Design and synthesis of a gonadotropin-releasing hormone (GnRH) analogue, [Tyr(OMe)5,d-Glu6,Aze9]GnRH: Receptor binding, gonadotropin release and ovulation studies

Summary Gonadotropin-releasing hormone (GnRH) stimulates the release and synthesis of gonadotropin hormones (GtH) and is the key regulator of reproduction. The present study was carried out to design a potent GnRH analogue containing Tyr(OMe) at position 5 and ad-amino acid at position 6. This was based on a previous study in which [Tyr(OMe)⁵]GnRH was shown to have reduced potency compared to GnRH. A novel GnRH peptide containing Tyr(OMe)⁵ andd-Glu⁶ in combination with other substitutions at positions 9 and 10 was synthesized in the present study and tested for binding to the rat pituitary as well as potency in terms of gonadotropin (GtH) release in the goldfish pituitary and ovulation in sea bass. The results demonstrate that the replacement of the glycine residue at position 6 with ad-Glu in combination with the substitution of proline at position 9 with azetidine (Aze) increased the binding and biological activity of [Tyr(OMe)⁵]GnRH. The observed increased potency is likely to be related to the improved resistance to degradation. The present findings may lead to the development of a more potent GnRH agonist for inducing ovulation in fish.     

Patterns of folliculogenesis in ducks following the administration of a gonadotropin-releasing hormone 1 (GnRH) analogue

The efficacy of synthetic gonadotrophin-releasing hormone (GnRH) analogue in improving the folliculogenesis of ducks has not been established. The aim of the study was to investigate the effect of oral administration of GnRH analogue as luteinizing hormone releasing hormone A₂ (LHRH-A₂) on expression of relevant genes, egg production, changes of hormone levels and an ovarian follicle development in ducks. Five hundred ducks at 220 days old were randomly allotted to five groups, where each bird received daily in food 0, 5, 10, 15, or 20 μg LHRH-A₂ for 60 days. In all treated groups, a non-significant increase in the level of GnRH receptor was noticed as compared to the corresponding control. Interestingly, the egg product in the 10 and 15 μg LHRH-A₂ groups was profoundly increased (P < 0.05) if compared to 0 and 5 μg LHRH-A₂ groups or control. A reduction in circulating prolactin (PRL) levels occurs concurrently with an increase in progesterone (P₄) and estradiol (E₂) particularly in 5, 10 and 15 μg LHRH-A₂ groups. Maximal apoptotic percentage for the granulosa cells was obtained in 20 μg LHRH-A₂ group as compared to control or 5, 10 and 15 μg treatment groups. Finally, these data suggest that the oral administration of 10 and 15 μg LHRH-A₂ may induce ovarian cycle and play vital gonadotrope role during the folliculogenesis process in ducks. This study also demonstrated a need to concentrate further research on the potential effect of GnRH during the early period to improve the reproductive performance.     

Hormonal secretion in male rats chronically treated with [D-Trp6, Pro9-NEt]-LRF.

Chronic daily administration of the LRF agonist [DTrp⁶, Pro⁹NEt]LRF (A) was accompanied by a blunting of the LH, FSH and testosterone responses. However, plasma LH levels continued to be elevated with each injection, while plasma FSH values remained within control range. As a result, LH/FSH ratios of treated rats were higher than those of control animals. Preinjection plasma testosterone levels were significantly lowered. Repeated injections of A were usually accompanied by increases in plasma progesterone levels which were largest on the second day of qd administration schedules. The intermittent administration of A every 2nd, 3rd or 4th day resulted in continued large increases in plasma progesterone concentrations. These results suggest that the periodically elevated secretion of progesterone and/or LH in the presence of lowered levels of circulating testosterone, may play a role in the paradoxical antigonadal effects of LRF agonists.     

Testicular modulation of luteinizing hormone response to gonadotropin-releasing hormone (GnRH)-agonist treatment

