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.

Evidence that luteinizing hormone-releasing hormone statin from ovine rete testis fluid is immunologically related to alphaC inhibin.

LHRH Statin is a putative gonadal protein that increases the interval between two consecutive LHRH pulses. The present work was aimed at analyzing the immunological homology between LHRH Statin and the N-terminal region of the alphaC subunit of inhibin. Thus, rete testis fluid (RTF) proteins were purified by immunoaffinity chromatography using antibodies against residues 1-7 plus 7-30 (experiment 1, A-fractions) and 14-28 of the alphaC inhibin subunit (experiment 2, B-fractions), and the LHRH Statin activity of the fractions was examined by intracerebroventricular administration in castrated rams followed by RIA of plasma LH levels in 15-min blood samples. Fractions that bound to the immunoaffinity column with low affinity were eluted with 0.5 M NaCl, pH 7.4 (-F2); then highly bound fractions were eluted sequentially in acidic (pH 2.5, -F3) followed by basic conditions (pH 11.5, -F4). In experiment 1, RTF (40 microg, n = 4) and highly bound fractions (A-F3, 30 ng, n = 8, 150 ng, n = 3; A-F4, 120 ng, n = 5) decreased LH mean plasma levels between 4 and 6 h after injection by 39%, 29%, 43%, and 37%, respectively (P<0.001 to 0.01), while the weakly bound fractions (A-F2, 180 ng, n = 4) and albumin control (40 microg, n = 4) had no activity. In experiment 2, RTF (100 microg, n = 4) and B-F3 (100 ng, n = 3) decreased plasma LH levels by 48% and 38%, respectively (P<0.001 to 0.05), whereas B-F4 (100 ng, n = 4) and albumin control (100 microg, n = 4) had no effect. A fraction obtained from B-F3 by gel filtration had significant LHRH Statin activity (63%, n = 6, P<0.001). PAGE with colloidal gold staining revealed 3 high molecular weight bands and 5 low molecular weight bands in B-F3. The 3 high molecular weight bands were shown to belong to the clusterin family and did not appear to have LHRH Statin activity. The 5 low molecular weight bands were all labeled by anti-alphaC inhibin antibodies. Collectively, these results strongly suggest that LHRH Statin has some homology with the 14-28 alphaC inhibin sequence.

Anastomosis of the ovarian vein to the hepatic portal vein in sheep induces ovarian hyperstimulation associated with increased LH pulsatility, but only in the absence of the contralateral ovary.

In this study, two experiments were performed, the first of which examined the ovarian response in ewes that were subject to unilateral ovariectomy (ULO) at different intervals (0-14 days) after surgical anastomosis (AN) of the ovarian vein to the mesenteric vein (n=7 ewes), or sham operation (SO; n=4 ewes). Hypertrophy and development of multiple follicular and luteal structures on AN ovaries were observed after ULO, while SO ovaries remained of normal size and appearance after ULO. The second experiment involving 11 ewes (five AN; six SO) aimed to clarify the mechanism by which AN following ULO-induced ovarian hypertrophy and increased follicle development. The results confirmed that there were more large (>5 mm) follicles on AN compared with SO ovaries; however, their rate of atresia was similar. Oestradiol and progesterone concentrations in follicular fluid of class 1 follicles (5-9 mm) were higher in AN ovaries than those in control follicles of the same size collected in the late follicular phase of an induced oestrous cycle. In AN ewes, intrafollicular progesterone concentrations increased while follicular aromatase activity and intrafollicular oestradiol, inhibin A, follistatin and activin A concentrations all decreased as follicle size increased. Oestradiol and progesterone concentrations were substantially higher in ovarian venous blood than in hepatic venous blood, both in AN and SO ewes, whereas inhibin A levels were not significantly modified by passage through the liver in either group. Mean plasma LH concentration, and LH pulse frequency and amplitude increased markedly after AN but were not affected by SO. Plasma FSH showed only a small transient increase after AN, presumably due to the maintenance of inhibin feedback. Injection of prostaglandin F(2)(alpha) 4 days later did not further modify LH or FSH secretion in either group. Full ovariectomy (FO) 9-14 days after AN or SO increased LH secretion markedly in SO ewes but to a lesser degree in AN ewes; FO induced a large and rapid increase in FSH levels in both groups. In conclusion, AN of the ovary to the liver via the mesenteric vein provides a useful model for studying the feedback between the ovary and the hypothalamo-pituitary system and the mechanisms controlling follicle development. The present results indicate that the pattern of LH secretion is an important factor controlling the terminal phase of follicle development in the ewe.

