Evidence that histaminergic neurons are devoid of estrogen receptor alpha in the ewe diencephalon during the breeding season.

In sheep as in rat, it has been highly suggested that neuronal histamine (HA) participates to the estradiol (E2)-induced GnRH and LH surges, through H1 receptor. With the aim of determining if E2 could act directly on HA neurons, we examined here whether HA neurons express estrogen receptor alpha (ERα) in the ewe diencephalon during the breeding season. We first produced a specific polyclonal antibody directed against recombinant ovine histidine decarboxylase (oHDC), the HA synthesizing enzyme. Using both this anti-oHDC antibody and an anti-ERα monoclonal antibody in double label immunohistochemistry, we showed that HA neurons do not express ERα in diencephalon of ewes with different hormonal status. This result diverges from those obtained in rat, in which around three quarters of HA neurons express ERα in their nucleus. This discrepancy between these two mammal species may reflect difference in their neuronal network.

Short-term effect of oestradiol on neurokinin B mRNA expression in the infundibular nucleus of ewes.

In sheep, essentially all the neurokinin B (NKB) neurones of the infundibular nucleus express oestradiol receptor alpha, and analysis of female and male brains has revealed an exceptionally marked female-dominant sex difference in the numbers of NKB neurones in the infundibular nucleus. This neuronal population is located in an oestradiol-sensitive brain area involved in the control of gonadotropin-releasing hormone (GnRH) secretion and oestrous behaviour, but its physiological role is poorly documented. The aim of the present study was to analyse NKB mRNA expression at a crucial time when the steroid has stimulated the pathways leading to the induction of these two events. After cloning a specific ovine NKB antisense riboprobe, we examined the effects of a short oestradiol treatment (4 h subcutaneously) on the expression of NKB mRNA in the caudal part of the infundibular nucleus of progesterone-primed ovariectomized ewes. We demonstrated that oestradiol decreased both the level of NKB mRNA expression (34%) and the number of cells containing NKB mRNA (43%). Oestradiol acts strongly on these NKB cells in the short term. We suggest that this early change in NKB mRNA expression during the preovulatory period might be involved in the control of the induction of GnRH secretion or oestrous behaviour.

Prolonged neurogenesis during early development of gonadotropin-releasing hormone neurones in sheep (Ovis Aries): in vivo and in vitro studies.

Gonadotropin-releasing hormone (GnRH) neurons involved in controlling the reproductive function in vertebrates are derived from the olfactory placode. However, in the sheep and the rat species, GnRH-immunoreactive (GnRH-IR) neurons could not be detected in the olfactory region during the earliest phase of GnRH system development. Using in situ hybridization (ISH) and immunohistochemistry (IHC) in sheep embryos ranging from 26 to 53 days' gestational age (G26-G53), the present work confirmed that GnRH expression could not be detected during the earliest steps of migration. The first ISH+ cells were detected in the nasal septum and at the entrance of the telencephalon at G33 stage. [(3)H]-thymidine pulses applied to in vitro olfactory explant cultures showed that GnRH neuron precursor cells have an extended multiplication period corresponding to G26-G36 before entering the neuronal differentiation process. Therefore, the lack of GnRH neuron detection during the early phase of development in the sheep compared to the mouse and other vertebrates represents a major difference in the early development of GnRH neurons. In the mouse, GnRH neuron precursors have a limited multiplication period in the vomeronasal pit and only postmitotic neurons start migration, whereas in the sheep embryo, the multiplication period is extended to about 10 days as demonstrated in olfactory explant cultures.

[Metabolism-reproduction interactions in domestic animals]. / Les interactions métabolisme-reproduction chez les animaux domestiques.

In farm animals as in human, reproduction is affected by changes in the level of energetic balance. The main factors that link metabolism and reproduction, like insulin, IGFs, glucose, FFA, leptin and NPY, act on hypothalamo-pituitary axis as well as on gonads by directly altering gametogenesis.

Evidence that the mediobasal hypothalamus is the primary site of action of estradiol in inducing the preovulatory gonadotropin releasing hormone surge in the ewe.

Although a neural site of action for estradiol in inducing a LH surge via a surge of GnRH is now well established in sheep, the precise target(s) for estrogen within the brain is unknown. To address this issue, two experiments were conducted during the breeding season using an artificial model of the follicular phase. In the first experiment, bilateral 17beta-estradiol microimplants were positioned in either the medial preoptic area (MPOA) or the mediobasal hypothalamus (MBH), and LH secretion was monitored. An initial negative feedback inhibition of LH secretion was observed in ewes that had estradiol microimplants located in the MPOA (6 of 6 ewes) or caudal MBH in the vicinity of the arcuate nucleus (4 of 4). In contrast, a normal LH surge was only found in animals bearing estradiol microimplants in the MBH (5 of 10). Detailed analysis of estradiol microimplant location with respect to the estrogen receptor-alpha-immunoreactive cells of the hypothalamus revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants located bilaterally within or adjacent to the area of estrogen receptor-expressing cells of the ventromedial nucleus. Two of these ewes exhibited a LH surge without showing any form of estrogen negative feedback. In the second experiment, we used the technique of hypophyseal portal blood collection to monitor GnRH secretion directly at the time of the LH surge induced by estradiol delivered either centrally or peripherally. Central estradiol implants induced the GnRH surge. The duration and mean plasma concentration of GnRH during the surge were not different between animals given peripheral or central MBH estradiol implants. Cholesterol-filled MBH microimplants did not evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.

