Opioid modulation of LH secretion in the ewe

Administration of opioid agonists and antagonists and measurement of resulting hormone changes were used to study the possible effects of opioids on reproductive function in the ewe. Intravenous administration of the long-acting methionine-enkephalin analogue FK33-824 (250 micrograms/h for 12 h) to 3 ewes during the follicular phase of the oestrous cycle depressed episodic LH secretion. This effect was reversed by administration of the opiate antagonist naloxone (25 mg/h) in combination with the FK33-824 treatment; in fact LH secretion was enhanced by the combined regimen. Naloxone (25 mg/h for 12 h) administered alone to 3 ewes in the follicular phase also enhanced LH secretion. In 3 animals treated with FK33-824 during the follicular phase, progesterone remained basal for 14 days after treatment, suggesting that ovulation was blocked. Jugular venous infusion of naloxone (25, 50 or 100 mg/h for 8h) into 5 ewes during the early and mid-luteal phase of the cycle resulted overall in a significant increase in mean plasma LH concentrations and LH episode frequency. To investigate whether endogenous opioids suppress LH release in seasonally anoestrous sheep, naloxone was infused intravenously into mature (25, 50 or 100 mg/h for 8 h) and yearling ewes (12 . 5, 25 or 50 mg/h for 8 h) during early, mid- and late anoestrus and plasma LH concentrations were measured. In the mature ewes, there was a trend for naloxone to increase LH values during the early anoestrous period but naloxone was without effect during mid- and late anoestrus. In the yearlings, naloxone infusion consistently increased plasma LH concentrations as a result of a significant increase in LH episode frequency. These experiments indicate that endogenous opioid peptides probably modulate gonadotrophin secretion during both the follicular and luteal phases of the oestrous cycle. However, the follicular phase of the sheep cycle is of short duration, and there may be residual effects of luteal-phase progesterone during this period. Secondly, there may be an age-dependent effect of naloxone on LH secretion during seasonal anoestrus in the ewe, with opioids playing a part in the suppression of LH in young but not in mature animals.     

Opioid modulation of LH secretion during the oestrous cycle of heifers

Injections of an opioid agonist (bremazocine) and/or an antagonist (quadazocine) were given to heifers during the luteal or follicular phase of the oestrous cycle. Quadazocine was injected (210 mg/injection) three times at 2-h intervals, and bremazocine was injected (0.45 mg/injection) every 15 min for 6 h. Blood samples were taken every 15 min beginning 6 h before treatments started and continued for 18 h. LH secretion patterns were not affected by quadazocine in the luteal-phase heifers, but quadazocine and bremazocine had marked effects during the follicular phase. Quadazocine increased LH secretion by increasing peak height but not peak frequency. Bremazocine decreased LH secretion through both peak height and frequency. This decrease was of greater magnitude than the increase due to quadazocine. When quadazocine and bremazocine were given together, these effects were cancelled and none of the effects carried over into the bleeding period after treatments stopped. No apparent interruption of follicular maturation was detected since all follicular-phase heifers were detected in oestrus at normal intervals. We conclude that heifers in this experiment did not have an opioid-mediated mechanism for progesterone suppression of LH but that an opioid mechanism for modulating LH does exist during the follicular phase.     

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 micrograms cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 micrograms/ml was needed. Cortisol also inhibited the 17 beta-estradiol-induced increase in GnRH response. ACTH-(1-24), ACTH-(1-39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Ovulation-inducing factor (OIF) induces LH secretion from pituitary cells

A substance in the seminal plasma of llamas and alpacas has been discovered that induces ovulation and growth of the corpus luteum (CL) in the female of the same species. The ovarian effects of the ovulation-inducing factor (OIF) are associated with a surge release of LH into circulation. We hypothesize that OIF stimulates LH release from gonadotroph cells in the anterior pituitary gland. Four experiments were done to determine if purified OIF isolated from llama seminal plasma stimulates LH secretion in pituitary cells using tissue from an induced ovulator (llama) and spontaneous ovulator (cattle). Anterior pituitary cells were cultured in vitro for two days, and on the third day, wells were incubated for 2 h with media containing no treatment (control), GnRH or OIF. Concentrations of LH in the culture medium were measured using radioimmunoassay and compared among groups by analysis of variance. In all experiments, GnRH and OIF treatments induced more LH secretion than untreated controls (P<0.05). A dose-related effect was evident in the llama pituitary cell cultures in that mean LH concentrations were greater (P<0.05) in wells treated with a higher dose of OIF (5.41 ± 0.28 ng/mL) compared to wells treated with a lower dose (2.70 ± 0.50 ng/mL), both of which were higher (P<0.05) than in wells with no treatment (0.87 ± 0.18 ng/mL). Although OIF stimulated LH release in bovine cell cultures, a dose-related effect was not detected. We conclude that OIF stimulates LH secretion from pituitary gonadotrophs in vitro.     

