the induction of EGR-1 may be a more sensitive marker for mating-induced neural activation in these areas than is FOS.

c-Fos proto-oncogene activity induced by mating in the preoptic area, hypothalamus and amygdala in the female rat: role of afferent input via the pelvic nerve.

In order to identify brain areas which receive afferent genitosensory input important for mating-induced prolactin release, we compared numbers of Fos-immunoreactive (Fos-IR) cells in brains of intact estrous females 1 h after differential mating stimulation. Numbers of Fos-IR cells were approximately 3-fold higher in the preoptic area (POA), medial amygdala (mAMYG) and bed nucleus of the stria terminalis (BNST) when females received intromissions (I) from males than when they received mounts-without-intromission (M) or were taken directly from their home cage. In the ventrolateral portion of the ventromedial nucleus (VL-VMN), the paraventricular nucleus (PVN) of the hypothalamus and the midbrain central tegmental field (CTF) numbers of Fos-IR cells were significantly higher than home cage levels in groups of females exposed to males regardless of type of mating stimulation received. Bilateral transection of the pelvic nerve eliminated the increases in Fos-IR in POA and mAMYG which occurred in sham-transected females in these areas after intromissions from males. These data demonstrate that afferent input via the pelvic nerve activates cell groups within the POA, mAMYG and BNST and suggests that these areas may be involved in initiation of mating-induced prolactin surges.

Paced copulation in rats: Effects of intromission frequency and duration on luteal activation and estrous length

When estrous female rats regulate or pace (P) the timing of vaginal intromissions received from males during mating, the stimulation is more effective in inducing luteal function and abbreviating the period of receptivity than is nonpaced (NP) stimulation. The present studies examined whether the coital stimuli necessary for each of these functional consequences are similar. In Experiment 1, estrous females received either 5 or 10 intromissions from males in P or NP tests; control animals received mounts-without-intromission (MO). The duration of estrus was not affected by 5P, 5NP, or 10NP stimulation, but was significantly abbreviated in 10P animals. In contrast, activation of prolonged luteal function occurred in 70% of 5P females compared to only 10% of 5NP females; luteal activation was similar in 10P and 10NP females (74% for both groups combined). In Experiment 2, male copulatory behaviors were compared in tests with P and NP females. Males tested with P females exhibited significantly longer intromission durations (616 +/- 21 msec) than did males tested with NP females (527 +/- 30 msec). Other measures of male copulatory performance such as the number of intromissions to ejaculation and the ejaculation latency did not differ between groups. These studies demonstrate that luteal activation is more readily induced by paced coital stimulation than is abbreviation of estrus. In addition, they suggest that differences between P and NP females in the behavioral and neuroendocrine responses to coital stimulation may result from differences in intromission duration displayed by males under these test conditions.

Mating-induced increases in FOS protein in preoptic area and medial amygdala of cycling female rats

Genitosensory stimulation received during mating initiates neural and endocrine changes necessary for pregnancy. The present study examined sites of induction of the proto-oncogene, c-Fos, after mating in the cycling female rat to determine neural sites activated by such stimulation that might be involved in pregnancy initiation. Two groups of cycling female rats were exposed to males on the evening of proestrus and remained with the male until receiving either 14 mounts-with-intromission including ejaculations (Intromissions) or a control number of mounts-without-intromission (Mounts-Only). These two mating treatments were previously shown to induce pregnancy/pseudopregnancy in 100% and 0% of the animals, respectively. Seventy-five minutes after mating, females were perfused intracardially with 2% paraformaldehyde/2.5% acrolein, and the brains were processed for FOS immunocytochemistry using standard procedures. FOS-immunoreactive cells (FOS-IR) were counted in standard template quadrilaterals in the preoptic area (POA), medial amygdala (mAMYG), and the paraventricular (PVN), ventromedial (VMN), and dorsomedial (DMN) nuclei of the hypothalamus at 50 x magnification using a camera lucida. Significantly higher numbers of FOS-IR cells were seen in Intromissions females above Mounts-Only females in the POA and mAMYG, demonstrating that c-Fos expression in these areas depended upon cervical-vaginal stimulation rather than on cutaneous somatosensory input received in Mounts-Only tests. The cells within the POA and mAMYG that are activated in response to the intromittive stimulus may be directly involved in pathways triggering one or several of the neuroendocrine responses to mating in the female rat.

