Melatonin binding sites in the brain of sheep exposed to light or pinealectomized.

The binding sites of [125I]melatonin were identified in the sheep brain using a specific and sensitive autoradiographical method. Rams were either untreated (controls) or exposed to light before slaughter or pinealectomized (px). In all animals labelling was intense in the pars tuberalis (PT) and absent in the suprachiasmatic nucleus (SCN). In light-treated and in px rams, but not in controls, we demonstrated melatonin binding sites in the intermediate and ventrolateral septum and in the stratum lacunosum of the hippocampus. Labelling was less marked in nervous tissues than in PT cells and did not seem to be different between treatments. These results indicate the presence of light-dependent melatonin binding sites in the septum and hippocampus of sheep, but rise the question of the involvement of melatonin in the SCN activity in this species.

Melatonin binding sites in the sheep pars tuberalis.

The localization of [125I]melatonin binding sites has been studied by autoradiography on frozen unfixed sections of the pituitary stalk, the suprachiasmatic area, the pineal and the pituitary glands in sheep. Dense specific labelling has been found exclusively in the pars tuberalis of the pituitary stalk but not in the part of the median eminence surrounded by the pars tuberalis. The labelling was completely excluded by a 200-fold excess of cold melatonin. No comparable labelling was found in the suprachiasmatic nucleus, the pineal and the pituitary glands. These results constitute the first report of melatonin-specific labelling in the ovine species.

Quick freezing of rat embryos.

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).

Sequential effects of FSH on the first stages of ovarian follicular development in normal and dwarf Snell mice.

The population of small growing ovarian follicles was divided into 4 classes according to the number of granulosa cells (from 15 to 95) surrounding the oocyte, and a comparison was made of normal and dwarf mice. Follicular cell proliferation was estimated by tritiated thymidine incorporation. In normal mice, most follicles in classes 1 (15 to 35 granulosa cells in their largest cross-section) and 2 (36 to 55 cells) were labelled (86 and 95%, respectively); FSH treatment increased the labelling index (L.I.) in all follicle classes. In dwarf mice, only 38 and 76% of follicles in classes 1 and 2, respectively, were labelled. However, FSH treatment increased the percentage of labelled follicles and the L.I. to levels which were similar to those in the ovaries of untreated, normal animals. FSH stimulation of the percentage of labelled follicles and L.I. was obvious as early as 3 h after injection. There was a major increase of the L.I. 24 h after FSH stimulation, specially in dwarf mice; several hypotheses are proposed to explain this finding. We conclude that FSH is necessary for the development of the population of small growing follicles in the mouse ovary.

Hemiovariectomy and follicular growth in the immature rat.

Female rats were hemiovariectomized on day 1 (T2) or day 10 (T3) after birth. The population of growing follicles in the remaining ovary and the plasma levels of FSH an LH were compared to controls (T1) on post-natal days 20, 30 and 38. There was a non significant trend towards higher FSH and LH levels in hemicastrates. Hemicastration had a significant (P less than 0.05) overall effect on the number of small (1 to 2 layers of granulosa cells) and preantral follicles (more than 2 layers of granulosa cells and diffuse antrum): small follicles were more numerous (P less than 0.05) in ovaries of the T3 group, whereas preantral follicles were more numerous in the T2 group. Atresia was somewhat lower in hemicastrates compared to controls. The observed increase in the whole population of follicles, in the remaining ovary, may be attributed to several factors including a decrease in steroids and inhibin, an increase in FSH and/or a neural discharge signal. The increased number of follicles may constitute a reserve to maintain the characteristic ovulation pattern of the species in later life.

Quantitative autoradiographic study of FSH binding sites in prepubertal ovaries of three strains of rats.

Iodinated FSH was injected to 18- and 36-day-old rats of 3 strains (03, 04 and 12) with different sensitivity to FSH (12 less than 03 less than 04) and autoradiography was performed on histological sections of the labelled ovaries. Specific labelling was quantified by microphotometry on histological slides, on granulosa cells of individual follicles with different sizes (greater than 80 micron diameter) and qualities. In small preantral follicles (less than 160 micron diameter) the labelling was low and homogeneous within the granulosa; it increased between 18 and 36 days of age in the 3 strains. At 36 days, ovaries were characterized by the presence of large preantral and antral follicles with a higher labelling in the outer layers of granulosa (near the theca), compared to the inner layers. In definitely atretic follicles, a loss of binding sites was detected in the outer layers. In rats of Strains 03 and 04, the number of binding sites for FSH in the outer layers of granulosa of follicles with a diameter of greater than 160 micron increased with follicular size; no change was detected in follicles of Strain 12 rats. The low number of binding sites for FSH and the lack of terminal maturation which characterize the follicles of strain 12 rats can be related to the poor and delayed follicular development, the low sensitivity to exogenous FSH and the low fertility of the animals of this strain.

