Do sensory calcitonin gene-related peptide nerve fibres in the rat pelvic plexus supply autonomic neurons projecting to the uterus and cervix?

Sensory nerve fibres containing calcitonin gene-related peptide (CGRP) innervate neurons of the paracervical ganglion (PCG) in the female rat pelvic plexus. We have combined retrograde tracing with immunocytochemistry to investigate whether CGRP-immunoreactive (-IR) fibres supply neurons targeting the genital tract. Of the total neurons projecting to either the uterine horns or the cervix, 38 and 41% received CGRP-IR innervation, respectively. All these neurons displayed choline acetyltransferase-IR, thus are cholinergic. They were found throughout the PCG and other pelvic plexus ganglia, namely accessory ganglia (AG) and hypogastric plexus (HP). Pelvic nerve section showed that afferent fibres in these nerves provided most of the CGRP-IR fibres supplying uterine- or cervical-related neurons in the PCG/AG, none in HP. It is suggested that such sensory-motor network may provide a local pathway for reflex control of genital tract activity, acting through cholinergic nerve projections.

Effect of short photoperiods on the in vitro GnRH release by hypothalamic explants in intact and castrated male Syrian hamsters: relation to testicular regression and recrudescence.

Prolonged exposure of adult Syrian hamsters to short days decreases LH and FSH circulating levels within 2-4 weeks, then induces testicular regression. After 18 weeks of short days, the testis size and gonadotropin levels increase spontaneously. This study investigated whether these phases of photosensitivity and photorefractoriness corresponded to variations of in vitro GnRH release. Male hamsters were either kept under long days (LD 16:8) or transferred to short days (SD 6:18) and sacrificed from 2-26 weeks after transfer. To separate the effects of testis feedback from a possible direct photoperiodic drive on the hypothalamus, males were bilaterally castrated, kept under LD or transferred to SD, and sacrificed from 2-14 weeks after transfer. Hypothalamic explants were incubated in a saline buffer for three periods of 15 min and exposed to KCl (60 mM) for 15 min. The return to basal values was followed for six periods of 15 min, then the explants were stimulated with copper complexed equimolarly with histidine (Cu/His, 200 microM) and prostaglandin E2 (PGE2, 10 microM). At the end of the incubation period, the concentration of GnRH remaining in the explants was measured. In intact males, GnRH release in vitro increased significantly between 2 and 4 weeks after transfer to short days; it returned to values similar to LD ones between 6 and 12 weeks, during the phase of testis involution. At the beginning of photorefractoriness (SD 14-18), it increased transiently and returned to values similar to LD ones from SD 20, during the testis spontaneous recrudescence. After castration, the in vitro GnRH release decreased significantly under LD and SD. The transfer of castrated hamsters to SD resulted in transient increases of GnRH release (SD 4, 8 and 14), and in a progressive loss of the explant's ability to release GnRH in vitro. These results showed a photoperiodic regulation of in vitro GnRH release and a testis feedback effect on this release. They demonstrated an inverse relationship between the readily releasable pool of GnRH and the circulating levels of gonadotrophins at the beginning of photosensitive and photorefractory phases and after castration.

Effect of melatonin on the release of gonadotropin-releasing hormone and cyclic AMP from the rat hypothalamus: an in vitro study.

Using an in vitro static incubation system of adult male rat hypothalami, we have studied the effect of melatonin on the release of gonadotropin-releasing hormone (GnRH) and cyclic adenosine monophosphate (cAMP). Mediobasal hypothalamus (MBH) and preoptic area (POA) were incubated separately in Minimum Essential Medium (MEM) for 6 h. The release of GnRH was measured by radioimmuno-assay in the incubation medium sampled every 7.5 min. In the MBH and POA incubation medium, the mean amount of GnRH released was 8.9 +/- 1.1 and 3.4 +/- 0.6 pg GnRH/7.5 min, respectively (P < 0.01). The mean number of GnRH pulses under basal conditions was 2 +/- 0.3 per 2 h in the MBH and 1.6 +/- 0.3 per 2 h in the POA (P > 0.05). Melatonin (10(-8) M) did not alter the release of GnRH in the presence or absence of forskolin (10(-4) M). Melatonin, which was without effect on basal cAMP, inhibited forskolin-stimulated cAMP accumulation in the medium by 50% in the MBH and 40% in the POA. These results suggest that in our incubation system, melatonin does not modify GnRH release, but probably acts through the melatonin binding sites located in the hypothalamus to inhibit forskolin-stimulated cAMP.

Annual variations of in vitro GNRH release by hypothalamic explants in intact and castrated male mink: relations with LH, FSH and testosterone circulating serum levels.