Central effect of rete testis fluid, inhibin 32K, and follicular fluid on plasma gonadotropin concentrations in sheep: inhibin is not the rete testis fluid protein able to suppress luteinizing hormone pulses.

We have previously shown that peripheral administration of rete testis fluid (RTF) proteins was able to suppress LH pulses through the suppression of LHRH pulses. This activity was named "LHRH Statin." The aims of the present work were to analyze LH inhibition after an intracerebroventricular injection of RTF and to determine whether inhibin is the factor responsible for this inhibition. Castrated rams (experiment 1) or ewes (experiment 2) received an intracerebroventricular injection of RTF, purified bovine inhibin 32K, bovine follicular fluid, or human serum albumin as control. Animals were bled every 15 min for 5 h before injection and for 7 h after injection. LH mean levels were significantly lowered (p < 0.01) only in the RFT-treated groups. FSH levels were not affected irrespective of group, source, or dose of inhibin. These experiments show first, that protein(s) present in ovine RTF can suppress LH secretion in sheep; second, that bovine follicular fluid or purified bovine inhibin 32K have no effect on LH secretion. Furthermore, the results suggest that centrally administered inhibin has no effect on FSH secretion under our experimental conditions. Together, these experiments clearly demonstrate that inhibin 32K does not exert any "LHRH statin" activity.

Evidence for a protein-like factor from "rete testis" fluid that suppresses luteinizing hormone-releasing hormone (LHRH) pulses (LHRH statin): a new hormonal activity?

Signals that modulate LH-releasing hormone (LHRH) pulse frequency are fundamental mechanisms for regulating important reproductive processes. Gonadal steroids are presently considered to account for the entire gonadal feedback mechanism that modulates LHRH secretion. However, we have previously suggested that a testicular protein(s) present in charcoal-treated rete testis fluid (ctRTF) can suppress LH pulsatility in the ram. The present experiments were aimed at determining whether the disappearance of LH pulses induced by ctRTF administration implicate a hypothalamic or a pituitary site of action. Thus, we have examined the effects of ctRTF peripheral administration on 1) the LH response to LHRH, 2) LHRH portal blood levels, and 3) LHRH content in hypothalamic tissue. Finally, the effects of ctRTF administered into the third ventricle on plasma LH levels were assessed. The present results show that a testicular protein(s) is able to suppress LHRH pulse frequency without affecting amplitude and without any effect on the LH response to LHRH (LHRH Statin). The observation that an active dose administered by the intracerebroventricular route is 0.0005 the active dose needed by the peripheral route reinforces this evidence. These data lead to the new concept that the testicular signals that govern LHRH pulse frequency may be not only steroids, but also proteins.

Evidence for a gonadal nonsteroidal factor that specifically inhibits release of luteinizing hormone in ewes.

Bilaterally ovariectomized ewes were used to investigate the effect of systemic administration (i.v.) of charcoal-treated aqueous luteal extracts from ovine corpora lutea on plasma concentrations of pituitary gonadotrophins. Jugular blood samples were taken every 15 min at least 5 h before (control period) and 5 h after (treatment period) injection. In Expt 1, the administration of luteal extract from corpora lutea of days 70-76 of pregnancy, but not of the extract prepared from muscular tissue, resulted in a significant decrease of mean concentrations of luteinizing hormone (LH) (P < 0.02) and frequency of LH pulses (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations were not affected by injections of either extract. These findings provide the first demonstration of the presence of a nonsteroidal factor in the corpus luteum of midpregnancy that selectively suppresses the secretion of LH. In Expt 2, mean concentrations of LH and FSH and frequency of LH pulses were unaffected by injections of luteal extracts from ovine corpora lutea of days 10-12 of the oestrous cycle or day 15 of pregnancy. These data suggest that some factor(s), probably from the fetoplacental endocrine unit, is required to ensure the production of a significant quantity of the luteal LH-inhibiting factor after day 15 of pregnancy. In Expt 3, treatment of luteal extract from corpora lutea of day 70 of pregnancy with proteolytic enzymes destroyed the LH-inhibiting activity, suggesting the proteic nature of the luteal LH-inhibiting factor. In Expt 4, plasma concentrations of LH were not affected by injection of charcoal-treated extract prepared from fetal cotyledonary tissue of days 110-120 of pregnancy suggesting that the LH-inhibiting factor exclusively originates from the corpus luteum during pregnancy. These experiments provide the first direct evidence for the existence of a potent nonsteroidal factor of luteal origin that specifically inhibits pulsatile secretion of LH, without influencing FSH release in female animals. We propose the term LH-release-inhibiting factor (LH-RIF) to describe this activity.

