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R. P. MILLAR

U.C.T./M.R.C. Protein Research Unit, Department of Chemical Pathology, Medical School, Observatory 7925, Cape Town, Republic of South Africa

(Received 26 January 1978)

Higher molecular weight (HMW) immunoreactive forms of somatostatin have been reported in extracts of ovine hypothalami (Vale, Ling, Rivier, Villarreal, Rivier, Douglas & Brown, 1976), rat pancreas and stomach (Arimura, Sato, Dupont, Nishi & Schally, 1975) and human pancreatic somatostatinoma (Larsson, Hirsch, Holst, Ingemansson, Kühl, Jensen, Lundquist & Rehfeld, 1977). However, the possibility that the HMW immunoreactive substances were oligomers of somatostatin or somatostatin bound to larger molecules was not excluded. The present study was undertaken to establish that authentic HMW immunoreactive somatostatin is present in the ovine hypothalamus and to glean information on the structural relationship of the HMW species with somatostatin.

Sheep hypothalami were extracted and subjected to gel permeation chromatography as described previously by Millar, Aehnelt & Rossier (1977). Fractions were assayed for somatostatin immunoreactivity

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JUDY A. KING and R. P. MILLAR

Ovine pineal gland extracts were examined throughout the year for content and molecular form of immunoreactive (IMR) LH releasing hormone (LH-RH). The content of IMR LH-RH in pineal glands collected in spring and summer was 160–2230 pg/gland, while the content in pineal glands collected in autumn and winter was lower (31–39 pg/gland). Pineal gland IMR LH-RH, purified by affinity and Sephadex G-25 chromatography, yielded displacement curves parallel to those of hypothalamic IMR LH-RH and synthetic LH-RH in radioimmunoassays employing four antisera which require different regions of the decapeptide for effective binding, suggesting considerable similarities in the structure of the molecule. The majority of pineal gland IMR LH-RH behaved identically to hypothalamic IMR LH-RH and synthetic LH-RH on gel filtration, cation exchange chromatography, high-pressure liquid chromatography and cellulose thin-layer chromatography. However, a small amount of a less positively charged LH-RH species was also present in all pineal gland extracts. Our findings indicate that hypothalamic decapeptide LH-RH occurs in the ovine pineal gland.

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J T Smith, A Roseweir, M Millar, I J Clarke and R P Millar

Kisspeptin signalling is indispensable for fertility, stimulating gonadotropin-releasing hormone (GnRH) secretion and mediating gonadal steroid feedback on GnRH neurons. Moreover, kisspeptin neurons have been implicated in other non-reproductive neuroendocrine roles. Kisspeptin appears to also regulate growth hormone secretion but much of the data appear contradictory. We sought to clarify a potential role of kisspeptin in growth hormone (GH) regulation by examining the effect of kisspeptin antagonists on GH secretion in ewes under various physiological conditions. Our data show clear and robust increases in GH secretion following lateral ventricle or third ventricle infusion of kisspeptin antagonists p-234 and p-271 in either ovariectomized or anestrous ewes. Central infusion of kisspeptin-10 had no effect on GH secretion. To determine the level at which kisspeptin may influence GH secretion, we examined expression of the cognate kisspeptin receptor, GPR54, in pituitary cells and showed by immunocytochemistry that the majority of somatotropes express GPR54 while expression was largely negative in other pituitary cells. Overall, we have demonstrated that blocking kisspeptin signalling by antagonists stimulates GH secretion in ewes and that this is likely mediated by inhibiting endogenous kisspeptin activation of GPR54 expressed on somatotropes. The findings suggest that endogenous kisspeptin inhibits GH secretion through GPR54 expressed on somatotropes.

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J. A. King, J. S. Davidson and R. P. Millar

ABSTRACT

The presence of two endogenous forms of gonadotrophin-releasing hormone (GnRH) in the chicken hypothalamus (chicken GnRH-I ([Gln8]GnRH) and chicken GnRH-II ([His5,Trp7,Tyr8]GnRH)), and the stimulation of gonadotrophins by both forms, suggests the possible existence of GnRH receptor subtypes and gonadotroph subtypes in the chicken pituitary. This question was investigated by assessing the effects of various combinations of the two known forms of chicken hypothalamic GnRH and antagonist analogues of GnRH on LH release from dispersed chicken anterior pituitary cells in both static and perifused systems. The relative inhibition of chicken GnRH-I-stimulated and chicken GnRH-II-stimulated LH release by 12 GnRH antagonists did not differ significantly, suggesting a single GnRH receptor type. Chicken GnRH-II was approximately sixfold more potent than chicken GnRH-I in releasing LH. Release of LH in response to maximal doses of chicken GnRH-I and chicken GnRH-II and to a mixture of both was similar and the two peptides were not additive in their effects, consistent with the presence of a single type of LH gonadotroph and a GnRH receptor which binds both forms of GnRH. Each form of GnRH desensitized cells to subsequent stimulation with the other form, providing additional evidence for a single type of LH gonadotroph. These findings suggest that chicken GnRH-I and -II stimulate gonadotrophin release through a single GnRH receptor type on a single class of LH gonadotroph in the chicken pituitary.

