Cervicovaginal Stimulation Research Articles

Vaginal Lubrication after Cervicovaginal Stimulation Is Facilitated by Phosphodiesterase Type 5 Inhibition in Ovariectomized Mice

IntroductionNitric oxide synthases (NOSs) and estrogen receptors are expressed in the vagina. AimWe aimed to assess the impact of sildenafil on vaginal lubrication according to the hormonal status and to determine the role of the neuronal isoform of NOS (nNOS). MethodsFourweekold C57/BL6 female mice were sham operated or ovariectomized. At 10 weeks of age, they were injected intraperitoneally by any combination of sildenafil, 7nitroindazole (7NI)—a potent selective nNOS inhibitor—or the corresponding vehicles. Vaginal lubrication was induced in a physiological manner by cervical vaginal probing and quantified depending on the hormonal and pharmacological conditions. The animals were then sacrificed for vaginal histomorphometry. Main Outcome MeasuresThe main outcome measure is the quantification of vaginal transudate after cervicovaginal stimulation and vaginal histomorphometry. ResultsSildenafil increased cervicovaginal probinginduced vaginal lubrication in ovariectomized and shamoperated animals. Ovariectomized mice exhibited decreased vaginal lubrication as compared with shamoperated mice. When taking into account the presence of severe vaginal atrophy, a threefold increase in transudate per gram of vagina wet weight was revealed in ovariectomized animals. Castration markedly reduced the thickness of the vaginal wall. nNOS inhibition by 7NI had no impact on vaginal lubrication. ConclusionsIrrespective of the hormonal status, sildenafil increased vaginal lubrication. The vaginal effect of sildenafil was independent of the nNOS pathway and more pronounced in ovariectomized animals.

Genital sensory stimulation shifts estradiol intraoviductal signaling from nongenomic to genomic pathways, independently from prolactin surges.

Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.

A SINGLE DOSE OF MIFEPRISTONE INDUCES OVULATION IN PSEUDOPREGNANT RATS

We used mifepristone (M) to evalute the role of progesterone in maintaining pseudopregnancy. Cycling rats were made pseudopregnant (psp) by cervico-vaginal stimulation (CVS) on the day of estrus (day 0) and received 10 mg/kg M or vehicle (control groups) on day 3. Blood samples were taken at 06.00 h on days 4, 6 or 7 or at 18.00 h on days 3, 4, 6 or 10. M induced proestrus 2 days later (day 5), estrus on day 6, and a second prolonged diestrus afterwards. Prolactin and progesterone levels were similar in the control and M treated groups excepting on day 6, when both were reduced in the M-treated animals, and these rats were in estrus, suggesting a temporary impairment of luteal function. To demonstrate activated corpora lutea the endometrium was scratched on the fourth day of the first or second diestrus in additional control and M-treated groups. The deciduomal response was seen in the control and M groups after scratching the endometrium on day 4 of the first or second diestrus, respectively, but M blocked the deciduomal response in the first diestrus. Ovulation was confirmed by finding that 66.7% of the M-treated rats showed ova in the Fallopian tubes on the M-induced estrus and 4 out of 10 of the M rats placed with males on the M-induced proestrus showed spermatozoa in the vaginal smears. Half of these became pregnant, delivering 2 pups each. The results show that M can induce ovulation in psp rats, demonstrating that the anovulation observed after CVS is dependent on progesterone, yet luteal function persists after M in pseudopregnancy. Progesterone may act either by suppressing LH secretion or by permitting prolactin secretion, or both. Moreover, progesterone is required to maintain endometrial responsiveness.

Endogenous opioid peptides participate in the modulation of prolactin release in response to cervicovaginal stimulation in the female rat.

