Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor ...subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.
We wanted to determine if changes in the expression of serotonin 2A receptor (5HT2A receptor) gene in the premammillary hypothalamus are associated with changes in reproductive neuroendocrine status. ...Thus, we compared 2 groups of ovariectomized-estradiol-treated ewes that expressed high vs low LH pulsatility in two different paradigms (2 groups per paradigm): (a) refractoriness (low LH secretion) or not (high LH secretion) to short days in pineal-intact Ile-de-France ewes (RSD) and (b) endogenous circannual rhythm (ECR) in free-running pinealectomized Suffolk ewes in the active or inactive stage of their reproductive rhythm.
In RSD ewes, density of 5HT2A receptor mRNA (by in situ hybridization) was significantly higher in the high LH group (25.3 +/- 1.4 vs 21.4 +/- 1.5 grains/neuron, P < 0.05) and 3H-Ketanserin binding (a specific radioligand) of the median part of the premammillary hypothalamus tended to be higher in the high group (29.1 +/- 4.0 vs 24.6 +/- 4.2 fmol/mg tissu-equivalent; P < 0.10). In ECR ewes, density of 5HT2A receptor mRNA and 3H-Ketanserin binding were both significantly higher in the high LH group (20.8 +/- 1.6 vs 17.0 +/- 1.5 grains/neuron, P < 0.01, and 19.7 +/- 5.0 vs 7.4 +/- 3.4 fmol/mg tissu-equivalent; P < 0.05, respectively).
We conclude that these higher 5HT2A receptor gene expression and binding activity of 5HT2A receptor in the premammillary hypothalamus are associated with stimulation of LH pulsatility expressed before the development of refractoriness to short days and prior to the decline of reproductive neuroendocrine activity during expression of the endogenous circannual rhythm.
Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The
aim of this study was to identify the high-affinity melatonin-receptor ...subtypes expressed in this structure. To achieve this,
we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By
using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis
(PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures
considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35 S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the
pattern of expression observed in the PT.
Abstract
Expression of melatonin receptor MTRNIA in the PMH of the ewe is subject to diurnal control
Melatonin transduces the effect of day length on LH secretion by acting on the hypothalamus. However, the precise hypothalamic site is unknown. Two studies were undertaken to clarify where melatonin ...acts in the hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes were screened to determine whether specific regional densities in melatonin binding existed. A higher density of binding was observed in the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher than the rest of the hypothalamus). This binding area is delimited rostrally by the infundibular recess, caudally by the mammillary bodies, dorsally by the fornix, and ventrally by the base of the brain; and it encompasses the premammillary and tuberomammillary nuclei. To test the functional importance of the identified area, 3 groups of animals received bilateral melatonin microimplants: 1) in the PMH (n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH; n = 8); and 3) sham-operated animals received empty microimplants in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated s.c. with a 20-mm SILASTIC brand capsule of estradiol and exposed to long days (16-h light, 8-h dark). At the end of the 80-day experiment, no animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group displayed a stimulation of LH secretion. In contrast, melatonin implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on day 44.5 +/- 5.3 (mean +/- SEM). ANOVA revealed that the changes in LH secretion were not different between the SHAM and the AH/MBH groups but the PMH group differed from the other 2 groups (P < 0.0001). This study suggests that the PMH is an important target for melatonin to regulate reproductive activity.
Seasonality of ovulatory activity is observed in European sheep and goat breeds, whereas tropical breeds show almost continuous ovulatory activity. It is not known if these tropical breeds are ...sensitive or not to temperate photoperiod. This study was therefore designed to determine whether tropical Creole goats and Black-Belly ewes are sensitive to temperate photoperiod. Two groups of adult females in each species, either progeny or directly born from imported embryos, were used and maintained in light-proof rooms under simulated temperate (8 to 16 h of light per day) or tropical (11 - 13 h) photoperiods. Ovulatory activity was determined by blood progesterone assays for more than two years. The experiment lasted 33 months in goats and 25 months in ewes.
Marked seasonality of ovulatory activity appeared in the temperate group of Creole female goats. The percentage of female goats experiencing at least one ovulation per month dramatically decreased from May to September for the three years (0%, 27% and 0%, respectively). Tropical female goats demonstrated much less seasonality, as the percentage of goats experiencing at least one ovulation per month never went below 56%. These differences were significant. Both groups of temperate and tropical Black-Belly ewes experienced a marked seasonality in their ovulatory activity, with only a slightly significant difference between groups. The percentage of ewes experiencing at least one ovulation per month dropped dramatically in April and rose again in August (tropical ewes) or September (temperate ewes). The percentage of ewes experiencing at least one ovulation per month never went below 8% and 17% (for tropical and temperate ewes respectively) during the spring and summer months.
