Relation of endogenous opioid peptides and morphine to neuroendocrine functions.

Morphine and the endogenous opioid peptides (EOP) exert similar effects on the neuroendocrine system. When adminstered acutely, they stimulate growth hormone (GH), prolactin (PRL), and adrenocorticotropin (ACTH) release, and inhibit release of luteinizing hormone (LH), follicle stimulating hormone (FSH),and thyrotropin (TSH). Recent studies indicate that the EOP probably have a physiological role in regulating pituitary hormone secretion. Thus injection of naloxone (opiate antagonist) alone in rats resulted in a rapid fall in serum concentrations of GH and PRL, and a rise in serum LH and FSH, suggesting that the EOP help maintain basal secretion of these hormones. Prior administration of naloxone or naltrexon inhibited stress-induced PRL release, and elevated serum LH in castrated male rats to greater than normal castrate levels. Studies on the mechanisms of action of the EOP and morphine on hormone secretion indicate that they have no direct effect on the pituitary, but act via the hypothalamus. There is no evidence that the EOP or morphine alter the action of the hypothalamic hypophysiotropic hormones on pituitary hormone secretion; they probably act via hypothalamic neurotransmitters to influence release of the hypothalamic hormones into the pituitary portal vessels. Preliminary observations indicate that they may increase serotonin and decrease dopamine metabolism in the hypothalamus, which could account for practically all of their effects on pituitary hormone secretion.     

Monolayer cultures of dispersed cells from bovine anterior pituitary: responses to synthetic hypophysiotropic peptides.

Cells were dispersed from bovine anterior pituitary glands, by digestion with collagenase, and cultured. After 4 days the cell monolayers were incubated with fresh medium containing synthetic hypophysiotropic peptides for 2, 6, or 20 h, and hormone released into the medium was estimated by radioimmunoassay. After 2 h, thyroid releasing hormone (TRH) stimulated the release of thyroid-stimulating hormone (TSH) up to eightfold, and of prolactin (PRL) and follicle-stimulating hormone (FSH) about twofold at a minimal effective concentration of 1 ng/ml; enhanced growth hormone (GH) release was not apparent until 20 h, and release of luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) was unaffected. Luteinizing hormone releasing hormone (LH-RH) enhanced release of LH maximally (three- to fourfold) during a 2 h incubation and was effective at 0.1 ng/ml; FSH release was significantly enhanced by about 50% above control level. Growth hormone release inhibiting hormone (GH-RIH)(somatostatin) showed significant effects only in the 20 h incubation; GH release was inhibited by 50% and release of PRL was slightly, but significantly, enhanced. Pituitary cell monolayers apparently permit maximal expression of releasing activities inherent in the hypothalamic hormones.     

Effects of anterior pituitary hormones on hepatic corticosterone metabolism in rats

Experiments were conducted to determine the effects of anterior pituitary hormones on hepatic corticosterone metabolism in rats. Hypophysectomy lowered A-ring reduction but did not affect sidechain metabolism. Administration of prolactin, FSH, LH or FSH + LH to hypophysectomized rats affected neither process. Similarly, ACTH or growth hormone, when given alone, did not affect corticosterone metabolism. However, combined treatment with ACTH and growth hormone significantly reduced the rate of ring A metabolism, suggesting that hormonal interactions may be important in the control of hepatic steroid metabolism.     

Localization of Carboxyl Ester Lipase in Human Pituitary Gland and Pituitary Adenomas