We and others have observed that the response of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to chronic gonadotropin-releasing hormone-agonist (GnRH-A) treatment is substantially different in normal compared to hypogonadal males. These data suggested that products of the testes determine the gonadotropin response to GnRH-A. The present studies were designed to determine whether this effect is mediated by products of the interstitial (steroids) or the tubular compartment. To create experimental states with selective impairment of interstitial, tubular, or both compartments, 100 male sexually mature Wistar rats were divided into five groups: I, intact; II, castrated; III, castrated with 20-mm testosterone (T) implants; IV, bilaterally cryptorchid; and V, ketoconazole-treated animals. Cryptorchid animals have been shown to have impairment of tubular function while ketoconazole inhibits T biosynthesis. Each of the 5 groups was divided into 2 subgroups to receive daily injections of either saline or 1 microgram of a potent GnRH agonist, D-leu6 des-Gly10 GnRH N-ethylamide, for 4 wk. Unlike the intact animals, which showed an elevation of basal serum LH concentration after 4 wk of GnRH-A treatment, the castrated animals showed significant suppression below baseline. Animals with preferential impairment of tubular function (cryptorchid and castrated + T) also showed significant suppression of LH after GnRH-A treatment. However, the ketoconazole-treated animals (with inhibition of T biosynthesis and intact tubular function), behaved similarly to intact animals and demonstrated an elevation of LH after GnRH-A treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrine and reproductive responses of beef cattle to a synthetic gonadotropin-releasing hormone agonist (fertirelin acetate)

A synthetic gonadotropin-releasing hormone (GnRH) agonist (fertirelin acetate, FA) was administered to beef cattle within 12 h after onset of estrus (Day = 0) to study effects on subsequent endocrine responses and fertility. In Study 1, 16 crossbred beef heifers were injected with either 100 mug FA (n = 8) or saline (n = 8) at 6 or 12 h (n = 7; n = 9) after onset of estrus. Concentrations of luteinizing hormone (LH) over time were affected (P<0.01) by the interaction of treatment and interval from onset of estrus to treatment. Heifers treated with FA at 6 h after onset of estrus exhibited the greatest increase in LH after treatment. There was no effect of treatment, interval from onset of estrus to treatment or treatment by interval interaction on duration of the estrous cycle, on concentrations of progesterone from Days 1 through 14 posttreatment, or on concentrations of progesterone prior to subsequent estrus (Day -10 through 0, posttreatment estrus). In summary, FA administered to beef cattle within 12 h after onset of estrus effectively increased peripheral plasma concentration of LH, but this increase had no effect on subsequent luteal function as measured by duration of the estrous cycle or concentrations of plasma progesterone. In Study 2, 86 parous beef cows were bred artificially to one of two bulls following natural or prostaglandin F(2)alpha induced estrus. Cows received either no treatment or 50 or 100 mug FA at the time of AI. There was no effect of treatment, breed, parity, technician, service sire or interactions on conception rate (mean = 76.7%). Although not significant, the numerical pattern of conception rate among experimental groups (control = 71.4%, 50 mug FA = 76.7%, 100 mug FA = 82.1%) supports further investigation of this GnRH agonist with larger numbers of cattle.     

Gonadotropin secretion in ovariectomized ewes: effect of passive immunization against gonadotropin-releasing hormone (GnRH) and infusion of a GnRH agonist and estradiol

Gonadotropin secretion was examined in ovariectomized sheep after passive immunization against gonadotropin-releasing hormone (GnRH). Infusion of ovine anti-GnRH serum, but not control antiserum, rapidly depressed serum concentrations of luteinizing hormone (LH). The anti-GnRH-induced reduction in serum LH was reversed by circhoral (hourly) administration of a GnRH agonist that did not cross-react with the anti-GnRH serum. In contrast, passive immunization against GnRH led to only a modest reduction in serum concentrations of follicle-stimulating hormone (FSH). Pulsatile delivery of the GnRH agonist did not influence serum concentrations of FSH. Continuous infusion of estradiol inhibited and then stimulated gonadotropin secretion in animals passively immunized against GnRH, with gonadotrope function driven by GnRH agonist. However, the magnitude of the positive feedback response was only 10% of the response noted in controls. These data indicate that the estradiol-induced surge of LH secretion in ovariectomized sheep is the product of estrogenic action at both hypothalamic and pituitary loci. Replacement of the endogenous GnRH pulse generator with an exogenous generator of GnRH-like pulses that were invariant in frequency and amplitude could not fully reestablish the preovulatory-like surge of LH induced by estradiol.     

Using gonadotropin-releasing hormone (GnRH) and GnRH analogs to modulate testis function and enhance the productivity of domestic animals

Gonadotropin releasing hormone (GnRH) controls the activity of the gonadotrope cells of the pituitary gland and, as a consequence, is a critical component of the endocrine cascade that determines the growth, development, and functional activity of testicular tissue. The use of GnRH and GnRH analogs is common in domestic animal production systems. Although GnRH and GnRH analogs are most commonly used to control the fertility and reproductive events in female animals, GnRH agonists and antagonists are increasingly used to modulate the fertility, behavior, and productivity of male animals as well. This review will focus on recent advances in this use of GnRH agonists and antagonists.     

