Three gonadotropin releasing hormones, seabream GnRH (GnRH-I), chicken GnRH-II (GnRH-II) and salmon GnRH (GnRH-III) cDNAs were isolated from the brain of the red drum, Sciaenops ocellatus. The GnRH cDNA sequences of red drum showed more similarity to those of Atlantic croaker, sea bass and sea bream. The real-time quantitative RT-PCR study revealed expression of GnRH-I and GnRH III mRNAs in the olfactory bulb plus telencephalon (OB+TEL), and preoptic-anterior hypothalamic area (POAH), indicating an overlap of the GnRH-I and GnRH-III systems in these forebrain regions. However, GnRH-II mRNA expression was detected only in the midbrain tegmentum (MT). The GnRH-I mRNA expression in the POAH in fish undergoing gonadal recrudescence was significantly higher than that in gonadally immature individuals, suggesting involvement of the POAH GnRH-I in gonadal maturation. On the other hand, GnRH-III mRNA expression was significantly higher in the OB+TEL region compared with that in the POAH. Moreover, the demonstration of GnRH-I mRNA and peptide expression in red drum lymphocytes indicates that the GnRH-I is synthesized in these cells of the immune system similar to the situation in mammals.
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F. Jockenhövel, S. A. Khan, and E. Nieschlag
Serum FSH levels in fertile and infertile men were determined by applying the Sertoli cell in-vitro bioassay and six different immunoassays. Bioassay and immunoassay estimates were significantly correlated (r ranging from 0·78 to 0·86; P<0·01). On average, all immunoassays measured lower FSH concentrations in samples with low FSH levels and higher FSH concentrations in those with high FSH levels compared with the bioassay. Ratios of bioactivity to immunoreactivity (B/I) were highest in fertile men and lowest in men with severe disturbances of testicular function. Depending on which immunoassay was used these differences were either significant or only marginal. Dose–response characteristics for WHO FSH standard preparation 78/549, used in the bioassay as well as in the immunoassays, were different between immunoassays and the bioassay, suggesting that decreasing B/I ratios with increasing FSH serum levels were method-related and reflected different slopes of the dose–response characteristics of the assays, rather than being true changes in the molecular composition of FSH. The present investigation underlines the necessity of choosing the immunoassay used for comparison with the bioassay carefully and of validating the system in regard to parallelism between dose–response characteristics. B/I ratios must be interpreted with great caution and previous studies which report changing B/I ratios in various endocrine situations may have to be reevaluated.
Journal of Endocrinology (1990) 127, 523–532
J. G. Schofield, A. I. Khan, and A. Wood
Acetylcholine is known to stimulate the secretion of growth hormone and prolactin and the efflux of 86Rb from bovine anterior pituitary cells: dopamine prevents the stimulation of 86Rb efflux and of prolactin but not growth hormone secretion. The sensitivity of these responses to pertussis toxin has been determined.
Treatment of bovine anterior pituitary cells in primary culture with pertussis toxin (18 h, 100 ng/ml) did not modify the stimulation of prolactin secretion by acetylcholine, but prevented its inhibition by dopamine. In lactotrophs, dopamine but not acetylcholine receptors are therefore coupled to secretion through a pertussis toxin substrate. The stimulation of 86Rb efflux by acetylcholine was also unaffected by pertussis toxin and, again, its inhibition by dopamine was prevented.
Treatment of the cells with pertussis toxin enhanced the secretion of growth hormone in response to acetylcholine. Nitrendepine (1 μmol/l) prevented the cholinergic stimulation of growth hormone but not prolactin secretion from these cells. Acetylcholine increased the cytoplasmic calcium concentration and this rise was enhanced by treatment of the cells with pertussis toxin. Nitrendepine partially inhibited the rise in calcium caused by acetylcholine, and prevented the enhancement of the rise following pertussis toxin treatment.
Cholinergic stimulation of growth hormone therefore depends on calcium entry through nitrendepine-sensitive channels, whereas stimulation of prolactin secretion does not, and in somatotrophs a pertussis toxin substrate may limit calcium entry through these channels. These different sensitivities of somatotrophs and lactotrophs to pertussis toxin and nitrendepine may reflect differences in the properties of the predominant calcium currents in the two cell types.