J. Endocr. (1988) 117,43–49

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G Majdic, M R Millar and P T K Saunders

Abstract

Androgens are required for the development of male internal and external genitalia. Androgen action is mediated by an intracellular receptor which acts as a transcription factor following activation by ligand binding. The aim of the present study was to define the time of appearance of androgen receptor (AR) in the male fetal rat gonad using immunohistochemistry. Intact fetuses (days 13·5–16·5) or testicular tissue (days 16·5–20·5 and days 3–7 postnatal) were fixed in Bouins' solution and processed into paraffin wax. On day 16·5 nuclear AR were present in mesenchymal cells surrounding the Wolffian duct but those around the Mullerian duct were receptor negative. During the following day (17–18) the abundance of nuclear staining increased, becoming detectable in the epithelial cells of the Wolffian ducts. Within the testis some nuclear staining was apparent at day 17 but was confined to interstitial cells surrounding the seminiferous cords. As development of the testis proceeded the abundance of nuclear AR in peritubular and elongated mesenchymal cells increased. AR were not detected in fetal Leydig cells expressing 3β-hydroxysteroid dehydrogenase nor in the ovaries or associated ducts of female fetuses at the same ages.

In conclusion, in the rat we have found AR expression detectable by immunohistochemistry in mesonephric mesenchyme to be confined to that underlying the Wolffian ducts and to be absent from the area around the degenerating Mullerian duct. On and after day 17 of gestation AR is present in Wolffian duct epithelial cell nuclei and within the testis it is confined to peritubular and interstitial cells which may have migrated from the mesonephros. Fetal Leydig cells were receptor negative. Within the seminiferous cords AR in Sertoli cells remained low until after birth and some perinuclear staining was detected in cells thought to be gonocytes. We believe this to be the first report of immunolocalisation of AR to fetal testicular interstitial cells.

Journal of Endocrinology (1995) 147, 285–293

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J. S. Davidson, I. Wakefield, J. A. King and R. P. Millar

ABSTRACT

Barium is known to elicit secretion in a number of cell systems. The mechanism of Ba2+ stimulation of LH release in cultured chicken pituitary cells was investigated in the present study. Barium-stimulated LH release was inhibited by extracellular Ca2+, indicating that Ba2+ does not act by stimulating Ca2+ entry. Simultaneous stimulation of the cells with Ba2+ and phorbol ester produced a synergistic response, similar to the synergism obtained with phorbol ester and treatments which increase cytosolic Ca2+. Both Ba2+-stimulated LH release and the synergism of Ba2+ with phorbol ester were inhibited by calcium channel blockers (Co2+, methoxyverapamil and nifedipine) and by calmodulin antagonists (trifluoperazine and chlorpromazine). These results indicate that the actions of Ba2+ are dependent on its entry through Ca2+ channels, and suggest that calmodulin activation is necessary for the synergism between Ba2+ and phorbol ester. Thus, synergism does not result from a direct effect of divalent cations on C-kinase.

J. Endocr. (1987) 114, 11–16

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T Sweeney, P T K Saunders, M R Millar and A N Brooks

Abstract

Anti-Mullerian hormone (AMH) and androgenic steroids are key factors regulating the masculinisation of the internal and external genitalia during fetal development. AMH is produced in Sertoli cells and causes regression of the Mullerian ducts in the male. 3β-Hydroxysteroid dehydrogenase (3β-HSD) is one of the key steroidogenic enzymes regulating testosterone production in Leydig cells. The objective of this experiment was to elucidate the development of the ovine fetal testes by identifying the spatio-temporal expression of AMH, 3β-HSD and androgen receptor expression within them. Fetuses from days 30 and 40 of gestation were fixed intact, while the gonads were dissected from the fetuses on days 70, 100 and 130 of gestation. Tissue was fixed in Bouin's fixative for 6 h, processed into paraffin wax and sections immunostained using rabbit anti-human AMH, 3β-HSD or androgen receptor antibodies.

While seminiferous cords were absent on day 30 of gestation, pre-cord organisation was apparent and the gonad could be clearly distinguished from surrounding tissue by the presence of AMH and 3β-HSD immunopositive cells. Androgen receptor expression was not apparent at this stage. By day 40 of gestation the testis was organised into distinct seminiferous cords and intense immunostaining for AMH and 3β-HSD was present in Sertoli cells within the cords and Leydig cells in the interstitium respectively. Androgen receptor immunopositive cells were present in the interstitium but cells destined to develop into rete testis were immunonegative. By day 70 of gestation, the rete testis was organised in the centre of the testis and was strongly androgen receptor immunopositive. AMH and 3β-HSD expression was present in Sertoli and Leydig cells respectively. The expression of AMH, 3β-HSD and androgen receptor in the 100 and 130 day gestation fetuses was similar to that identified in the 70 day fetuses. In conclusion, Sertoli and Leydig precursor cells are present in the gonad prior to seminiferous cord formation and contain AMH and 3β-HSD at all stages of gestation examined. While androgen receptor immunoexpression was present in nuclei of interstitial cells from day 40 of gestation and in the rete testis from day 70 of gestation, Sertoli cells were immunonegative for androgen receptor at all of the stages examined.