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor: structure, distribution and regulation of expression

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.     

Characterization of gonadotropin-releasing hormone (GnRH) binding to pituitary receptors in goldfish (Carassius auratus)

Goldfish pituitary gonadotropin-releasing hormone (GnRH) receptors were characterized by using a superagonist analog of teleost GnRH (tGnRH-A; [D-Arg6, Trp7, Leu8, Pro9-NHEt]-GnRH). Equilibrium binding of 125I-tGnRH-A to a goldfish pituitary membrane preparation was achieved after a 30-min incubation at 4 degrees C; binding was significantly reduced after increasing incubation temperature to 22 degrees C. Binding of the radioligand was a function of tissue concentration, with a linear correlation over the range of 0.5-2 pituitary per tube. Incubation of the pituitary membrane preparation with increasing concentrations of 125I-tGnRH-A indicated saturable binding at radioligand concentrations of 470 pM and above. The binding of 125I-tGnRH-A was found to be reversible after addition of the cold analog, and the dissociation curve could be resolved into two linear components; slower rates of dissociation of 125I-tGnRH-A were observed after the addition of excess unlabeled tGnRH than after the addition of tGnRH-A, indicating that the analog is more effective in displacing the label than the native peptide. Addition of the cold analog displaced bound 125I-GnRH-A, and Scatchard analysis suggested the presence of at least two classes of binding sites: a high-affinity/low-capacity site and a low-affinity/high-capacity site. Bound 125I-GnRH-A was displaced by tGnRH from both sites in parallel to that observed with tGnRH-A, indicating that both peptides bind to the same classes of binding sites; however, tGnRH-A had a greater affinity for the receptors than the native tGnRH. These results demonstrated the presence and provided characterization of GnRH receptors in goldfish pituitary.     

Danazol versus [d-Trp6]GnRH agonist for the management of infertility due to atypical endometriosis

Summary The aim of this study is to assess the impact of atypical endometriosis on fertility as well as the effect of treatment in endometriosis-related infertility. A prospective randomized study comparing the effects of triptorelin (Arvekap, Ipsen) and danazol (Danatrol, Winthrop) was completed recently. The 103 patients (aged 30.9±4.92, range 21–41) who finished the study were divided into three comparable (infertility duration, age) groups. Group A (n=37) received danazol (600 mg/day) for 6 months, Group B (n=32) received triptorelin (3.75 mg) injections, one every 28 days for 6 months, and Group C (n=34) did not receive any kind of treatment. More pregnancies were observed in Group B (65.62%) compared with Group C (20.58%, p=0.00012) but not compared with Group A (54.05%, p=0.05857). The overall monthly fecundity rate in Group B was 0.11, and differences concerning spontaneous abortion rates were not observed. Our results indicate that danazol and triptorelin are equally effective in the treatment of atypical endometriosis-related infertility.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the brain and infundibulum of the sheep

Summary To determine how neural influences control the function of the pineal gland, morphological and biochemical relationships after pharmacological treatment have been studied in rat pineal cells in monolayer cultures. Norepinephrine (NE) and dibutyryl cyclic 3,5-adenosine monophosphate (dBcAMP) treatment of cells that had been in culture for 5 and 21 days produced a stimulation in the enzyme activity of serotonin N-acetyl transferase, an enzyme important in indole synthesis. NE and dBcAMP also produced morphological changes which were dependent on the time of cells in culture. When 5 day-cultures were treated with NE and dBcAMP, light and dark cells were noted and endoplasmic reticulum increased and became more organized. Only dBcAMP treatment at 5 days produced an increase in dense granules and an elongation of cytoplasmic processes. Treatment of 21 day-cultures with dBcAMP also produced an increase in cytoplasmic processes while treatment with NE produced an increase in the synaptic ribbons and clear vesicles within the processes.     

Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.     

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.     