J. Endocr. (1988) 116, 393–401
MUSTAQ A. KHAN, W. M. DICKSON, and K. M. MEYERS
Exposure of newborn Holstein and Jersey calves to −4 °C did not significantly increase the plasma corticosteroids (cortisol and corticosterone) concentrations compared with calves kept at 16 °C. Two Holstein calves exposed to −12 °C showed a slight decrease of plasma corticosteroid concentrations and one Holstein calf at −18° C responded with a marked increase in both hormones during cold exposure. In the animals at 16 and −4 °C the plasma cortisol and corticosterone concentrations fell steadily during the sampling period. There was also a marked, and almost linear, decrease in the packed cell volume during the sampling period; this occurred in all groups. That this was not due entirely to the withdrawal of blood was shown by a similar decrease in two calves from which only small quantities of blood had been taken. Thus, the decrease in plasma corticosteroids may have resulted to some extent from haemodilution.
An increase in glucose concentration was observed in both the control and cold-exposed calves. There was no correlation between the changes in plasma glucose and plasma corticosteroids.
A A Al-Habsi, A Y A AlKindi, I Y Mahmoud, D W Owens, T Khan, and Aisha al-Abri
Circulating estradiol (E2), progesterone (Pro), testosterone, and corticosterone (B) levels were monitored in the green turtles Chelonia mydas during different nesting phases. Successful nesting includes emergence from sea, chamber and nest excavation, oviposition, burying the nest, and returning to sea. Unsuccessful nesting includes chamber and nest excavations but without oviposition. Blood samples were taken from the cervical sinus and collected within 5-min of capture to minimize stress. The samples were collected between 2000 and 0100 h during the peak season (May–October). High-performance liquid chromatography using a u.v. detection system coupled with tandem quadrupole mass spectrometry was used to measure B. Plasma B levels were significantly higher in successful and unsuccessful phases over emergence and excavation phases. However, B levels in successful versus unsuccessful or emergence versus excavation phases were not significantly different. Plasma steroid levels were measured by the Coat-A-Count RIA technique. Pro levels were significantly higher (P<0.005) in successful over unsuccessful turtles and also successful turtles over turtles in the other phases (P<0.01). The Pro levels immediately after nesting were found to be higher than that reported previously. Plasma testosterone values were higher in successful turtles but not significantly different from the turtles in other phases. Estrogen levels were undetected in all phases. Overall, the hormone values during different phases of nesting may play a major role in formulating the nesting behavior and physiology of the nesting activities in the green turtle.
N A Khan, N Wiernsperger, V Quemener, and J-P H Moulinoux
In this study, metformin (N, N1 dimethylbiguanide) was found to potentiate insulin-induced Xenopus laevis oocyte maturation, a phenomenon of transition from late G2 to M phase of the cell cycle. These cells also accumulated exogenous metformin (130 ± 6·5 nmol/oocyte). Metformin covalently-coupled to Sepharose 4B beads failed to potentiate the insulin-induced oocyte maturation which suggests that these cells did not take up metformin from the extracellular medium. Addition of metformin alone to Xenopus laevis oocytes did not induce the maturation process, though these cells took up exogenous metformin. Micro-injection of metformin (120 nmol/oocyte) to oocytes accelerated the insulin-induced maturation, but it was lower than in cells which were incubated with free metformin together with insulin. Interestingly, insulin had no effect on metformin uptake by the oocytes. Methylglyoxal-bis(guanylhydrazone), MGBG, an apparent analogue of metformin, induced oocyte maturation. Addition of metformin, either free or Sepharose-bound, did not influence the MGBG-induced 60% maturation of Xenopus laevis oocytes. These results suggest that the internalization of metformin is necessary for its action and its effects are specific on insulin activity.
Journal of Endocrinology (1994) 142, 245–250
P. L. Storring, S. A. Khan, Y. G. Mistry, and E. Diczfalusy
The LH biological potency of the International Reference Preparation (IRP) of Human Pituitary LH for Immunoassay (IRP 68/40) relative to that of the 2nd IRP of Human Pituitary FSH and LH for Bioassay (IRP 78/549) is markedly greater when estimated by in-vitro interstitial cell testosterone production (TICT) bioassay than by in-vivo bioassay, and by the 4-h ovarian ascorbate depletion (OAAD) assay than by the 4-day seminal vesicle weight gain assay. Other preparations of human LH which, like IRP 68/40, were highly purified, showed a similar spectrum of bioactivity in these assay systems and also contained a higher proportion of more basic LH isoforms than are found in crude pituitary extracts such as IRP 78/549.