Naloxone, a specific opiate receptor antagonist, was used to evaluate the influence of endogenous opiate peptides on PRL secretion during various stages of the estrous cycle and after cervicovaginal stimulation. Naloxone (0.2 mg/kg, iv) administered at 0930 h suppressed basal 1000 h PRL levels on each day of the estrous cycle, but had no effect on the PRL surges occurring on the afternoon of proestrus and estrus. These afternoon surges could only be suppressed when naloxone was given immediately before the onset of the surge at 1330 h, suggesting a critical period for naloxone action. Later injections (at 1530 and 1730 h) had no further suppressive effects, although these injections suppressed the basal PRL secretion occurring during diestrous days I and II to near undetectable levels. In contrast, the immediate (1000 h) and afternoon PRL discharges triggered by cervicovaginal stimulation at 0930 h on proestrus, estrus, and diestrous day I were significantly suppressed by a single injection of naloxone, but only when it was administered before application of the stimulus. Moreover, this single naloxone injection significantly inhibited the long term PRL surges occurring during days 2 and 4 of pseudopregnancy in animals stimulated on estrus and diestrous day I. Naloxone treatment significantly shortened the duration of pseudopregnancy but did not prevent it, indicating that only minimal levels of PRL may be necessary to initiate and maintain pseudopregnancy. This striking difference in the time at which naloxone suppressed the stimulus-induced PRL discharges (compared to the spontaneous surges) suggests that endogenous opiate peptides also process the sensory information necessary for establishment of PRL surges during pseudopregnancy. The marked dependence of the spontaneous as well as the stimulus-induced PRL discharges on the stage of the estrous cycle support established evidence that ovarian steroids enhance endogenous opiate activity or facilitate the transfer of information along opiatergic pathways for the generation of PRL surges.

Estrogen but not progesterone facilitates the lordosis reaction to cervicovaginal stimulation of ovariectomized rats

Ovariectomized rats were treated sequentially with 1, 10 or 100 μg/kg of estradiol benzoate and 8 mg/kg of progesterone (or vehicles) and were tested for lordosis reactivity. A male was used to apply cutaneous stimulation only, or cutaneous and cervicovaginal stimulation, in females with masked or unmasked vaginal orifices, respectively. Graded cervicovaginal stimulation alone was obtained with a manually operated penile dummy. Progesterone caused a significant increase in the lordosis quotient to male stimulation after the doses of 10 and 100 μg/kg of estrogen but not after 1 μg/kg of the hormone. This effect of progesterone was unaffected when the vaginal orifice was masked to avoid penile intromission and the resulting cervicovaginal stimulation. Lordosis reactions to artificially graded cervicovaginal stimulation following progesterone only or vehicle only were both infrequent and weak. The three estrogen doses employed increased markedly the reactivity to cervicovaginal stimulation, both when the percent frequency of lordosis and when lordosis intensity ratings were considered. In contraposition with the effect on male stimulation, progesterone did not increase the reactivity to cervicovaginal stimulation, as measured by the percent incidence of lordosis. This was also the case when lordosis intensity scores were considered, with the sole exception of a significant difference when 1 μg/kg of estradiol and the strongest stimulation were used. Our results suggest that the sensitivity of the afferent pathways to elicit the lordosis reaction are under different hormonal modulations: cutaneous afferents seem to be facilitated by estrogen and progesterone, while cervicovaginal afferents would be facilitated only by estrogen.

Modulation of sensitivity to cervicovaginal stimulation during the estrous cycle: Evidence for an extrapituitary action of LH-RH

Single stimulations of the vagina and cervix were performed between proestrus and the first day of diestrus with a stimulator designed to grade the intravaginal penetration of a rod. The percent incidence of pseudopregnancy after this stimulation was exponentially related to the extent of intravaginal penetration and was also affected by the stage of the cycle at which the stimulation was performed. At 10.00 h on proestrus, an exponential increase in the incidence of pseudopregnancy was observed with shallow penetrations, while an exponential decrease was found when deeper penetrations were applied. Such negative exponential correlation had disappeared at 22.00 h on proestrus. At that time, also, some responses were elicited by very shallow penetrations (17 mm) and all the animals responded to penetrations of 20 mm or more. Sensitivity to cervicovaginal stimulation at 10.00 h on estrus was lower than that at 22.00 h on proestrus and it was even lower at 10.00 h on the first day of diestrus. The response to 18 mm of penetration was studied every 3 h between 10.00 h on proestrus and 10.00 h on estrus, and then every 12 h until 10.00 h on the first day of diestrus. This stimulation was usually ineffective to induce pseudopregnancy, except for a brief period encompassing the night between proestrus and estrus, when a peak in the incidence of responses was reached. This peak sensitivity could be advanced following the s.c. administration of 250 and 500 ng of LH-RH at 11.00 h on proestrus. Other doses were ineffective. The peptide (500 ng) was unable to induce pseudopregnancy in rats that received no cervicovaginal stimulation. Also, the number of ova shed on that cycle was not affected by this treatment. However, several doses of LH, FSH or their combinations failed to mimic the facilitation of the response to cervicovaginal stimulation by LH-RH.