Role of the corpus luteum of pregnancy in controlling pituitary gonadotrophin secretion during the early post-partum period in the ewe.

No difference was found between 5 intact ewes and 5 ewes from which the CL had been excised at Day 70 of pregnancy in the plasma concentration of progesterone at Day 140, and concentrations of progesterone remained below 0.2 ng/ml during the first 20 days post partum. Plasma concentrations of LH, frequency and amplitude of LH pulses were low at Day 140 and increased considerably, particularly in the CL-excised ewes, as early as Day 5 post partum. No significant differences were found between the two groups of ewes in the mean plasma concentrations of FSH for any of the 5 stages examined. Taken together, these results suggest that some factor, other than progesterone, associated with the CL of pregnancy is involved in the inhibition of pulsatile LH secretion during the early post-partum period.

Evidence that the corpus luteum of pregnancy contributes to the control of tonic secretion of LH in the ewe.

Concentrations of LH and FSH were measured in blood samples collected from the jugular vein at 20-min intervals for 7 h (09:00-16:00 h) on Days 60, 80, 100 and 120 of pregnancy in 5 intact ewes and 5 from which the CL had been excised on Day 70. In the 5 intact ewes, plasma LH concentrations remained low and unchanged between Days 60 and 120. During this period, pulsatile release of LH occurred irregularly and infrequently. Removal of the CL resulted in an increase in the basal values of LH and in the frequency and amplitude of LH pulses. Concentrations of FSH were relatively constant in all stages of pregnancy examined and were similar in both groups of ewes. These results show that (1) LH concentrations are low during the second half of pregnancy; and (2) LH, but not FSH, increases after CL excision, presumably by removing some luteal factor inhibitor of LH secretion.

Season influences FSH concentration in ovariectomized Ile-de-France ewes.

Ile-de-France ewes were ovariectomized during anoestrus or the mid-luteal phase of an oestrous cycle (day of ovariectomy = Day 0). In a short-term study, FSH concentrations were measured in blood samples collected hourly the day before and on Days 1, 3, 7 and 15 after ovariectomy (10 ewes per group). FSH concentrations increased significantly from 6.1 to 16.5 ng/ml within 1 day of ovariectomy and increased further to 47.1 ng/ml by Day 15. Differences between seasons of ovariectomy were not significant. In a long-term study, FSH concentrations were measured in blood samples collected hourly on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy in anoestrus or the breeding season (10 ewes per group). Further samples were taken (5 ewes/group) at 240 and 365 days after ovariectomy. The pattern of change in FSH after ovariectomy differed between the two seasons and the interaction between season and sampling day was significant. For ewes ovariectomized during anoestrus, FSH concentrations increased to a maximum by Day 180 and remained high thereafter. In contrast FSH increased more slowly in ewes ovariectomized in the breeding season and differences between the groups were significant from Day 90 to Day 270. However, both groups had similar FSH concentrations at Day 365. These results show that FSH concentrations increase rapidly after ovariectomy. There are seasonal differences in FSH concentrations in the absence of ovarian feedback with increases in FSH concentration around the time of the onset of the breeding season. Once FSH concentrations had reached a maximum, major seasonal changes were no longer apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of the final phase of ovulation has no effect on the post-ovulatory surge of serum FSH in the rabbit.

Mating induces a surge of both LH and FSH in the blood of female rabbits, followed 10-12 h later by a surge of FSH only, which begins at the time of ovulation. We have studied the effect of suppression of ovulation on the post-ovulatory surge of FSH. In the first experiment, follicular fluid and oocytes were withdrawn from the largest follicles 8 h after coitus. In the second experiment, ovulation was inhibited by injecting the rabbits with 25 mg indomethacin/kg body weight 7.5 h after mating. Levels of serum FSH and LH were measured for 24-48 h after mating. Control rabbits ovulated normally in both experiments. The treatments did not significantly affect the levels of serum FSH in either experiment, although the second surge of FSH was slightly higher after fluid had been aspirated from the preovulatory follicles. These observations show that the post-ovulatory surge of serum FSH is not dependent upon the completion of ovulation and that it is programmed before 7.5-8 h post coitum.

Highly specific antisera against native ovine follitropin (oFSH) obtained from crude antisera by affinity chromatography on solid-phase undissociable lutropin (LH) column.