Carboxyl ester lipase (CEL) is an enzyme that hydrolyzes a wide variety of lipid substrates, including ceramides, which are known to show inhibitory regulation of pituitary hormone secretion in experimental models. Because no studies on CEL expression in human pituitary and pituitary adenomas have been reported in the literature, we investigated CEL expression in 10 normal pituitary glands and 86 well-characterized pituitary adenomas [12 FSH/LH cell, 17 α-subunit/null cell, 6 TSH cell, 21 ACTH cell, 11 prolactin (PRL) cell, and 19 GH cell adenomas] using IHC, immunoelectron microscopy, Western blotting, and quantitative RT-PCR. In normal adenohypophysis, CEL was localized in GH, ACTH, and TSH cells. In adenomas, it was mainly found in functioning GH, ACTH, and TSH tumors, whereas its expression was poor in the corresponding silent adenomas and was lacking in FSH/LH cell, null cell, and PRL cell adenomas. Ultrastructurally, CEL was localized in secretory granules close to their membranes. This is the first study demonstrating CEL expression in normal human pituitary glands and in functioning GH, ACTH, and TSH adenomas. Considering that CEL hydrolyzes ceramides, inactivating their inhibitory function on pituitary hormone secretion, our findings suggest a possible role of CEL in the regulation of hormone secretion in both normal and adenomatous pituitary cells. (J Histochem Cytochem 58:881–889, 2010)     

Validation of the mechanisms proposed for the stimulatory and inhibitory effects of progesterone on gonadotropin secretion in the estrogen-primed rat: a possible role for adrenal steroids.

The stimulatory and inhibitory effects of progesterone on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion were found to be dependent on the length of estrogen exposure in ovariectomized estrogen-primed rats. Progesterone suppressed LH and FSH secretion when administered 16 hours after a single injection of estradiol to ovariectomized rats. If the estradiol treatment was extended over 40 hours by two injections of estradiol 24 hours apart, progesterone administration led to a highly significant elevation of both serum LH and FSH levels 6 hours later. In addition to the direct stimulatory effect on LH and FSH release, progesterone, when injected 1 hour before, was able to antagonize the suppressive effect of a third injection of estradiol on LH and FSH release. In the immature ovariectomized estrogen-primed rat, 10 IU of ACTH brought about a release of progesterone and corticosterone 15 minutes later and LH and FSH 6 hours later. Progesterone, but not corticosterone, appeared to be responsible for the effect of ACTH on gonadotropin release. The synthetic corticosteroid triamcinolone acetonide brought about LH and FSH release in the afternoon, while cortisol, similar to corticosterone, was unable to do so. Nevertheless, triamcinolone acetonide and cortisol brought about increased secretion of FSH the following morning.     

Adenohypophyseal hormone response to chronic stress in dexamethasone-treated rats.

The influence of dexamethasone treatment on the basal values of corticosterone, GH, prolactin (PRL), LH and FSH, as well as on the adenohypophyseal hormone response to chronic stress was studied in female rats. Dexamethasone acetate (25 micrograms/100 b.w.), given by gavage twice daily for 10 days, decreased the resting plasma levels of corticosterone, GH, LH and PRL, whereas the FSH titers remained normal. The secretion of ACTH (evaluated indirectly through corticosterone concentrations) and of GH appeared to be most sensitive to the suppressive effect of dexamethasone. The same hormonal response pattern was induced by 8 h of daily immobilization for 10 days, except that ACTH release was enhanced and the plasma LH titers dropped more drastically. Dexamethasone administration in combination with restraint did not alter the characteristic hormonal profile of chronic stress, despite the fact that ACTH secretion was completely blocked. These data suggest that the inhibition of PRL, LH and GH secretion following severe, chronic stress is not causally related to the sustained elevation of plasma ACTH.     