Reassessment of LRF radioimmunoassay in the plasma and hypothalamic extracts of rats and rams.

A highly sensitive and specific radioimmunoassay for LRF was applied to the measurement of endogenous LRF in various hypothalamic extracts. Specific antiserum was obtained by injecting LRF conjugated to human serum albumin with glutaraldehyde. Thyrotropin-releasing hormone, lysine vasopressin, oxytocin, noradrenaline, LH, FSH and cortical extracts did not appear to affect the assay, and the maximum cross-reaction observed with the LRF analogs tested was 8.5 p. 100 with LRF 2-10. The best detection limit (0.4 pg/tube) was usually obtained when the labelled LRF had been purified by polyacrylamide gel electrophoresis. Within and between-assay coefficients of variation were 8.0 and 12.6 p. 100, respectively (from B/Bo = 20 to 80 p. 100). Synthetic LRF administered to rams by intravenous injection was readily detectable in the peripheral plasma. However, the direct measurement of plasma endogenous LRF may give misleading results due to non-specific interference by plasma factors. No endogenous LRF could be detected in plasma methanol or acetone extracts obtained from rats and rams in various physiological conditions. The inhibition curves parallel to the synthetic LRF curve were obtained by diluting the crude hypothalamic extracts of rams and rats, and a good correlation (r = 0.997) with the Ramirez-McCann bioassay resulted, indicating that using radioimmunoassay to determine hypothalamic LRF content may be fruitful in studying hypothalamo-pituitary gonad interactions. The LRF content of rat and ovine hypothalami ranged from 2-8 to 20-80 ng of LRF, respectively.

The sheep pars tuberalis: an immunohistochemical study. Demonstration of the presence of glycoprotein and lipotropin hormones.

Using indirect immunofluorescence with fourteen different antisera raised against pituitary hormones and peptides, we characterized immunochemically the cells of the sheep pars tuberalis. The presence of LH- and FSH-containing cells, shown in previous studies, was also observed in the present investigation. In addition, we found TSH-containing cells, never observed in sheep, and beta LPH-containing cells. The latter hormone has never been found in any studied species. It appeared that a small amount of perikarya (less than 20%) were immunolabelled and, that the sheep pars tuberalis contained a majority of immunonegative cells as in the guinea-pig rabbit and rhesus monkey. This study may contribute to a better knowledge of the function of the sheep pars tuberalis.

Characterization of melatonin receptors in the ram pars tuberalis: influence of light.

The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20 degrees C, but unstable at 28 or 37 degrees C; 3. melatonin and [127I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 10(10) l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25 +/- 0.30 vs 1.01 +/- 0.17 fmol/mm2 pars tuberalis, p less than 0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.

Localization of luteinizing hormone beta-mRNA by in situ hybridization in the sheep pars tuberalis.

The localization of luteinizing hormone beta (LH beta)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded 32P- or 35S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH beta-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by in situ hybridization followed by immunohistochemistry using a specific LH beta-antiserum indicated that the labelling of LH beta-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH beta subunit, but that the level of translation greatly varies according to the location of the cells.

Localization and quantification of melatonin receptors in the diencephalon and posterior telencephalon of the sheep brain.

In an attempt to better understand the mechanisms by which melatonin controls neuroendocrine activity, we tried to define with accuracy the brain areas where the density of melatonin receptors is the highest in sheep and to establish their characteristics. The specific labelling of 125I-melatonin was first revealed by autoradiography on brain sections of the posterior telencephalon and diencephalon in three ewes. The extent and position of the five structures where the binding was found to be the highest (i.e., the pars verticalis and pars horizontalis of the nucleus tractus diagonalis, the septal area, the bed nucleus of the stria terminalis, and the ventromedial hypothalamic area) were then accurately defined by image analysis. In comparison to the landmarks given by image analysis, photographs of coronal sections of another ewe permitted the accurate definition of the limits of the structures to be punched in a second step. In six ewes, each of the five structures previously identified were punched from frozen coronal sections and binding of 125I-melatonin to membrane preparations was studied individually by Scatchard analysis. The correlation coefficient between the B/F ratio and binding (B) was in the range of 0.96-0.98, indicating that a precise quantification was possible in these different structures. The Bmax was the highest in the bed nucleus of the stria terminalis, the septal area, and the ventromedial hypothalamic area (1.38, 1.25, and 0.95 fmol/mg protein, respectively). All Kd values were less than 10 pM and the Hill coefficient was close to 1, indicating the presence of a single class of receptor to 125I-melatonin. These results indicate the reliability of a method used to measure with accuracy low concentrations of melatonin receptors in brain structures. In addition, the ventromedial hypothalamic area was found to be rich in melatonin receptors. This region is known to be involved in the central gonadotrope control in sheep.