Journal of Endocrinology (1997) 153, 27–32

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P. V. Kaye, P. A. van der Merwe, R. P. Millar and J. S. Davidson

ABSTRACT

The mechanism of arachidonic acid (AA)-induced LH release was characterized using sheep pituitary cells in primary culture permeabilized with Staphylococcal α-toxin. In intact cells, exogenous AA evoked release of LH in a manner which was partially dependent on extracellular Ca2+. At similar concentrations, AA also caused cell permeabilization as monitored by efflux of [3H]2-deoxyglucose metabolites. In α-toxin-permeabilized cells where cytosolic Ca2+ was clamped at resting levels, AA retained its ability to cause LH release. Unlike the stimulation of exocytosis produced by Ca2+, phorbol ester or cyclic AMP, AA-evoked release was independent of ATP and was not inhibited by pretreatment with N-ethyl maleimide. These findings indicated that exogenous AA does not cause LH release by Ca2+ influx or mobilization or by activating protein kinase C. The results suggest that LH release induced by exogenous AA is probably due to its detergent-like properties, and does not represent true exocytosis.

Journal of Endocrinology (1992) 132, 77–82

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J S Fisher, M R Millar, G Majdic, P T K Saunders, H M Fraser and R M Sharpe

Abstract

The sites of action and the physiological role of oestrogens in the male reproductive tract are poorly understood. We have undertaken a systematic study of the immunoexpression of oestrogen receptor-α (ERα) in the male rat from late fetal life through to adulthood and compared the findings with results obtained in the marmoset monkey (Callithrix jacchus) from neonatal to adult life. The testes, rete testis, efferent ducts and epididymis were examined from normal male rats (aged 4, 8, 10, 15, 20, 25, 38, 48 and 90 days) and from male rat fetuses on days 17·5 and 18·5 of gestation; comparable tissues were examined from neonatal, infantile, peripubertal and adult marmosets aged 8, 18–24, 54–62 and 92–112 weeks respectively. Immunolocalisation of ERa used antigen retrieval and a monoclonal antibody directed to the N-terminus, which had proved superior to six other antisera tested. ERa was immunoexpressed in interstitial cells, including the fetal/neonatal generation of Leydig cells, in both the rat and marmoset. In the rat, the adult generation of Leydig cells were also immunopositive for ERa whereas the comparable cells in the marmoset were only weakly immunopositive. ERa was not expressed in Sertoli cells, peritubular myoid cells, blood vessels or germ cells at any time in either species. In late fetal life in the rat, ERa was immunoexpressed in cells surrounding the mesonephric tubules, whereas postnatally it was expressed in the epithelium of the rete testis and efferent ducts at all ages from 4 to 90 days; this immunoexpression was most pronounced in the efferent ducts. In the marmoset, the efferent ducts, but not the rete testis, also showed intense immunoexpression of ERa. Apart from sporadic immunostaining for ERa in the epididymal duct of the rat in the neonatal period, the caput, corpus and cauda epididymis were negative for immunoexpression of ERa at all ages in both species. These findings suggest that the main actions of oestrogens in the male reproductive tract, mediated by ERa, are related to the development and function of the efferent ducts and the Leydig cells. In consideration of data from this and previous studies of oestrogen binding, we predict possible sites of expression of other oestrogen receptors (e.g. ERβ) in Sertoli cells and the epididymis. Interactive effects, related to the relative levels of androgens and oestrogens, could be physiologically important in the excurrent ducts of the adult testis.

Journal of Endocrinology (1997) 153, 485–495

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K. B. Smith, M. R. Millar, A. S. McNeilly, P. J. Illingworth, H. M. Fraser and D. T. Baird

ABSTRACT

The localization of inhibin α-subunit within the human corpus luteum was investigated. The antiserum used was raised in sheep against the first 1–23 amino acid sequence of the N-terminus of the human inhibin α-subunit. Using the avidin-biotin immunoperoxidase technique, intense immunostaining was localized within the granulosa-lutein cells of the corpus luteum, with absence of staining in the theca-lutein cells and surrounding ovarian tissue. Similar distribution of inhibin α-subunit immunostaining was observed in 12 corpora lutea obtained during the early, mid- and late-luteal phases and no changes in intensity were apparent at these different stages. Negative controls were obtained by applying antiserum which had been preabsorbed overnight with excess inhibin peptide in place of primary antiserum and also normal non-immune sheep serum as a substitute for primary antiserum. These results provide further evidence that the human corpus luteum is a significant source of immunoreactive inhibin during the normal human menstrual cycle. The specific localization within the granulosalutein cells of the corpus luteum suggests that inhibin α-subunit production may originate from a discrete cell population within the human corpus luteum.

Journal of Endocrinology (1991) 129, 155–160