Sensory mechanisms involved in the induction of pseudopregnancy by progesterone: increased sensitivity to stimulation of the pudendal sensory field.

The sensory mechanisms that participate in the induction of pseudopregnancy after a single injection of progesterone were investigated. Unless otherwise indicated, rats were kept in group cages and vaginal smears were taken daily. Progesterone evoked pseudopregnancy in a dose-dependent manner when administered to proestrous or estrous rats that received no cervicovaginal stimulation. The probability of pseudopregnancy after progesterone was higher on estrus. Cervicovaginal stimulation of proestrous rats that received 5 mg progesterone 10 h before was performed with a rod with a sliding stop attached to regulate its intravaginal penetration. Progesterone facilitated responsiveness to this stimulus, although the amount injected was not significantly effective in increasing the incidence of pseudopregnancy in nonstimulated rats. However, the mere application of the stop of the stimulator on the perineal skin was followed by a significantly higher incidence of pseudopregnancy in progesterone-injected rats than in their vehicle-injected controls, which suggested an action of the steroid on perineal sensitivity. Accordingly, the pseudopregnancy-evoking effect of progesterone was clearly inhibited by refraining from taking vaginal smears for 5 days after steroid injection on estrus. No further inhibition was observed after isolating the animals in single rat cages. However, daily finger stimulation of the perineal skin of nonsmeared rats restored to a normal level response to progesterone. Furthermore, this response was severely impaired by transecting the pudendal nerves before the injection. It is concluded that pseudopregnancy is induced in progesterone-treated rats through sensory stimulation of the pudendal receptive field and it is suggested that the pudendal nerve may subserve as a secondary afferent system to elicit the pseudopregnancy response. The possibility progesterone also acts on other afferent systems including the main afferent system constituted by the pelvic nerve is discussed.

A study of the sensory requirements for eliciting the pseudopregnancy response

Stimulations of the vagina and cervix were performed with a stimulator designed to grade intravaginal penetration of a rod. Single cervicovaginal stimulations performed in estrus or in diestrus II elicited immediate or delayed luteal responses, respectively. The probabilities of both types of response increased as a function of the strength (penetration) and duration of the stimulus. Immediate responses were significantly more probable than delayed responses. The other observed parameters of the response (plasma progesterone and diestrus length) were not related to stimulus magnitude. The probabilities of immediate or delayed responses were related to rod penetration by power functions, implying an exponential transformation of the received stimulus. An exponential transformation of rod penetration into pressure on the cervix, which occurs at the vaginal level, seems not to contribute substantially to the final exponential transformation. The relationship between stimulus duration and response probability could not be defined mathematically. No further increase in probability of response was caused by prolonging a deep penetration (24 mm) beyond 5 s (immediate responses) and 15 s (delayed responses). The novel stimuli from an unusual procedure to immobilize the rat may interfere with the response to cervicovaginal stimulation. However, the extravaginal stimuli the female receives during coitus did not seem to facilitate or inhibit the response to a deep (24 mm) penetration of the rod. The results establish some quantitative relationships between stimulus and response parameters and indicate the importance of cervicovaginal stimuli, among the multiple sensory stimulation of mating, to induce the luteal response.

TSH Release during the Estrous Cycle of the Rat: Variations in Responsiveness to Cervicovaginal Stimulation

Serum TSH, LH and FSH were measured at various times during the day, during the estrous cycle of the rat. Proestrous surges of FSH and LH were detected as previously reported. TSH values fell to low levels in the late afternoon on each day of the cycle. There was a significant elevation of TSH at 13.00 h on both proestrus and estrus. The elevation of TSH on proestrus was accompanied by a significant fall in pituitary TSH stores. LH stores were also significantly depleted by 19.00 h on proestrus. Cervicovaginal stimulation by a glass rod provoked a significant rise in serum TSH 3 h after stimulation at 10.00 h on estrus but not on other days of the cycle. Responsiveness to a test dose of TRH varied during the cycle. It was maximal on proestrus and declined to minimum values on diestrus day II. Values were further lowered after ovariectomy but were restored to diestrus day II levels by treatment with estradiol. It is concluded that there are important cyclic variations in TSH release in the female rat, that cervicovaginal stimulation can provoke TSH release on estrus and that these changes are associated with altered responsiveness to TRH. We conclude that ovarian steroids modulate the release of TSH from the pituitary and affect the responsiveness to cervicovaginal stimulation. These changes are presumably brought about by alterations in TRH release and in responsiveness of the pituitary to the neurohormone.