An important seasonality in ovulatory activity of tropical Creole goats was observed when females were exposed to a simulated temperate photoperiod. An unexpected finding was that Black-Belly ewes and, to a lesser extent, Creole goats exposed to a simulated tropical photoperiod also showed seasonality in their ovulatory activity. Such results indicate that both species are capable of showing seasonality under the photoperiodic changes of the temperate zone even though they do not originate from these regions.
Serotonin and serotonin receptors of class II (5HT2-R) are thought to be involved in the neural mechanisms which regulate the LH release associated with photoperiodic changes in sheep. A specific ...premammillary hypothalamic area displaying a significant binding of 3H-ketanserin, a potent 5HT2-R antagonist, was previously identified. The aim of the present study was to ascertain by in situ hybridization (ISH) that 5HT2-R mRNA-containing cells were also present in this specific hypothalamic area. Total RNA was prepared from sheep pars tuberalis/median eminence, and a cDNA fragment of 546 bp was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerated primers deduced from the human and rat 5HT2A-R sequences. After cloning and sequencing, the sheep nucleotide sequence had the highest homology (85.1-92.3%) with the other known mammalian 5HT2-R or 5HT2A-R sequences. Homology with other 5HT-R subtypes or other monoamine receptors was much lower, 60% at maximum. After ISH using sense and antisense 35S-riboprobes, specific labelling was found in different parts of the hypothalamus, especially in the mammillary bodies where the binding was higher. Within the hypothalamus, the density of labelled cells, mainly neurons, varied considerably. It was maximal in the mammillary bodies and also in a restricted ventral region of the premammillary hypothalamus located from about 500/700 micrometer to 1200/1400 micrometer in front of the mammillary recess, where 3H-ketanserin binding was previously reported.
(1) the structural study of the sequence indicated that the new cloned cDNA corresponds to the sheep 5HT2-R class and, probably, to the 5HT2A-R subtype and (2) the ISH studies revealed that a restricted area of the premammillary hypothalamus shows a large number of 5HT2-R mRNA-containing neurons.
Melatonin transduces the effect of day length on LH secretion by acting
on the hypothalamus. However, the precise hypothalamic site is unknown.
Two studies were undertaken to clarify where melatonin ...acts in the
hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes
were screened to determine whether specific regional densities in
melatonin binding existed. A higher density of binding was observed in
the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher
than the rest of the hypothalamus). This binding area is delimited
rostrally by the infundibular recess, caudally by the mammillary
bodies, dorsally by the fornix, and ventrally by the base of the brain;
and it encompasses the premammillary and tuberomammillary nuclei. To
test the functional importance of the identified area, 3 groups of
animals received bilateral melatonin microimplants: 1) in the PMH
(n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH;
n = 8); and 3) sham-operated animals received empty microimplants
in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated
sc with a 20-mm SILASTIC brand capsule of estradiol and exposed to long
days (16-h light, 8-h dark). At the end of the 80-day experiment, no
animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group
displayed a stimulation of LH secretion. In contrast, melatonin
implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on
day 44.5 ± 5.3 (mean ± sem). ANOVA revealed
that the changes in LH secretion were not different between the SHAM
and the AH/MBH groups but the PMH group differed from the other 2
groups (P < 0.0001). This study suggests that the
PMH is an important target for melatonin to regulate reproductive
activity.
This study was undertaken to assess the influence of photoperiod on growth hormone (GH) secretion in rams and its possible influence on body weight. Twenty young adult rams were divided into two ...groups. One was subjected to an annual (AR) and the other to a semestral (SR) light regime during the same 18-month period. In both groups, daylength (DL) varied gradually between 8 to 17 hr. Plasma prolactin (PRL) and GH profiles consisting of 6 hr samples were determined and animals were weighed throughout the course of the experiment. Maximal PRL secretion was observed with largest DL. In contrast, GH secretion increased during increasing DL but it began to decrease before maximal DL was reached in both light regimes. Mean GH secretion was maximal when the DL was about 11 hr in SR and between 8 to 12 hr in AR. Similarly, body weight increased when DL increased and plateaued during decreasing DL in both AR and SR animal groups. Significant (P less than 0.05) differences were observed throughout the course of the experiment according to the effects of decreasing or increasing DL in each group. Analysis of variance showed that the effect of DL on plasma PRL and GH levels and weight velocity (WV) was significant (P less than 0.05) in both light regimes. This suggests that in SR, plasma PRL and GH levels and WV vary according to a six month period.
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