Localization of the preprosomatostatin-mRNA by in situ hybridization in the ewe hypothalamus.

The peptidergic neurohormone somatostatin (SRIF) derives from a precursor called preprosomatostatin (PPS) by proteolysis. We have isolated by RT-PCR and sequenced a partial cDNA coding for the ovine PPS. It contains a 348 base pairs coding sequence that shares strong similarities with previously cloned mammalian cDNAs. The ovine cDNA was used to synthesize radiolabeled cRNA to probe the PPS mRNA in the ewe hypothalamus by in situ hybridization. The PPS mRNA-containing cells are widely distributed in the hypothalamus. According to the number of silver grains over a cell, they show various staining intensities. The distribution of the PPS mRNA is in good agreement with that of the peptide previously determined using immunohistochemistry. The strongest labeled areas include the periventricular region of the paraventricular nucleus and the lateral division of the ventromedial nucleus. The difference in labeling intensity observed in the diverse populations of labeled neurons could reflect various levels of neuronal activity.

Chromosomal localization of the human and mouse histidine decarboxylase genes by in situ hybridization. Exclusion of the HDC gene from the Prader-Willi syndrome region.

Using a rat histidine decarboxylase (HDC) cDNA probe, we have mapped the HDC gene by in situ hybridization to the q15-q21 region of human chromosome 15 and to the E5-G region of murine chromosome 2. These localizations strengthen a syntenic group conserved between human chromosome 15 and mouse chromosome 2. The localization of the HDC gene on the human chromosome 15 map shows that it is not included within the Prader-Willi Syndrome region (PWCR).

Effect of interleukin-1 receptor antagonist (IL-1RA) on histamine and serotonin release by rat basophilic leukemia cells (RBL-2H3) and peritoneal mast cells.

Recently, it has been appreciated that cultured mast cells are significant sources of cytokines. However, the role of interkeukin-1 (IL-1) on mast cells and/or basophil degranulation is still unclear. In this report we provide evidence that rat basophilic leukemia cells (RBLC) cultured with a natural inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1RA) (500 ng/ml) for 48 h, strongly inhibited the spontaneous release of serotonin (5HT) and histamine (from 22.50 to 43.49%), compared to untreated cells (control). When IL-1RA-treated and untreated RBLC were stimulated with a secretagogue (anti-IgE), no difference was found in the percent of 5HT and histamine release. Moreover, in another set of experiments using rat peritoneal mast cells (RPMC) treated and untreated with IL-1RA, we found that IL-1RA did not affect the release of 5HT or histamine, even when the secretagogue anti-IgE or compound 48/80 (C48/80) were used. The present studies describe an additional biological activity of IL-1RA, inhibiting histamine and 5HT release from RBLC cultures.

Monocyte chemotactic protein-1 provokes mast cell aggregation and [3H]5HT release.

Monocyte chemotactic protein-1 (MCP-1) and MCP-3, the most active and representative compounds of the CC chemokine family, are proinflammatory cytokines that attract and activate specific types of leucocytes. We have used highly purified isolated rat peritoneal mast cells (RPMC) cultured for different lengths of time with and without MCP-1 (200, 100, 50 and 25 nM). Our data clearly show that MCP-1 (200 nM) causes a marked release of [3H]serotonin ([3H]5HT and histamine, which reach a peak at 40 min of incubation (56.6 +/- 5.3 and 34.7 +/- 6 above the control, respectively). In dose-response experiments, MCP-1 (200, 100, 50, 25, 12.5, 6.25 and 3.12 nM) provoked a dose-dependent release of [3H]5HT and histamine from RPMC, which was maximum at 200 nM. After preparation of the histidine decarboxylase (HDC) probe, a Northern blot analysis was determined for HDC mRNA. After 4 hr, steady-state levels of HDC mRNA were induced in a dose-dependent manner by MCP-1 (200-25 nM), compared to the controls. However, MCP-1 failed to prime RPMC in [3H]5HT and histamine release when C48/80 (0.05 micrograms/ml) or anti-IgE was used. In contrast, murine interleukin-3 (IL-3) in combination with MCP-1 (200 and 100 nM) provoked a greater release of histamine and [3H]5HT than the compounds alone. Moreover, RPMC treated with MCP-1 (200 nM) showed, under light microscopy (20x), greater clump formation, a phenomenon absent in the controls (untreated cells). The electron microscope studies revealed that treatment with MCP-1 (200 nM) promoted binding of RPMC and clearly demonstrated a communication between the cytoplasms of adjacent mast cells. Our report describes additional biological activities for MCP-1, suggesting for the first time that this human monocyte chemoattractant plays a fundamental role in histamine and serotonin release and cell aggregation in rat peritoneal mast cells.