Catecholamine inhibition of luteinizing hormone secretion in isolated pig pituitary cells

This study investigated the direct effect of catecholamines, epinephrine (EPI), and norepinephrine (NE) on basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of luteinizing hormone (LH) from dispersed pig pituitary cells in vitro. Pig pituitaries were dispersed into cells with collagenase and DNAase and then cultured in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) pretreated with dextran-coated charcoal for 3 days. EPI and NE did not affect basal LH secretion after 4 h of incubation. When pituitary cells were incubated with EPI or NE (1 microgram/ml) for longer than 30 min, GnRH-stimulated LH secretion was reduced. The degree of this reduction was dependent on EPI and NE, and a concentration of EPI and NE higher than 1 ng/ml and 100 ng/ml, respectively, was needed. L-isoproterenol, a nonselective beta-agonist, also inhibited the LH response to GnRH. Propranolol, a beta-antagonist, blocked the inhibitory effect of EPI, whereas phentolamine, an alpha-antagonist, had no effect. These results suggest that catecholamines, acting by a beta 2-adrenergic receptor, may play a role in the control of the porcine pituitary gonadotrope's response to GnRH.     

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influences of GnRH, oxytocin and vasoactive intestinal peptide on LH and PRL secretion by porcine pituitary cells in vitro.

The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n = 8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n = 8) and Group 3 (n = 8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h before slaughter; and in Group 4 (n = 9) with progesterone (P4) at the dose 120 mg/ 100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (10(6)/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37 degrees C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37 degrees C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p < 0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p < 0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p < 0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals.     

Effects of in vivo pretreatment with D-Trp-6-LH-RH on prolactin and LH secretion by pituitary glands in vitro

In order to study a possible direct action of LH-RH analogs on the pituitary lactotrophs, we investigated the effect of long-term in vivo pretreatment with D-Trp-6-LH-RH on in vitro secretion of PRL and luteinizing hormone (LH) by the pituitary glands from male and female rats. In vivo pretreatment with D-Trp-6-LH-RH (50 micrograms/day, SC) for 15 days greatly reduced basal in vitro PRL release (p less than 0.01) in female, but not in male pituitary glands. TRH-stimulated PRL secretion was not affected by pretreatment with D-Trp-6-LH-RH in female rats, but was impaired in male pituitaries. Acute in vitro exposure to D-Trp-6-LH-RH did not modify PRL secretion by female pituitary glands pretreated in vivo with the analog. However, this same in vivo pretreatment greatly decreased PRL release from male pituitaries (p less than 0.01). Basal in vitro LH release by male pituitary glands was partially lowered by in vivo pretreatment with D-Trp-6-LH-RH, as compared to controls (p less than 0.01), while basal LH release in female pituitaries remained at control levels. Finally, D-Trp-6-LH-RH-induced stimulation of in vitro LH release was severely impaired in female pituitaries (p less than 0.01) but only slightly reduced in the males.     

Regulation of galanin secretion from pituitary cells in vitro by estradiol and GHRH

The effects of estradiol and growth hormone-releasing hormone (GHRH) on galanin release from anterior pituitary cells were examined in vitro. 17-β-Estradiol (0.001–10 nM) increased galanin secretion from anterior pituitary cells in a concentration-dependent manner. Estradiol (10 nM) increased galanin release 300 and 600% from pituitary cells of ovariectomized and male rats, respectively. Immunocytochemical studies demonstrated that estradiol (10 nM) increased the number of galanin-containing cells twofold after 4 days in culture. Growth hormone-releasing hormone (1 and 10 nM) increased and SRIF (1 and 10 nM) decreased galanin release from pituitary cells of ovariectomized and male rats. We conclude that estradiol increases galanin release by a direct effect on pituitary cells, in part by increasing the number of pituitary cells synthesizing galanin. In addition, GHRH stimulates galanin release when estradiol levels are low.     

Inhibitory control of the pituitary LH secretion by LH-RH in male rats.

