Proceedings of the New Zealand Society of Endocrinology
Annual Meeting held as part of the
MedSciNZ Congress
30 Nov - 3 Dec 2004
Millennium Hotel, Queenstown, New Zealand
Contents:
NZ01 2004 Nancy Sirett lecture: Oocyte-related genes affecting ovulation rate K.P.McNatty
NZ02 Characterisation of the pseudopregnant rat as a model of pregnancy-induced leptin resistance. R.A. Augustine, S.J. Bunn, D.R. Grattan
NZ03 Pregnancy-induced leptin resistance is not associated with reduced activation of the JAK/STAT3 signalling pathway in POMC neurons. S.R. Ladyman, D.R. Grattan
NZ04 Dietary phytoestrogen exposure reduced fertility of male rats. A. Glover, H.D. Nicholson, S.J. Assinder
NZ05 Expression of estrogen and progesterone receptors within tuberoinfundibular dopaminergic (TIDA) neurons during diestrus, pregnancy and lactation. F.J. Steyn, G.M. Anderson, D.R. Grattan
NZ06 The effects of 11-ketotestosterone on hypophysectomized short-finned eels (Anguilla Australis). M. Algie, K.A.N. George, S.L. Divers, P.M. Lokman
NZ07 Advancement of seasonal reproductive activity in stoats by use of artificial lighting. D.N. McLane, J.A. Duckworth, S. Scobie, J.E. Turner, G.K. Barrell
NZ08 Evidence for paracrine actions of oxytocin in the rat adrenal gland. B. Huang, S.J. Bunn, S.J. Assinder
NZ09 Placental ABC transporter expression: Regulation and implications for trophoblast cell survival. D.A. Evseenko, J.W. Paxton, J.A. Keelan
NZ10 Osteoprotegrin (OPG) mutations that cause idiopathic hyperphosphatsia impair OPG protein secretion and biological activity. D. Naot, C.A. Middleton-Hardie, H. Cundy, I.R. Reid, T. Cundy, J. Cornish
NZ11 Cardiotrophin-1: Circulating levels, cardiac secretion and molecular forms in cardiovascular disease. C.J. Pemberton, S.D. Raudsepp, T.G. Yandle, A.M. Richards
NZ12 Influence of sex hormones and angiotensin II on secretion of adrenomedullin from individual human endothelial cells. L.J. Pearson, C. Rait, K. Wright, T.G. Yandle, J.J. Evans
NZ13 Mechano-growth factor (MGF) reduces ischaemic injury in acute myocardial infarction. K.G. Matthews, C.D. McMahon, G.P. Devlin, P.H. Goldspink, S.Y. Yang, J.V. Conaglen, J.J. Bass, G. Goldspink
NZ14 Erythropoietin enhances cardiac contractility and secretion of vasoactive factors and protects against ischemia-reperfusion injury. J. Piuhola, C.J. Pemberton, J.I. Keenan, M.B. Hampton, A.M. Richards
NZ15 Reactive oxygen species (ROS) may initiate cachexia. S.J. Falconer, J.M. Oldham, F. Jeanplong, K.G. Matthews, C.D. McMahon
NZ16 Nuclear expression of §C-activin in ovarian cysts from mice with varying ovulation number. M.J. Richardson, C.R. BeaugiŽ, H.J. McQuillan, J.S. Fleming
NZ17 Can a model describing follicle growth tailor individual gonadotrophin treatments to women presenting for ART? A.J. Peterson, T.K. Soboleva, J. Peek, A.B. Pleasants
NZ18 Gonadal steroid hormones during early development in the brushtail possum. D.C. Eckery, G. Shuttleworth, B. Mester, B.P. Thomson
NZ19 Expression of mRNA encoding steroidogenic receptors in the developing brushtail possum ovary. L.J. Haydon, D.C. Eckery, B.P. Thomson, J.L. Juengel
NZ20 Modelling peptide interactions and the involvement of CAMP in the regulation of luteinising hormone. T.J. Connolly, J.J. Evans, D.J.N. Wall
NZ21 Effect of changing the ratio of androgen:oestrogen in normal and malignant epithelial prostate cells. K.J. King, H.D. Nicholson, S.J. Assinder