Antibodies recognizing determinants of the alpha-subunit, which is common to all glycoprotein hormones, were eliminated from antisera against native oFSH by affinity chromatography. Since the free alpha-subunit is immunologically different from the alpha-subunit in the intact hormone, we did not use an alpha-subunit affinity column. Instead, the antisera were applied to an LH affinity column. However, because intact LH dissociates in the conditions used to elute the purified antibodies, we prepared an LH derivative with covalently-linked subunits and coupled it to gel matrix. By this method oFSH antisera were freed from their non-specific antibodies. The cross-reaction of ovine lutropin in the oFSH radioimmunoassay (RIA) was lowered from 2% to less than 0.1%. Moreover, as the columns can be used repeatedly over long periods with no apparent loss of efficiency, large volumes of antiserum can be treated in this manner.

Variations in the plasma levels of gonadotrophin and testosterone and in Leydig and Sertoli cell populations between birth and adulthood in Romanov lambs born in spring or autumn.

Plasma gonadotrophin and testosterone levels during the prepuberal period have been compared in spring and autumn-born Romanov lambs; the somatic and germ cell populations of these lambs have also been compared at birth and at adulthood. At 1 and 3 months of age, the number of LH pulses per hour and mean plasma LH levels were significantly higher in spring than in autumn lambs. In adults, no such differences were observed. Similarly, at 1 and 3 months of age, the number of testosterone pulses per hour was higher in spring than in autumn animals. In the prepuberal period, the ratio of the levels of mean plasma testosterone to LH was higher in autumn than in spring lambs. The highest ratio was observed in adults but there was no variation according the season of birth. At 1 month of age, mean plasma FSH levels were higher in autumn than in spring lambs; this difference did not persist later on. Despite these endocrinological differences, testis weight, total Sertoli and Leydig cell numbers per testis, total number of gonocytes per testis at birth and daily production of round spermatids per testis in adult rams were similar in spring and autumn-born animals.

A comparison of the changes in LH, FSH and testosterone in spring-born ram lambs of two different breeds.

LH, FSH and testosterone variations were compared during the prepubertal period in Romanov and Ile-de-France ram lambs born in the spring. Mean plasma LH levels increased significantly between the 1st and 8th week of age in the Romanov and between the 1st and 12th week of age in the Ile-de-France. At 1 month of age, the number of LH and testosterone pulses per hour was higher in Romanov than in Ile-de-France lambs. The mean plasma testosterone levels, higher in the Romanov, increased from 1 week of age onwards. Mean plasma FSH levels increased from the first neonatal week till the 8th week in the Romanov and till the 12th week in the Ile-de-France. The levels of FSH did not differ significantly between the two breeds. The higher and earlier secretion of LH and production of testosterone in the Romanov might be a cause of its better reproductive performance.

[Variations in the plasma concentration of prolactin, LH and FSH and in testicular activity during the first year of life in the roe deer (Capreolus capreolus L.)]. / Variations de la concentration plasmatique en prolactine, LH, et FSH et de l'activité testiculaire au cours de la première année de vie chez le Chevreuil (Capreolus capreolus L.)

Plasma prolactin, LH, FSH, testosterone patterns, and spermatogenic activity were determined during the first year of life in roe deer. A high gonadotrophic activity was observed a few months after birth, in autumn, along with an increase of testis volume and spermatogenic activity. Some spermatozoa could be seen in seminiferous tubules in December. A second increase of pituitary gonadotrophic activity appears in early spring when testicular volumes comparable to the adult are achieved. The first period of gonadotrophic activity coincides with the autumnal decrease in photoperiod, whereas the second coincides with the progressive increase in day length. It is suggested that the precocious LH and FSH release induces the first pubertal testicular activity.

Hormonal levels after ovarian X-irradiation of ewes.

This paper reports the effects of X-irradiation on ovulation rate, cyclicity and progesterone and FSH levels in Ile-de-France ewes (4 control and 16 irradiated) after they were treated during the breeding season. The doses used (800 and 2 400 R) destroyed 50% of all size classes of the follicular population. Ovulation occurred in 87% of the treated ewes (ovulation rate = 1) when they were irradiated 24 h after luteolysis; 78% of the corpora lutea resulting from these ovulations were normal as to length and progesterone production. FSH in treated ewes started to increase 20 h after treatment and remained higher than in the controls until ovulation time. Later, while these levels were similar between groups on the day of ovulation, high ovulatory levels persisted in irradiated ewes. In the next cycles, the length of the follicular phases, ovulation rate and progesterone and FSH levels were similar between groups.