Active immunization of gilts against gonadotropin-releasing hormone: effects on secretion of gonadotropins, reproductive function, and responses to agonists of gonadotropin-releasing hormone

Sexually mature gilts were actively immunized against gonadotropin-releasing hormone (GnRH) by conjugating GnRH to bovine serum albumin, emulsifying the conjugate in Freund's adjuvant, and giving the emulsion as a primary immunization at Week 0 and as booster immunizations at Weeks 10 and 14. Antibody titers were evident by 2 wk after primary immunization and increased markedly in response to booster immunizations. Active immunization against GnRH caused gonadotropins to decline to nondetectable levels, gonadal steroids to decline to basal levels, and the gilts to become acyclic. Prolactin concentrations in peripheral circulation were unaffected by immunization against GnRH. The endocrine status of the hypothalamic-pituitary-ovarian axis was examined by giving GnRH and two agonists to GnRH and by ovariectomy. An i.v. injection of 100 micrograms GnRH caused release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in control animals, but not in gilts immunized against GnRH. In contrast, administration of 5 micrograms D-(Ala6, des-Gly-NH2(10] ethylamide or 5 micrograms D-(Ser-t-But6, des-Gly-NH2(10] ethylamide resulted in immediate release of LH and FSH in both control and GnRH-immunized gilts. Circulating concentrations of LH and FSH increased after ovariectomy in the controls, but remained at nondetectable levels in gilts immunized against GnRH. Prolactin concentrations did not change in response to ovariectomy. We conclude that cyclic gilts can be actively immunized against GnRH and that this causes cessation of estrous cycles and inhibits secretion of LH, FSH, and gonadal steroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pattern of gonadotropin-releasing hormone (GnRH)-like stimuli sufficient to induce follicular growth and ovulation in ewes passively immunized against GnRH.

The pattern of GnRH-like stimuli capable of inducing follicular growth, ovulation, and luteal function was evaluated in ewes passively immunized against GnRH. The estrous cycles of 30 regularly cyclic sheep were synchronized using vaginal pessaries impregnated with a synthetic progestogen. Animals were passively immunized against GnRH (groups 2-5, n = 6) or the carrier protein, keyhole limpet hemocyanin (KLH; group 1, n = 6), at the time of pessary removal (PR). Circhoral delivery of saline (groups 1, 2, and 5) or low amplitude GnRH agonist (des-Gly10 GnRH ethylamide [100 ng/hourly pulse]; groups 3 and 4) was initiated at PR and continued for 3 (groups 4 and 5) or 12 days (groups 1-3). In groups 4 and 5, the amplitude of the GnRH-like stimulus was increased to 800 ng/hourly pulse (stimulus-shift) during the 24-h period beginning 72 h after PR. The amplitude of the hourly stimulus was adjusted to 100 ng/pulse 96 h after PR and continued at that level to Day 12. The endocrine changes associated with follicle growth and maturation (serum concentrations of estradiol [E2] above 10 pg/ml), ovulation (surge-like secretion of LH and FSH), and normal luteal function (serum concentrations of progesterone [P] above 2 ng/ml) were evident in ewes passively immunized against KLH (group 1). In this group, the preovulatory surge of gonadotropins was noted 48.7 +/- 1.2 h after PR. These endocrine events were blocked by passive immunization against GnRH (group 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal variation in hypothalamic content of gonadotropin-releasing hormone (GnRH), pituitary receptors for GnRH, and pituitary content of luteinizing hormone and follicle-stimulating hormone in the mare

Seasonal changes in the hypothalamic-hypophyseal axis were investigated using tissue from 49 light-horse mares, of mixed breeding. Hypothalamic and pituitary tissues were collected at 5 intervals throughout the years 1981 and 1982, representing midbreeding season (July, n = 10), transition out of the breeding season (October, n = 11), midanestrus (December, n = 8), transition into the breeding season (March, n = 10), and again in the following midbreeding season (July, n = 10). The hypothalamic region was dissected into preoptic area, body and median eminence. Gonadotropin-releasing hormone (GnRH) was extracted from hypothalamic samples with methanol-formic acid and quantified by radioimmunoassay. The anterior pituitary was homogenized and receptors for GnRH were quantified in a crude membrane fraction. Concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in the resulting supernatant. Content of GnRH in each of the 3 hypothalamic areas varied with season (P less than 0.01) and was lowest during midanestrus (P less than 0.05). There was no effect of season (P greater than 0.01) on either concentration or total number of receptors for GnRH, or concentration of FSH in the anterior pituitary. Concentrations of LH in the anterior pituitary varied with season (P less than 0.001). Means (+/- SEM) for the 5 collection times were 15.5 +/- 2.7, 9.7 +/- 2.4, 2.3 +/- 0.5, 2.7 +/- 0.4 and 11.7 +/- 1.5 microgram LH/mg anterior pituitary, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)