Changes in LHbeta-gene and FSHbeta-gene expression in the ram pars tuberalis according to season and castration.

Luteinizing hormone beta (LHbeta) and follicle stimulating hormone beta (FSHbeta) subunits and their mRNAs were studied in the ram pars tuberalis following different seasonal (winter vs summer) and experimental (intact vs castrated animals) conditions. Hormone-containing cells were identified by immunohistochemistry, using homologous double-stranded 35S-cDNAs. The labelling was quantified by image analysis. Immunohistochemical staining showed that cells containing LHbeta and FSHbeta were localized mainly in the ventral part of the pars tuberalis but that, in the summer, additional LHbeta containing cells were present in the dorsal part in intact rams. On the other hand, LHbeta-mRNA labelling was found in the whole pars tuberalis in wethers but only in the ventral part in intact rams. The magnitude of LHbeta-mRNA labelling was significantly greater in summer than in winter rams, and in castrated than in intact animals (P<0.001). However, the number of labelled cells was found to be the greatest in the winter (P<0.001) and was not affected by castration. FSHbeta-mRNA expression was similar to that of LHbeta-mRNA except that the level and extent were considerably lower. Thus, our results show an increase in the magnitude of gonadotropin beta subunit-mRNA in the summer and following castration; this increase appears to involve the entire pars tuberalis.

Seasonal variation in LH and testosterone release in rams of two breeds.

Blood was collected hourly for 24 h in December, February, April, June and September from Préalpes du sud and Ile-de-France rams. Coincidence of the LH and testosterone peaks was found for 96.4% of a total of 670 LH peaks and 647 testosterone peaks. The number of LH and testosterone peaks increased by 66% in Ile-de-France rams and 200% in Préalpes du Sud rams between December and June (P less than 0.001). Values in June and September were similar in Préalpes du Sud rams. There were no differences between breeds in December, but in June, Préalpes du Sud had significantly more peaks than did Ile-de-France rams (P less than 0.025). The numbers of LH and testosterone peaks increased significantly (P less than 0.05) in Préalpes du Sud rams between December and February or April. These results indicate that, although numbers of peaks of LH and testosterone increase when the animals pass from the non-breeding to the breeding season, the genotype influences the pattern of release through the year.

A reinvestigation of hypothalamic-pituitary testicular interactions: simultaneous changes in tissue and plasma levels of gonadotrophins, prolactin, testosterone and hypothalamic LH-RH after bilateral orchidectomy and cryptorchidism.

Plasma concentrations of LH, FSH, prolactin and testosterone, pituitary concentrations of gonadotrophins and prolactin, and hypothalamic LH-RH were measured in normal, sexually mature male rats at regular intervals from 1 to 30 days after castration or cryptorchidism. Bilateral castration resulted in a marked decrease in testosterone levels 24 h after surgery. On the contrary, plasma testosterone was increased (at days 4 and 8) or unaffected by cryptorchidism when compared to intact controls. From day 1, castrated rats showed a rapid, marked increase in plasma LH and FSH (7 and 5 times higher at day 30 for LH and FSH, respectively) and a delayed, progressive increase in pituitary gonadotrophins (significant at day 8 and day 30 for LH and FSH, respectively). The cryptorchid animals showed similar but slower and less marked changes in plasma and pituitary FSH and LH levels. Unlike plasma prolactin levels, which were lowered at day 8 or days 4, 8 and 30, respectively, after castration or cryptorchidism, no change was observed in pituitary prolactin content. After castration, hypothalamic LH-RH content was significantly lower at day 4 and gradually decreased until day 30. On the other hand, no changes were observed after cryptorchidism. The present observations indicate that factors other than testosterone, associated with the presence of an active seminiferous epithelium, are involved in the regulation of gonadotrophin secretion. These factors do not seem to change the hypothalamic LH-RH content.