Effect of cryptorchidism in the ram: changes in the concentrations of testosterone and estradiol and receptors for LH and FSH in the testis, and its histology.

Cryptorchidism was induced in 5 pre-pubertal lambs and 7 adult rams, 5 months after surgery, testicular weight and membrane protein content were 4-fold lower than in the control. The total number of Leydig cells per testis was markedly decreased but their size was not changed. In contrast, the total number of Sertoli cells per testis was not affected but their nuclear size was smaller. Induced cryptorchidism had no effect on the length of seminiferous tubules; blood vessel volume was reduced; and the production of germ cells was completely disrupted. The number of LH receptors estimated per Leydig cell was not changed in pre-pubertal lambs but decreased 4-fold in adult rams. The number of FSH receptors calculated per Sertoli cell was reduced by 95% in both pre-pubertal and adult animals. No effect on the binding affinities of LH (Ka = 1 X 10(10) M-1) and FSH (Ka = 4.5 X 10(9) M-1) to their testicular receptors was observed. Although testicular concentrations of testosterone and estradiol-17 beta were increased, the total content of testosterone within the testis was increased only in pre-pubertal lambs. The estimated ratio of testosterone per Leydig cell was higher in cryptorchid animals than in controls, suggesting that, despite their reduction in number and the decrease of LH receptors, the Leydig cells of cryptorchid rams have an enhanced steroidogenic capacity. This study also confirms the important dysfunction of the Sertoli cells in cryptorchid rams.

Testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) in plasma of rabbit from birth to adulthood. Correlation with sexual and behavioural development.

Plasma testosterone, LH and FSH levels were determined and correlated with reproductive organs growth, testicular differentiation, fighting and mounting behaviour in maturing rabbit. An infantile phase of development extends from birth to 40 days, characterized by low testosterone and FSH levels, decreasing LH levels (until 20 days) and by a slow growth of testis and seminal vesicle. The peripubertal phase starts abruptly around day 40. It is marked by simultaneous events: the appearance of mature Leydig cells in the testis, a striking increase in testosterone and FSH levels, a small rise in LH levels and an acceleration of testicular growth. The phase of rapid growth of seminal vesicle and the first meiotic divisions start around day 70, in presence of high circulating levels of FSH and testosterone. Fighting (3 months) and mounting behaviour (146 +/- 13 days) occur lately after a long period of high circulating testosterone levels.

FSH and LH variations in beef cows during the postpartum period.

To study the plasma gonadotrophin profiles of 9 cows after parturition, blood samples were obtained every 20 min for 12 hrs on three occasions between 5 and 50 days postpartum and analysed by RIA techniques. The time of the first ovulation, as judged by plasma progesterone levels, varied from 30 to more than 60 days postpartum. Variations in mean levels of FSH and LH were not significantly correlated with the postpartum interval. However, the mean levels of plasma FSH and number of LH pulses were lower in females which had not ovulated than in those which had. The cows could be classified into four groups: group 1 with less than 4 LH pulses in 12 hrs and a mean plasma FSH level less than 138 ng/ml; group 2 with more than 4 LH pulses in 12 hrs and varying plasma FSH levels; group 3 with less than 4 LH pulses in 12 hrs and a mean plasma FSH level greater than 138 ng/ml; group 4 which had ovulated. This classification indicated that the LH and FSH levels progressed significantly (2.46 to 3.56 ng/ml, P less than 0.05; 120 to 159 ng/ml, P less than 0.01, respectively) from groups 1 to 3, and that they decreased in the females which had ovulated (group 4). Since the time of the first ovulation after parturition varied, it was not possible to demonstrate any relationship between that interval and the mean plasma gonadotrophin profiles. However, when ovulation was considered as time zero there was a clear increase in plasma gonadotrophin before ovulation.

Seasonal variations in rete testis fluid secretion and sperm production in different breeds of ram.

Production of spermatozoa and secretion of rete testis fluid (RTF) in rams was assessed by a rete testis cannulating technique. Four breeds (Ile-de-France, Romanov, Préalpes du Sud and cross-breed Romanov) were studied throughout the year. Inhibitory effects of the cannulation process on spermatogenesis were observed for some animals. Between-breed differences were found in sperm concentration and flow rate of the RTF. The seasonal variations in the daily sperm production of the testis were more pronounced for Ile-de-France rams than for the other breeds. There was a seasonal variation in the flow rate of RTF in Ile-de-France rams, the minimum flow being in February (winter) and the maximum in August-September (autumn).

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.