Participation of voltage-sensitive calcium channels in pituitary hormone release

The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of rat anterior pituitary cells. The basal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH), and adrenocorticotropin (ACTH) secretion were independent of extracellular Ca2+ concentration ([Ca2+]e). In contrast, the basal levels of growth hormone (GH) and prolactin (PRL) release showed dose-dependent increases with elevation of [Ca2+]e, and were abolished by Ca2+-channel antagonists. Under Ca2+-deficient conditions, BaCl2 mimicked the effects of calcium on PRL and GH release but with a marked increase in potency, and also increased basal LH and FSH release in a dose-dependent manner. In the presence of normal [Ca2+]e, depolarization with K+ maximally increased cytosolic [Ca2+] ([Ca2+]i) from 100 to 185 nM and elevated LH, FSH, TSH, ACTH, PRL, and GH release by 7-, 5-, 4-, 3-, 2-, and 1.5-fold, respectively. These effects of KCl were abolished in Ca2+-deficient medium or in the presence of the Ca2+-channel antagonist, Co2+, and were diminished by the dihydropyridine Ca2+-channel antagonist, nifedipine. The Ca2+-channel agonist BK 8644 (100 nM) enhanced the hormone-releasing actions of 25 mM KCl upon PRL, LH, FSH, GH, TSH, and ACTH by 2.3-, 2.0-, 1.8-, 1.7-, 1.6-, and 1.4-fold, respectively. The dose- and voltage-dependent actions of BK 8644 were specific for individual cell types; BK 8644 enhanced GH, PRL, TSH, LH, and ACTH secretion in the absence of any depolarizing stimulus, with ED50 values of 8, 10, 150, 200, and 400 nM, respectively. However, in the presence of 50 mM KCl, the ED50 values for BK 8644 were 1.5, 2, 3, 5, and 7 nM for GH, PRL, ACTH, TSH, and LH, respectively. [3H]BK 8644 bound specifically to pituitary membranes with Kd values of 0.8 nM and concentrations of about 900 channels per cell. These observations provide evidence for the presence and participation of voltage-sensitive calcium channels in the secretion of all five populations of anterior pituitary cells.     

Cellular associations of pituitary gonadotrophs in a rodent (Lagostomus maximus maximus) with photoperiod-dependent reproduction

The morphological characteristics and percentage of the cellular associations between gonadotrophs (LH- and FSH-secreting cells) and other cellular types were studied in pituitary pars distalis of adult male viscachas (Lagostomus maximus maximus) by double immunohistochemistry using specific antibodies to LH, FSH, PRL, GH, ACTH, TSH and S-100 protein (by folliculostellate cells; FSC), during long and short photoperiods. Bihormonal gonadotrophs were observed in ventro-medial and dorsal regions, interspersed between monohormonal gonadotrophs, and their number increased in short photoperiod. LH- and FSH-gonadotrophs were found around lactotrophs, enclosed by somatotrophs in the dorsal region, and associated with irregular corticotrophs. Gonadotrophs and thyrotrophs were associated along blood vessels and follicular structures. The cytoplasmic prolongations of FSC were in contact with both gonadotrophs. The percentage of LH–FSH, LH–ACTH, LH–FSC, FSH–LH, FSH–PRL, FSH–GH, FSH–ACTH, FSH–TSH and FSH–FSC associations decreased, whereas LH–PRL increased in short as compared to long photoperiod. The most abundant associations were LH–GH and LH–TSH during long photoperiod, but LH–GH and LH–PRL during short photoperiod. FSH–GH and FSH–PRL were the most numerous associations, and LH–FSC and FSH–FSC were the less abundant ones in both photoperiods. These results provide the morphological evidence for specific cellular associations between gonadotrophs and other cellular types of viscacha pituitary.     

Discrimination of LH,FSH, TSH and ACTH in dissociated porcine anterior pituitary cells by light and electron microscope immunocytochemistry

Summary Using the PAP unlabelled antibody method, LH, FSH, TSH and ACTH were localized at light microscope level in cultured cells dissociated from the porcine adenohypophysis. Antisera were shown to be specific for the subunits of the porcine glycoprotein hormones by radioimmunoassay and absorption studies. Using these antisera, it was found that LH and FSH were contained within the same cell, with TSH in a separate cell. When absorbed with LH, anti-porcine ACTH stained a separate distinct population of ACTH cells.Adjacent ultra-thin sections stained with anti-pLH and anti-pFSH, and examined at electron microscope level, showed that the ovoid, 150–400 nm secretory granules of the LH/FSH gonadotrophs contained both LH and FSH.The authors gratefully acknowledge the technical assistance of Carole Smith and Adrian Walsh     