Luteinizing hormone-releasing hormone (LH-RH) was administered to prepubertal male rats (intact, castrate or castrate-adrenalectomized, 60 g body weight) for 28 days (1 microgram LH-RH/day, s.c.), at a 10-fold physiological dose, as compared to the minimal FSH-releasing dose of 100 ng/rat s.c. In intact rats, serum LH and weight of androgen-dependent organs (vented prostate, seminal vesicles) were reduced after 14 days of treatment. In castrate rats, the postcastration rise in serum LH was abolished by treatment. Pituitary LH content, FSH secretion and prolactin secretion were not suppressed. Hypothalamic LH-RH was increased at 14 and 21 days. In castrate adrenalectomized male rats, LH secretion was also suppressed by 1 microgram LH-RH s.c. x 28 days. The hypothalamic LH-RH content did not increase. The pituitary LH-RH receptor level was not down-regulated after 14 days treatment either in intact or castrate male rats. Pituitary inhibition (LH release) in rats by a supraphysiological dose of LH-RH given for 28 days indicates that the optimal regime for chronic treatment has to be determined by monitoring LH release at regular intervals. Direct pituitary inhibition by LH-RH may explain some of the unexpected antifertility effects observed with high doses of LH-RH.     

Opioid modulation of thyrotropin releasing hormone induced prolactin secretion

It is known that opioids stimulate prolactin (PRL) secretion by an action on hypothalamic neurons, but in vitro studies have suggested a direct action on the lactotrophs. The present study was performed on male rats known to have little or no PRL response to TRH. A beta-endorphin (beta EP) injection in the third ventricle stimulated PRL secretion and induced furthermore a PRL secretory reaction to TRH injected intravenously 20 min later. Pretreatment with naloxone 10 min before beta EP injection abolished not only the PRL response to beta EP but also the conjugated effect of beta EP and TRH. Pretreatment with naloxone methyl bromide (Br-naloxone), a quaternary naloxone derivative, which does not cross the blood-brain barrier, had no effect on the PRL response to beta EP but prevented the conjugated effect of beta EP and TRH on PRL secretion. Pretreatment of the animals with -methyl-parathyrosine resulting in a dopamine depletion or with haloperidol, a dopamine antagonist, could not induce lactotroph responsiveness to TRH. These results suggest that beta EP in male rat sensitizes the PRL cell to TRH by a direct effect and not through an inhibition of the dopaminergic tone.     

Somatostatin affects morphology and secretion of pituitary luteinizing hormone (LH) cells in male rats

The somatostatin peptides (SRIH-14, SRIH-28) and their multiple receptors are generally associated with anti-proliferative and anti-secretory actions. This study compared, using standard morphometric measurements and terminal serum LH concentrations, effects of intracerebroventricular (icv) SRIH-14 and SRIH-28 in nanomolar amounts on immunohistochemically identified LH cells in pituitary glands of male rats. Rats received l microg/5 microl of SRIH-14 or SRIH-28 icv on days 1,3, and 5, whereas control rats received only icv saline. Animals were killed 5 days later for serum LH assays. Pituitarys were harvested for PAP immunohistochemistry and morphometry. Morphometric measurements were made by an observer blinded to the treatment group. Histochemically identified LH cells from both SRIH groups appeared smaller, often pycnotic and darkly stained compared to those from saline-treated rats. Both SRIH treatments reduced (p < 0.05) the quantitative morphometric measurements for cell volume, nuclear volume, and relative volume density. Both SRIH treatments also reduced serum LH concentration (p < 0.05), supporting the hypothesis that systemic physiology was altered. Collectively, the data support the opinion that nanomolar amounts of either SRIH peptide, acting on receptors reached from cerebrospinal fluid, exert an anti-secretory effect on LH cells of male rats. Modifications of central SRIH receptors may provide an approach for treatment of male sexual dysfunction and/or be of pathophysiologic significance in these disturbances.     

Opioid modulation of hypothalamic catecholaminergic neurotransmission and the pre-ovulatory LH surge in the rat

We have investigated the inter-relationship between the opioid and catecholaminergic systems in the control of LH secretion, and the involvement of &mgr;- and kappa-opioid subtypes in this process. Conscious female rats were intraperitoneally injected with either &mgr;- (diamorphine) or kappa-opioid agonists (U-50488H) alone or with their respective antagonists (naloxone and MR2266) before the critical period on pro-estrus. Hypothalamic catecholamine and plasma LH levels were determined by HPLC-ECD and RIA, respectively. Both &mgr;- and kappa-agonists significantly decreased concentrations of noradrenaline and its metabolite (DHPG) in all the hypothalamic regions examined concomitant with inhibition of the LH surge. Dopamine levels were selectively reduced only by the &mgr;-agonist in the MPOA. The inhibitory effects of both opioid agonists were mostly reversed following their co-administration with naloxone and MR2266 (except the kappa-antagonist on LH). These results indicate that both the &mgr;- and kappa-opioid subtypes may be involved in the inhibition of the LH surge by altering the hypothalamic noradrenaline content.     