In mink, a short-day breeder, testis growth begins in autumn (November), reaches a maximum in February, before matings occur, and decreases from March to very low volumes during spring and summer. To study the effects of season and testosterone feedback on gonadotrophin and GnRH secretion, the annual variations of LH, FSH, testosterone and GnRH were studied in intact and castrated mink. As portal blood sampling raised serious difficulties, an in vitro static incubation system was used for studying GnRH variations. In intact mink, serum LH concentrations did not vary significantly throughout the year, whereas FSH concentrations increased significantly between September and November then decreased to a minimum in January. Testosterone values rose significantly from November to a maximum from January to March, decreased very rapidly thereafter. Castration in November resulted in a significant increase in LH and FSH concentrations which remained higher than the values measured in intact males throughout the year. In long-term castrated mink, FSH concentrations did not fluctuate during the year, whereas LH concentrations showed an annual variation, with high values in April and August. For the study of in vitro GnRH liberation, medio-basal hypothalamic explants were incubated in Krebs-Ringer phosphate buffer for 3 periods of 15 min, and stimulated with copper complexed equimolarly with histidine (Cu/His, 200 microM) and prostaglandin E2 (PGE2, 10 microM). After Cu/His, the release of GnRH was 1 to 4 fold the basal release; after PGE2, the increase was 4-7 fold the basal release. The basal release of GnRH increased significantly between September and October to reach a maximum in November, decreased significantly in December to a minimum in February then increased progressively from May. The release of GnRH stimulated by Cu/His and PGE2 showed the same seasonal variation as the basal release. Castration 8 days before the sacrifice did not alter the release of GnRH, except in December: the release stimulated by PGE2 was then higher in intact than in castrated mink. Taken together, these results indicate that, with an in vitro static incubation system, it is possible to study the annual variations of GnRH release and to correlate these variations with those of serum gonadotrophin and testosterone concentrations. The synthesis and release of GnRH increased slightly from May, under long days, then more rapidly from September, resulting in an increased secretion of FSH in October, responsible for testis recrudescence. The annual pattern of basal and stimulated GnRH release was similar in intact and castrated mink, suggesting a direct effect of the season on the hypothalamus, rather than a negative feedback effect of the testis; however, testosterone seemed to feedback mainly at the pituitary level.

Influence of season of birth on onset of gonadotrophic and ovarian functions in young doe hares (Lepus europaeus).

The pituitary and ovarian responses to a monthly i.v. injection of 5 micrograms luteinizing-hormone-releasing hormone (LHRH) were studied in three groups of young doe hares, born in January-February (group I), in April (group II) or at the end of the breeding season (August-September, group III). The LHRH injection was always followed by a release of LH and progesterone, which did not differ among the three groups at 3 months of age. The pituitary and ovarian responses to LHRH increased gradually from the age of 3 months in groups I and III and from the age of 9 months in group II. One female of the ten born in January-February ovulated and reached puberty in June, at the age of 4 months, but with a weak pituitary response. The females born in April displayed a seasonally delayed puberty, at 9 months of age (two of five females ovulated in the next January). Four of the five females born at the end of the breeding season ovulated after LHRH when 5 months old (in February), with a full pituitary-ovarian response. The low pituitary response of group I in June-August, even if 10-20% of females ovulated after LHRH, suggests a need for a period of short days. Then, the most favourable conditions for the hare to reach puberty would be a period of short decreasing daylengths during the fall, followed by increasing daylengths after the winter solstice.

Gonadotrophins and sex steroids during pregnancy and natural superfoetation in captive brown hares (Lepus europaeus).

In brown hares, which are induced ovulators, sexual behaviour occurs episodically at the beginning of pregnancy. From Day 34 (length of pregnancy is 41 days), the frequency of sexual chases followed by mating, ovulation and fertilization increased and 59% of pregnant females presented a natural superfoetation. The pattern of circulating luteinizing hormone (LH), follicle-stimulating hormone (FSH), oestradiol and progesterone was studied in 13 pregnant females left permanently with a male, and in 10 females isolated from males around Day 20 of pregnancy. In the 2 groups FSH concentrations were high at the beginning and end of pregnancy. All females presented a peak value of FSH in the last 4 days of pregnancy, regardless of mating stimuli. This peak value was higher for females left permanently with a male than for isolated ones. Oestradiol concentrations fluctuated between 20 and 100 pg/ml, without any clear correlation with sexual behaviour, stage of pregnancy or profiles of other hormones. Prepartum matings occurred when progesterone values were still greater than 50 ng/ml; they were followed by a transient rise in LH and by a periovulatory progesterone secretion, with values above 100 ng/ml in the morning after mating. Such modifications of LH and progesterone were not detected before Day 34, suggesting that mating stimuli are not able to induce an LH surge at the beginning of pregnancy. After Day 34, mating can induce an LH surge, ovulation and superfoetation.

Pituitary and ovarian responses to luteinizing-hormone-releasing hormone during pregnancy and after parturition in brown hares (Lepus europaeus).

Female hares were given an i.v. injection of 5 micrograms luteinizing-hormone-releasing hormone (LHRH) between Days 7 and 19 (n = 21), 20 and 33 (n = 17) and 34 and 41 (n = 17) of pregnancy, and in the 3 days after parturition (n = 16). Whatever the stage of pregnancy, the LHRH injection induced a release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and an acute secretion of progesterone; these hormonal responses increased significantly during pregnancy, to reach values similar to those observed in nonpregnant, nonpseudopregnant females during the breeding season in the 3 days after parturition. However, the release of LH remained monophasic in pregnant and post-partum females, in contrast to the unmated females during the reproductive season, in which there was a biphasic profile. The proportion of ovulating females after LHRH treatment was approximately 60% at the beginning and end of pregnancy; and, after parturition, fell to 23% between Days 20 and 33. After Day 33, the pituitary response to LHRH was significantly higher in ovulating than in nonovulating females. At the beginning of pregnancy, 67% of females aborted after LHRH injection; after Day 20, the incidence of abortion decreased significantly and was 0% from Day 34. The amplitude and duration of progesterone secretion by the new corpora lutea resulting from ovulation after LHRH injection were similar to those of corpora lutea induced in nonpregnant females during the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)