Immunohistochemical study on adenohypophysial primordia in organ culture

Summary Rathke's pouches isolated from rat fetuses on day 12 were maintained in organ culture for 9 days and investigated immunohistochemically to test whether or not the hypothalamus is involved in the cytodifferentiation of the adenohypophysis. The unlabeled antibody enzyme method demonstrated that the cultured tissue contains different types of glandular cells, i.e., adrenocorticotropin (ACTH)-, growth hormone (GH)-, luteinizing hormone (LH)-, thyrotropin (TSH)-, and prolactin-producing cells. Indirect evidence was also obtained to indicate the presence of melanocyte stimulating hormone (MSH)-cells. These findings suggest that adenohypophysial primordial cells of rats start to synthesize their respective hormones without stimuli from neurosecretory substances of the brain which are known to be essential for the maintenance of the secretory activity of the adult gland.We wish to express our thanks to Dr. A. Kawaoi for providing anti-porcine ^1–39ACTH, to Dr. S.S. Spicer for the supply of anti-porcine ^17–39ACTH and to Dr. P. Petrusz for the gift of antisera against bovine GH, bovine TSH, HCG and rat prolactin. We should also like to thank Mr. Y. Okamura for technical help and Mr. I. Shimada for preparation of the photographs.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Immunohistochemistry of the hypothalamic neuropeptides and anterior pituitary cells in the Japanese quail

The pars distalis of the avian adenohypophysis consists of well-defined cephalic and caudal lobes which are distinct in their cellular constituents. Immunocytochemical investigations on the pituitary hormones of the pars distalis of the Japanese quail reveal five types of secretory cells, adenocorticotropin (ACTH) cells, prolactin (PRL) cells, thyroid-stimulating hormone (TSH) cells, growth hormone GH (STH) cells, and FSH/LH (gonadotropic) cells. The ACTH cells, TSH cells, and PRL cells are restricted to the cephalic lobe, and GH (STH) cells are confined to the caudal lobe, while FSH/LH cells are distributed throughout the cephalic and caudal lobes. The median eminence of birds has distinct anterior and posterior divisions, each with different neuronal components. The avian hypophysial portal vessels also consists of two groups, anterior and posterior. The peculiar arrangement and distribution of the avian hypophysial portal vessels are possibly related to the distribution of neuropeptides in the two divisions of the median eminence and to the cytological and functional differentiation of two lobes of the pars distalis. The localization of perikarya and fibers containing luteinizing hormone releasing hormone (LHRH), somatostatin, vasotocin, mesotocin, corticotropin-releasing factor (CRF), vasoactive intestinal polypeptide (VIP), glucagon, metenkephalin, and substance P in the hypothalamus and median eminence of the Japanese quail has been investigated by means of immunohistochemistry using antisera against the respective neuropeptides. LHRH-, somatostatin-, VIP-, met-enkephalin-, and substance P-immunoreactive fibers are localized in the external layer of the anterior and posterior divisions of the median eminence, while CRF- and vasotocin-reactive fibers are demonstrated only in the external layer of the anterior division of the median eminence. The metenkephalin fibers are thicker in the anterior median eminence but the substance P fibers are more abundant in the posterior division. Mesotocin fibers occur only in the internal layer of the median eminence and neural lobe.     

Immunocytochemistry of the pituitary glycoprotein hormones.

The storage sites of the pituitary glycoprotein hormones were identified with the use of electron microscopic immunocytochemical techniques and antisera to the beta (beta) chains of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating hormone (TSH). The TSH cells in normal rats is ovoid or angular and contains small granules 60-160 nm in diameter. In TSH cells hypertrophied 45 days after thyroidectomy, staining is in globular patches in granules or diffusely distributed in the expanded profiles of dilated rough endoplasmic reticulum. The gonadotrophs (FSH and LH cells) exhibited three different morphologies. Type I cells are ovoid with a population of large granules and a population of small granules. Staining for FSHbeta or LHbeta was intense and specific only in the large granules (diameter of 400 nm or greater). Type II cells are angular or stellate and contain numerous secretory granules averaging 200-220 nm in diameter. They predominate during stages in the estrous cycle when FSH or LH secretion is high. Type III cells look like adrenocorticotropin (ACTH) cells in that they are stellate with peripherally arranged granules. They generally stain only with anti-FSHbeta and their staining can not be abolished by the addition of 100 ng ACTH. In preliminary quantitative studies of cycling females, we found that on serial sections FSH cells and LH cells show similar shifts to a more angular population of cells during stages of active secretion. However, the shifts are not in phase with one another. Furthermore, there are at least 1.5 times more FSH cells than LH cells at all stages of the cycle. Our collection of serial cells shows that some cells (usually type I or II) stain for both gonadotropic hormones, whereas others (usually type II or III) contain only one.     

Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in human and primate hypothalamus

A 38 residue neuropeptide was recently isolated from ovine hypothalamus in our laboratory, and named pituitary adenylate cyclase activating polypeptide (PACAP38) based on its biological activity. Rabbit antisera against synthetic PACAP27 were characterized by ELISA for immunohistochemical use. PACAP-immunoreactive neuronal elements having similar distributions were demonstrated in both human and spider monkey hypothalami. Many PACAP-immunoreactive cell bodies were present in the supraoptic and paraventricular nuclei. Immunopositive nerve fiber networks were stained throughout the hypothalamus, including in both external and internal zones of the tuber cinereum, close to the transition of the pituitary stalk (median eminence). These results suggest that PACAP plays multifunctional roles as a hypophysiotropic hormone, neurohypophysial hormone, neurotransmitter or neuromodulator in higher vertebrate species including man.     

Prepubertal rat ovary: hormonal modulation of beta-adrenergic receptors and of progesterone response to adrenergic stimulation

We have previously shown that, in the rat, ovarian beta-adrenergic receptor content varies during the time of puberty, with values first increasing and then decreasing abruptly on the afternoon of the first proestrus, i.e., at the time of the preovulatory surge of gonadotropins and prolactin (Prl). In the present study, experiments have been conducted to determine: 1) if hormones other than follicle stimulating hormone (FSH) that are known to be involved in regulating prepubertal ovarian function can mimic the facilitatory effect of FSH on progesterone (P) response of granulosa cells to beta-adrenergic stimulation; 2) if beta-adrenergic receptor content of granulosa cells is under hormonal regulation; and 3) whether the facilitatory effect of hormones on the P response to beta-adrenergic stimulation is due to an increased cyclic AMP response to receptor activation. A 48-h in vitro preexposure of granulosa cells from juvenile, 29-day-old ovaries to the pituitary hormones Prl, luteinizing hormone (LH), or FSH showed that only the latter was able to facilitate the subsequent P response to Zinterol, a beta2-adrenergic agonist. Follicle-stimulating hormone also increased basal P release. Of the two nonpituitary hormones examined, the luteinizing hormone-releasing hormone (LHRH) agonist D-(Ala6,Pro9)-LHRH-ethylamide (LHRH-A) failed to affect P responsiveness, whereas corticosterone enhanced both basal P release and P response to Zinterol. This effect was less pronounced than that of FSH. Luteinizing hormone, Prl and corticosterone decreased beta-adrenergic receptor content to different extents, with corticosterone being the most effective and LH the least (50% and 15% decrease, respectively); LHRH-A was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitory action of corticotropin-releasing hormone on gonadotropin secretion in the ovariectomized rhesus monkey is not mediated by adrenocorticotropic hormone