Direct in vitro stimulation of pituitary LH release by alpha melanocyte stimulating hormone

The present $\text{in vitro}$ superfusion study demonstrates that synthetic α-MSH acts at the pituitary level, independent of the hypothalamus, to increase the release of LH in male but not in female rats.     

Cu2+对离体培养猪腺垂体细胞GH分泌的影响

目的：研究铜离子（Cu2＋）对离体培养猪腺垂体细胞生长激素（GH）分泌的影响。方法：试验选取32-35日龄仔猪腺垂体细胞,于10%小牛血清的Dulbecco MEM培养液（DMEM）中离体培养48 h。之后用含不同浓度Cu2＋（0 mg/L、0.025 mg/L、0.1 mg/L、0.4 mg/L、1.6 mg/L）的无小牛血清DMEM培养液培养36 h,于12 h、24 h、36 h收集细胞培养液,用Linco公司的试剂盒及放射免疫法测定培养液中GH浓度。结果：铜离子离体培养腺垂体细胞24 h,0.025 mg/L、0.1 mg/L、0.4 mg/L组培养液中GH浓度均高于0 mg/L组,其中0.1 mg/L组与0 mg/L组差异显著（P〈0.05）。结论：铜离子可促进离体培养猪腺垂体细胞分泌GH。     

Comparison of hyposmolar and hyperosmolar effects on in vitro luteinizing hormone secretion by anterior pituitary cells

It has previously been described that perifusion of acutely dispersed adenohypophyseal cells with hypotonic medium causes an immediate high-amplitude "on" burst of luteinizing hormone (LH) secretion. In the present report the converse study with hyperosmolar solutions has been made. Perifusion with hypertonic medium depressed LH secretion; return to isotonicity caused an immediate high-amplitude "off" burst of LH secretion closely resembling that induced by hypotonic perifusion. The data give further support to the theory that exocytotic secretion may involve expansion of the outer cell membrane, thus drawing secretory granules to the cell surface where their contents are extruded.     

Mechanism of LH release in cultured rat pituitary cells

To investigate the mechanisms of the synthesis and the release of gonadotropin, rat anterior pituitary cells were stimulated in vitro with luteinizing hormone releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin) and 12-0-tetradecanoyl phorbol-13-acetate (TPA), and then the LH and LH-beta subunit released into the medium were determined by radioimmunoassay. Buserelin showed its biological activity at a much lower concentration than LH-RH, but both of them caused the release of LH and LH-beta subunit in a dose-dependent manner. Furthermore, intracellular LH synthesis from LH-beta subunit by stimulation with LH-RH or Buserelin was also found. After inducing various degrees of desensitization by stimulation with LH-RH or Buserelin in a dose-dependent manner (the first stimulation), pituitary cells were stimulated with a fixed dose of TPA (the second stimulation) and the released LH was assayed. LH was released almost constantly by the second stimulation, regardless of the dose used for the first stimulation. These results suggest that the C-kinase pathway was unaffected by the desensitization induced with LH-RH or Buserelin.     

Ontogeny of pituitary gonadotrophin secretion in the fetal and post-natal pig in response to LHRH in vitro

Monolayer cultures of anterior pituitary cells from male or female pigs of 60, 80, 105 days of fetal life or of 60, 160 and 250 days of post-natal life were prepared and treated with LHRH (1 pM to 10 nM). Dose-related increases of LH were first seen at 80 days of gestation in both sexes, while only female fetuses responded to maximal LHRH at 60 days. Basal and stimulated LH release doubled in cultures from 105-day-old fetuses when compared with those at 80 days. Compared to late fetal stages LH release was 20- to 30-fold higher in cell cultures from 60-day-old (post-natal) donors without further change during the post-natal period. In all pre- and post-natal age groups basal and maximal LH release of pituitary cells from males was lower than that of females. FSH stimulation started in male and female cells at 80 days of gestation only at LHRH concentrations exceeding or equal to 0.1 nM. By 105 days FSH secretion was dose-related and pituitary cells of females responded with higher FSH values than did those of males. In general, post-natal cells released much higher amounts of FSH than did prenatal cells. Basal and maximal release of FSH decreased during post-natal development in both sexes. Basal as well as maximal FSH release of cultures from female donors was higher than that found in cultures from male donors. Determination of total LH and FSH content in fetal pituitary cell cultures indicated that the developmental increase in gonadotrophin release potential is a function of the total gonadotrophin content in vitro. We conclude that (1) the in-vitro release of gonadotrophins to LHRH is dose-, age- and sex-dependent; (2) in the female fetal pig LH responsiveness develops earlier than FSH responsiveness; (3) apparently, these maturational changes mainly reflect alterations in pituitary gonadotrophin content; and (4) there is no simple relationship between in-vitro release and circulating gonadotrophins.