Behavioral effects of 3a-androstanediol II: Hypothalamic and preoptic area actions via a GABAergic mechanism

We investigated whether 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol; 3 alpha-Diol), a neurosteroid whose effects are primarily inhibitory to sexual behavior, may act through interactions with gamma-aminobutyric acid (GABA) receptor complexes (GBRs) in the medial basal hypothalamus (MBH) and the preoptic area (POA). In Experiment (Exp.) 1, ovariectomized (ovx) rats were implanted with bilateral guide cannulae aimed above the MBH and were later treated with 17 beta-estradiol (E2, 2 injections of 1 microgram/0.2 ml in 10% ethanol) and either 3 alpha-Diol (3.0 mg/kg, s.c.) or vehicle. Progesterone (0.5 mg, s.c.) was given 24 h after the first E2 injection and a pre-test for lordosis responsiveness was carried out 4 h later. The GABAA agonist, muscimol (50 ng), then was infused into the MBH and rats were tested 10, 30 and 60 min later. Muscimol infusion facilitated lordosis behavior in vehicle-treated controls, but 3 alpha-Diol-treated animals failed to show this facilitation. To ascertain whether 3 alpha-Diol would also prevent muscimols action in the POA, a site in which muscimol inhibits, rather than facilitates, sexual receptivity, ovx animals in Exp.2 were implanted with bilateral guide cannulae aimed above the POA and were treated with E2, 3 alpha-Diol, and P and infused and tested as in Exp. 1. Muscimol and 3 alpha-Diol each significantly inhibited receptivity; when they were combined, the inhibition was more pronounced. In Exp. 3, POA infusions of the GABAA antagonist, bicuculline, counteracted muscimols and 3 alpha-Diols inhibition of sexual behavior. In Exp. 4, in vitro treatment of POA and MBH membrane fractions with 3 alpha-Diol (30 microM) enhanced maximal [3H]muscimol binding without altering the affinity of the binding sites for the agonist. These data suggest that 3 alpha-Diol inhibits E2 and progestin-induced lordosis behavior via actions at the GBR in both the MBH and POA.

Effects of paced mating and intromissive stimulation on feminine sexual behavior and estrus termination in the cycling rat.

The effects of differential mating stimulation on sexual behavior and estrus length were examined in cycling rats that could or could not self-regulate, or pace, the timing of sexual contact. Female rats (Rattus norvegicus) received 30 paced, 30 nonpaced, or 15 nonpaced followed by 15 paced intromissions during mating tests. Decreases in sexual responsiveness were seen during the second half of testing; pacing was associated with greater inter-intromission intervals, decreased proceptivity, and increased rejection behavior at this time. Female rats pacing during the second test half behaved similarly, regardless of prior treatment, showing that the number rather than the timing of prior intromissions affected subsequent behavior. However, estrus length was decreased by prior paced mating. These data suggest that changes in sexual responsivity occur throughout estrus and that the nature of these changes is differentially dependent on the type of mating stimulation received.

Prenatal and neonatal testosterone exposure interact to affect differentiation of sexual behavior and partner preference in female ferrets.

Implanting testosterone (T) subcutaneously over Postnatal Days 5-20 masculinized sexual behavior, reduced proceptive responsiveness, and shifted sexual preference more readily in male than in female ferrets gonadectomized on Day 5. This enhanced sensitivity of males to neonatal T was best duplicated in females exposed transplacentally to T over Embryonic Days (E) 27-39 (41-day gestation) and injected at birth with T (2.5 micrograms sc in oil: 10% ethanol). Extended exposure of male ferrets to high levels of T, beginning shortly after the onset of testicular steroidogenesis (E25) and continuing for several hours after birth (E41) normally sensitizes their brains to the subsequent organizational effects on coital performance and sexual motivation of the relatively low levels of T that circulate in male ferrets during the first 3 postnatal weeks.

Effects of paced and non-paced mating stimulation on plasma progesterone, 3α-diol and corticosterone

In the female rat, gonadal and adrenal progestins and androgens modulate sexual receptivity and in turn, their levels increase in response to mating stimulation. Paced mating, in which the female controls the timing of sexual contacts with the male, is particularly effective at eliciting acute increases in progesterone (P) and 5 alpha-Androstane-3 alpha, 17 beta-diol (3 alpha-Diol). Interestingly, restraint stress produces comparable increases in P and 3 alpha-Diol levels, as well as increases in corticosterone (CORT) levels. In this study, we explored the possibility that paced mating would be associated with increased CORT, in conjunction with mating-induced increases in P and 3 alpha-Diol. Ovariectomized rats primed with estradiol benzoate (10 micrograms in oil SC) and P (0.5 mg in oil s.c.) received a single ejaculatory series from males in paced or non-paced mating tests. Fifteen minutes post-mating rats were exposed to CO2 and rapidly decapitated for the collection of trunk blood and determination of P, 3 alpha-Diol and CORT via radioimmunoassay. As expected, P and 3 alpha-Diol concentrations were significantly elevated in serum obtained from animals allowed to pace their sexual contacts with males compared to those which did not pace their contacts. Importantly, although all mated animals had CORT levels between 10-20 micrograms/dl, there were no differences between paced and non-paced conditions. This suggests that the acute rises in P and 3 alpha-Diol in response to paced mating are not due to paced mating being more stressful than non-paced mating.