FSH, LH and prolactin levels, ovarian follicular development and ovarian responsiveness to FSH in the Snell dwarf mouse.

The homozygous Snell dwarf mouse is sterile. It has been shown that pituitary hormone levels are low in 3 month old animals except for FSH and LH whose pituitary contents and plasma concentrations are normal. In this study, the pituitary FSH, LH and prolactin (Prl) content, the FSH plasma concentration and the ovarian follicular development of the Snell dwarf mouse were studied at 18, 20, 24, 40 and 80 days of age. Normal mice were also studied at the same age and served as controls. Pituitary FSH was significantly lower in dwarf mice compared with controls during the period days 18 to 30, while plasma FSH was significantly lower during the period days 20 to 80. Pituitary LH was significantly lower in dwarf mice during the period days 18 to 40. In normal mice, pituitary Prl increased with age, but remained consistently low in dwarf mice. The normal number of growing follicles was similar in dwarf mice and controls up to day 30, but thereafter the total number of growing follicles was greater in the controls. In the dwarf mice, the production of antral follicles was low and there were no ovulations. The rates of atresia were similar in the two genotypes. The responsiveness of the dwarf mouse ovary to FSH was then examined. When dwarf and control mice were supplemented with FSH for 5 days starting at 24 days of age, the ovarian and uterine weights increase 6- and 5-fold, respectively, in the dwarf mice, and 2- to 3-fold in the normal mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Diurnal variation in release of LH and testosterone in the ram.

The time of appearance of plasma LH and testosterone peaks through the day determined in 75 Préalpes du Sud and 41 Ile-de-France rams in December and in 44 Préalpes du Sud and 11 Ile-de-France rams in June. The distribution of peaks throughout the day was non-random for the two hormones in the two breeds and for both times of the year (P less than 0.01 at least on each occasion; P less than 0.001 on pooled data from the two breeds). The most striking features were the occurrence of (1) a minimum of LH and testosterone peaks immediately after 'dawn' (lights on) in both months; (2) a maximum of peaks 3 h after 'dawn' in June and 4 h after 'dawn' in December. For several hours after the increase in frequency of peaks the probability of measuring peaks of LH values in December and June when adjusted for the time of 'dawn' suggest that dawn could act as a synchronizer of gonadotroph activity.

Testicular growth and hormonal parameters in the male Snell dwarf mouse.

Dwarf mice show delayed testicular growth and their adult testis weights are half the normal value. The aims of the present work were firstly, to compare the developmental profiles of plasma gonadotropins and of testicular cell multiplication and differentiation in dwarf vs normal mice and secondly, to determine the effect of hMG supplementation on dwarf mice. In the dwarf mice no pubertal rise in plasma FSH was observed, and the adult values remained very low when compared with those of normal mice; plasma LH decreased after 40 days of age and remained equal to half the normal values. In adults, testicular testosterone content was greatly increased in dwarf mice compared with normal mice, whereas plasma testosterone and accessory gland weights were reduced. At 24 days of age, the total numbers per testis of Leydig and Sertoli cells were reduced in dwarf vs normal mice, whereas in adult mice their differentiation, but not their total numbers, was reduced. This resulted in lower daily production of leptotene primary spermatocytes and of round spermatids in dwarf than in normal mice. hMG supplementation promoted Leydig and Sertoli cell multiplication, but did not produce full differentiation, resulting in increased daily production of round spermatids. In conclusion, in adult dwarf mice a deficiency in plasma gonadotropins prevents full differentiation of Leydig and Sertoli cells without affecting the number of these cells.

Influence of 2 Br-alpha-ergocryptine (CB 154) on the secretion of prolactin, LH, FSH and testosterone and on testicular growth in rams subjected to different photoperiods.

The influence of 2-Br-alpha-ergocryptine (CB 154) on the secretion of gonadotrophins and on testicular function has been studied in rams subjected to either a normal photoperiod or an abnormal photoperiod causing hyperprolactinaemia. The CB 154 treatment significantly lowered the mean frequency of LH and testosterone pulses in hyperprolactinaemic animals as compared to solvent-treated ones. Also, only those groups subjected to an abnormal photoperiod (groups 2 and 3) exhibited a significant rise in the frequency of LH and testosterone peaks after CB 154 was withdrawn. During treatment, plasma FSH concentrations increased significantly only in group 1 which was subjected to normal photoperiodic variations. Testicular growth was delayed in CB 154-treated rams compared to solvent-treated ones only in group 3 (hyperprolactinaemic).