[Neuroendocrine control of ovulation in sheep]. / Le contrôle neuroendocrinien de l'ovulation chez la brebis.

The preovulatory surge of LH, or positive feedback response to oestradiol, is related to the steroid's direct stimulating action on the pituitary gland, but we have demonstrated that, in sheep, the central nervous system plays an essential role in its action: a preovulatory GnRH surge. Since GnRH neurons appear to contain few, if any receptors for oestradiol the question is raised: Where in the central nervous system does oestradiol act to stimulate GnRH secretion? A series of experiments were conducted with a combination of techniques for sampling pituitary portal blood and stereotaxic brain micro-implantations of oestradiol. Castrated Ile-de-France breed ewes were treated during the pseudo follicular phase of successive artificial cycles, receiving either peripheral oestradiol implants or brain implants containing oestradiol or cholesterol. Administration of oestradiol implants into the ventromedial region of the hypothalamus induced a preovulatory GnRH surge in 5 out of 10 animals. The interval between estradiol and the GnRH surge was comparable with that observed in animals treated with a peripheral implant although the amplitude of the surge was smaller. No GnRH surges were observed in animals treated with cholesterol. Finally, there was an inverse correlation between the response intensity (amplitude of the GnRH/LH surges) and the distance separating implants from cells in the region carrying oestradiol receptors. These results show that the mediobasal hypothalamus is a site of action for the oestradiol-induced preovulatory GnRH surge in the ewe. Immunohistochemical tests should identify the nature of the cells forming the relay for the steroid action.

Assignment of the rat genes coding for dopa decarboxylase (DDC) and glutamic acid decarboxylases (GAD1 and GAD2).

By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5'-phosphate (PLP)-dependent decarboxylases--namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)--were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)--Tpl2--Gad2 on the rat Chr 17.

Preparation of a rat brain histidine decarboxylase (HDC) cDNA probe by PCR and assignment of the human HDC gene to chromosome 15.

The formation of histamine from its precursor histidine is catalyzed by histidine decarboxylase (HDC), a pyridoxal phosphate (PLP)-dependent decarboxylase. The knowledge of sequence similarities between various rodent HDCs permitted us to prepare a rat brain HDC cDNA probe. After reverse transcription of rat brain polyA + mRNA, the HDC cDNA obtained was amplified by the polymerase chain reaction using two specific primers. The resulting 1019-bp DNA was cloned in the p-MAL vector. Its sequence corresponds to the published data on rat fetal liver HDC. This 1019-bp rat probe detected two BamHI sequences in man; these were assigned to chromosome 15 by somatic hybrid cell analysis. According to the well-known homology between human chromosome 15 and mouse chromosome 2, the result obtained is in agreement with the published localization of HDC on mouse chromosome 2. The mapping of the human HDC gene on chromosome 15 is an original contribution to the chromosomal assignment of related PLP-dependent decarboxylases.

Mapping of the dopa decarboxylase gene to the 11A band of the murine genome.

A human DOPA decarboxylase (DDC) cDNA probe of 747 base pairs has been used to map the DDC gene by in situ hybridization on mouse metaphase chromosomes. This result indicates that the gene is located on band 11A, near the erythroblastosis oncogene B (erb b) locus. This provides evidence for a synteny group on mouse chromosome 11 and human chromosome 7.

Characterization of DOPA decarboxylase mRNA in rat pheochromocytoma.

Total poly (A+) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A+ mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.

Preparation of a human DOPA decarboxylase cDNA probe by PCR and its assignment to chromosome 7.

The reverse transcription of mRNA of a human pheochromocytoma using an oligonucleotide complementary to rat DOPA decarboxylase (DDC) sequence as a primer, gave rise to a single strand cDNA. This resultant cDNA permitted by PCR amplification the preparation and cloning in PUC 19 of a human DDC probe of 747 base pairs. The choice of primer was dictated by the presence of tryptophan codons in the DOPA decarboxylase sequence which were conserved in different species throughout evolution. The presence of mismatches on the primers was not an obstacle to a specific amplification and the probe sequence was found identical to the human DDC cDNA sequence. This probe, labelled by nick translation detected on Southern blot of human-rodent hybrids, bands on human DNA after EcoRI, BamHI or HindIII digestion. The results with EcoRI were explicit and drove to the conclusion that DDC is located on chromosome 7. Five bands were obtained on human DNA digested with EcoRI, indicating that either numerous introns could interrupt the coding sequence of DDC gene or duplicated sequences could be present in chromosome 7.