Earlier observations in our laboratory indicated that i.v. infusion of human/rat corticotropin-releasing hormone (hCRH) suppresses pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in ovariectomized rhesus monkeys. Since cortisol secretion increased significantly as well, it was not possible to exclude the possibility that this inhibitory effect of hCRH on gonadotropins was related to the activation of the pituitary/adrenal axis. The purpose of the present study was to determine the role of pituitary/adrenal activation in the effect of hCRH on LH and FSH secretion. We compared the effects of 5-h i.v. infusions of hCRH (100 micrograms/h, n = 7) and of human adrenocorticotropic hormone (ACTH) (1-24) (5 micrograms/h, n = 3; 10 micrograms/h, n = 3, 20 micrograms/h, n = 3) to ovariectomized monkeys on LH, FSH, and cortisol secretion. As expected, during the 5-h ACTH infusions, cortisol levels increased by 176-215% of baseline control, an increase similar to that observed after CRH infusion (184%). However, in contrast to the inhibitory effect observed during the CRH infusion, LH and FSH continued to be released in a pulsatile fashion during the ACTH infusions, and no decreases in gonadotropin secretion were observed. The results indicated that increases in ACTH and cortisol did not affect LH and FSH secretion and allowed us to conclude that the rapid inhibitory effect of CRH on LH and FSH pulsatile release was not mediated by activation of the pituitary/adrenal axis.     

The role of LH pulse frequency in ACTH-induced ovarian follicular cysts in heifers

The aim of the present study was to induce ovarian cysts experimentally in cattle using ACTH and to closely examine the role of LH pulse frequency in ovarian cyst formation. Five regularly cycling Holstein-Friesian heifers (15-18-month-old) were used. Ovaries were scanned daily using an ultrasound scanner with a 7.5 MHz rectal transducer. Daily blood samples were obtained via tail venepuncture for hormone analyses. Additional blood samples (for FSH and LH pulses) were obtained through an indwelling jugular vein catheters every 15 min for 8 h on Days 2 (early luteal phase; ELP), 12 (mid-luteal phase; MLP) and 19 (follicular phase; FP) of control estrous cycle and on alternate days during follicular cyst (FC) formation and persistence. Cysts were induced using subcutaneous injections of ACTH (Cortrosyn) Z; 1 mg) every 12 h for 7 days beginning on Day 15 of the subsequent estrous cycle. Plasma concentrations of progesterone (P4), estradiol-17beta, FSH and LH were determined by double antibody radioimmunoassay while cortisol concentration was determined by enzyme immunoassay (EIA). Ovarian follicular and endocrine dynamics were normal during the control estrous cycles. Ovarian follicular cysts were induced in four of the five heifers. Mean maximum size of cysts was larger (P<0.05) than that of ovulatory follicles (26.78+/-3.65 versus 14.1+/-0.90 mm), respectively. Cortisol levels were increased during ACTH treatment. High concentrations of estradiol and low progesterone were observed after cyst formation. LH pulse frequency was significantly reduced (P<0.05) during cyst formation and persistence compared to ELP (7.5+/-0.75) and FP (6.5+/-0.58), but was not significantly (P=0.23) different from MLP (2.8+/-0.29) pulses. Mean LH pulse amplitude and concentrations were not different. Similarly, the mean pulse frequency, amplitude and concentration of FSH were not different between control study and cystic heifers. These results suggest that the LH pulse frequency observed following ACTH treatment may interact with high estradiol concentration to induce ovarian cyst formation in heifers.     

Studies on human prolactin physiology

Although the clinical and experimental data were in favour of the existence of prolactin in humans like other vertebrates, as a pituitary hormone distinct from growth hormone, its presence remained contested until recent years. The predominant influence of the human hypothalamus on prolactin secretion is inhibitory. Circulating prolactin shows diurnal variations, which are not synchronized with that of TSH or ACTH; the prolactin rhythm is abolished during the last trimester of pregnancy and in patients with prolactin secreting tumors. Estrogens appeared to be less marked stimulators of prolactin secretion in man than in animals, although serum prolactin levels follow a pattern similar to that of endogenous estrogens during the normal menstrual cycle and during pregnancy. After delivery, basal prolactin levels declined progressively. In women under long term medroxyprogesterone acetate treatment, the immunoreactive serum prolactin was within the normal range of cycling women. Prolactin is found in appreciable amounts in amniotic fluid and in the serum of newborn infants. Synthetic LH and FSH releasing hormone did not change circulating prolactin levels in normal humans. A possible luteotrophic action of human prolactin in synergism with LH cannot be excluded.