In an attempt to explain these differences, a comparison was made of the plasma survival in rats of the LH bioactivity (by TICT assay) of these two preparations. Contrary to expectation, their relative plasma clearance rates over a 4-h period did not account for their differing bioactivities. The plasma half-life of the LH bioactivity (with 95% confidence limits) was estimated to be 42·4 (35·3–49·5) min for IRP 68/40 and 41·3 (31·5–51·0) min for IRP 78/549.
Furthermore the time-course of action in vivo of IRP 78/549 did not appear to be more prolonged than that of IRP 68/40. Thus their plasma testosterone responses during the course of these 4-h plasma clearance studies were similar, and estimates of the LH potency of IRP 68/40 relative to that of IRP 78/549 were no greater by 2-h than by 4-h OAAD assay.
The more basic isoforms of LH present in IRP 68/40 and in other purified human LH preparations may therefore differ from those in crude pituitary extracts, such as IRP 78/549, in their intrinsic activity to stimulate the different target cells in these assay systems rather than in their bioavailability in vivo.
J. Endocr. (1988) 119, 327–334
Saad A Amer, Nadia G Alzanati, Avril Warren, Rebecca Tarbox, and Raheela Khan
The purpose of this study was to investigate androgen production and the role of insulin and LH in its regulation in subcutaneous adipose tissue (SAT) of women with polycystic ovarian syndrome (PCOS). Protein and mRNA expression of androgen synthesis enzymes (cytochrome P450 17A1 (CYP17A1) and aldo-keto reductase 1C3 (AKR1C3)) were measured in SAT biopsies from women with PCOS, diagnosed according to the Rotterdam criteria (n = 15) and healthy controls (n = 15). Cultured mature adipocytes (differentiated from SAT biopsies) were treated with insulin ± phosphoinositol-3-kinase inhibitor (LY294002) or LH ± insulin. CYP17A1 and AKR1C3 mRNA expression and testosterone concentrations were measured in treated and untreated adipocyte cultures. AKR1C3 mRNA was significantly (P < 0.001) greater in PCOS vs non-PCOS SAT, but CYP17A1 was not significantly different between the two groups. AKR1C3 and CYP17A1 protein expression was not significantly different in PCOS vs non-PCOS SAT. In untreated adipocyte cultures, CYP17A1, AKR1C3 and testosterone levels were significantly higher in the PCOS vs the non-PCOS groups. Addition of insulin increased AKR1C3 mRNA and testosterone levels, but not CYP17A1 mRNA in non-PCOS with no effect on PCOS adipocytes. The stimulatory effects of insulin were not inhibited by LY294002. Addition of LH increased CYP17A1, AKR1C3 and testosterone in non-PCOS adipocytes with no effect in PCOS adipocytes. In conclusion, SAT of women with PCOS produces excess androgen, which may contribute to PCOS-related hyperandrogenaemia. This SAT androgen excess is independent of obesity and is not directly stimulated by inulin or LH.
R. J. FRANKEL, J. S. JENKINS, J. J. WRIGHT, and M. U. A. KHAN
The lateral hypothalamus, and various sites within the limbic system and frontal lobe of the rhesus monkey brain were electrically stimulated using chronically implanted electrodes. A considerable increase in plasma aldosterone levels was observed after stimulation of the lateral hypothalamic area, certain localized sites in the cingulate area, and lower medial parts of the frontal lobe. Inactive sites included most of the amygdala, hippocampus, and basal ganglia, together with other areas within the frontal lobe and cingulate gyrus. Stimulation of all active areas was followed by an increase in plasma renin activity. Plasma cortisol also increased considerably after hypothalamic stimulation but in the case of extra-hypothalamic sites the cortisol response was much less.
J. S. JENKINS, R. J. FRANKEL, J. J. WRIGHT, and M. U. A. KHAN
The increase in aldosterone and plasma renin activity (PRA) observed after stimulation of extrahypothalamic sites within the brain of the rhesus monkey was prevented by the prior administration of the β-adrenergic blocking agent propranolol. α-Adrenergic blockade by phentolamine had no inhibiting effect. Propranolol only partially reduced the response of aldosterone to lateral hypothalamic stimulation in spite of inhibition of PRA; a partial reduction in aldosterone was also obtained from this site after dexamethasone treatment without any effect on PRA. It was concluded that the increase in aldosterone observed after extra-hypothalamic stimulation was mediated mainly through the renin-angiotensin mechanism whereas in the case of the hypothalamus, release of ACTH was also a contributory factor.