Social behaviour between adult male and female new zealand fur seals, Arctocephalus forsteri (Lesson) during the breeding season.

Observations on social relations between territorial adult male and adult female A. forsteri were made on the Open Bay Islands, Westland, New Zealand, during the breeding season 1970-71. Threats comprised about 80% of all social encounters between males and females. Attempted and successful olfactory investigations of females by males, herding of females by males, and 'peace-keeping' by males accounted for about 18% of all encounters, and such interactions usually had agonistic overtones. Less than 2% of all encounters were not agonistic from their inception. Herding responses of males were vigorous and frequent, and are interpreted as serving two functions: containment of females and communication of certain of the herding males' characteristics to the females. Because herding occurs throughout the Otariidae, and is rarely effective in containing females, the second function is probably more important. Males showed much individual variation in their herding tendencies. The frequency of herding behaviour was depressed at warm temperatures. Olfactory investigations of facial and perineal regions of females were common and were not restricted to peri-oestrous females. Only oestrous females were sexually receptive to and showed little aggression toward territorial males, and males detected their physiological state mainly through olfaction. A few oestrous females solicited males with mock threats or by rubbing against them. Precopulatory and early copulatory behaviour was characterized by a moderate amount of mutual contact-seeking behaviour, multiple mounts by the male, and 'activation' of the female by the male biting her. Copulations appeared to be terminated through physical resistance by the female, which resulted in ejaculation and subsequent dismounting by the male. At the time of female resistance, the male commonly had to physically control her. The sudden return to aggressive behaviour typical of non-oestrous females may be mediated through a neural inhibitory process resulting from cervico-vaginal stimulation, as has been proposed for the guinea pig.

Mating behaviour in the chinchilla

Guinea pigs and chinchillas belong to the same suborder of rodents—the Hystrichomorpha; and their mating patterns have several significant similarities. In these common traits the two species differ from representatives of the Myomorpha such as the laboratory rat and mouse, deermouse and hamster. The differences have to do with inter relations between the male and female which result in the male's ejaculation. For the four myomorphic species ejaculation is preceded by a series of brief, closely-spaced penile insertions (rat, hamster, deermouse), or by a very large number of copulatory thrusts during a smaller number of intromissions (mouse). Female myomorphs in full behavioural oestrus are not often resistant to the male. Characteristically they receive multiple insertions and, in the rat, mouse and deermouse, successive ejaculations from the same or different males before losing their receptivity. Male chinchillas and guinea pigs frequently ejaculate during their first insertion after a few seconds of thrusting. It appears that when males take more than one intromission to ejaculate it is usually because the female has forced premature termination of coitus upon preceding insertions. The evidence reveals that many female chinchillas and guinea pigs are characteristically inonsistent in their willingness to receive the male. They often permit him to mount and then dislodge him just before or after the penis enters the vagina. In chinchillas and possibly in guinea pigs one ejaculation by the male frequently terminates the female's receptivity for several hours if not for the remainder of the oestrous period. It is here suggested that these differences in coital patterns may bear a functional relation to differences in female reproductive cycles. The guinea pig and chinchilla spontaneously exhibit a post-ovulatory luteal phase during which corpora lutea secrete progesterone and the uterus is prepared for implantation. Therefore insemination is the principal requirement for successful pregnancy. In contrast, for the rat, and probably for the other Myomorpha mentioned, secretion of effective amounts of progesterone depends upon cervicovaginal stimulation derived from the male's intromissions. Insemination without adequate stimulation leads to fertilization but not to pregnancy since secretion of progesterone is inadequate to permit implantation. It is proposed that consistent receptivity in myomorph females is essential for the male's successive pre-ejaculatory intromissions and these in turn are essential for successful impregnation. Since hystrichomorph females can be impregnated without extensive cervicovaginal stimulation there is in their case no obvious functional value to prolonged or persistent receptivity.