NZ22 Interferon-a stimulation of bovine adrenal medullary chromaffin cells results in phosphorylation and nuclear translocation of STAT-1, 2 and Ð3 protein.s S.J. Bunn, S. Douglas
NZ23 Do suppressors of cytokine signalling (SOCS) proteins cause hyperprolactinaemia during late pregnancy and lactation? G.M. Anderson, D.R. Grattan
NZ24 Prolactin alters STAT5a but not STAT5b signalling in GnRH neurons. M.A. Fenwick, F.Y. Ma, D.R. Grattan, G.M. Anderson
NZ25 Maternal nutrition during pregnancy determines responsiveness to peripheral leptin treatment in offspring. S.O. Krechowec, M.H. Vickers, A. Gertler, B.H. Breier
NZ26 Lysophospholipids: Localisation of receptors and activities in human placent and Bewo choriocarcinoma cells. E. Okuda, J.A. Keelan
NZ27 Effects of chronic pulsatile growth hormone (GH) infusion to the growth-restricted fetal sheep on mRNA levels of iGF-I and of the GH and IGF type 1 receptors. H.H. Phua, F.H. Bloomfield, M.K. Bauer, J.E. Harding
NZ28 A form of obesity independent of insulin resistance is determined by maternal nutrition during pregnancy. N.M. Thompson, M.H. Vickers, S.O. Krechowec, J. Miles, R. Shankar, B.H. Breier
NZ29 Cell proliferation in the epithelial compartments of mouse ovaries of varying age and ovulation number, measured by bromodeoxyuridine incorporation. C.R. BeaugiŽ, H.J. McQuillan, J.S. Fleming
NZ30 Pharmacogenomics and infertility treatment in polycystic ovarian syndrome. A.J. Umbers, N. Johnson, J. Falkiner, A.N. Shelling
NZ31 A new breed of fox hunting: Breast cancer and the forkhead dynasty. R.B. Sprague, K.J. Woad, W.J. Smale, A.N. Shelling
NZ32 Boys, barbeques, baked beans and babies. E. Podivinsky, B.M. Thompson
NZ33 Testicular oxytocin receptor expression is increased in oestrogen receptor alpha knockout mice. M. Gould, H.D. Nicholson
NZ34 Proteomic analysis of changes in structure and viability of fetal membranes overlaying the Cervical os prior to membrane rupture at term. J.A. Keelan, B. Nijmeijer, E. Parry, P. Stone
NZ35 Effects of leptin on eel (Anguilla Australis) previtellogenic oocyte growth in vitro. S.L. Divers, A.R. McLean, P.M. Lokman
NZ36 11-Ketotestosterone induces in vitro growth of previtellogenic oocytes of eel (Anguilla Australis). P.M. Lokman, K.A.N. George, G. Young
NZ37 Identifying the mechanism of a novel genetic mutation affecting fecundity in sheep. E.S. Feary, J.L. Juengel, P. Smith, B.J. Mcleod, P.A. Farquhar, G.H. Davis, K.P. McNatty
Abstracts:
NZ01 2004 NANCY SIRETT LECTURE
OOCYTE-RELATED GENES AFFECTING OVULATION RATE
K.P.McNatty
AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt,
New Zealand
Ovulation rate in mammals is determined by a complex exchange of hormonal signals between the pituitary gland and the ovary and by a localised exchange of hormones within ovarian follicles between the oocyte and its adjacent somatic cells. From examination of inherited patterns of ovulation rate in sheep, several breeds have been identified with point mutations in two growth factor genes and a related receptor (i.e. ALK6 otherwise known as BMPRIB) that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9B) gene, one in GDF9 gene and one in ALK6. Animals heterozygous for any of the aforementioned mutations, heterozygous for the above GDF9 mutation as well as one of the BMP15 mutations, homozygous for the above ALK6 mutation, heterozygous for the ALK6 as well as heterozygous for one of the BMP15 mutations, have higher ovulation rates (i.e. +0.6-10) than their wild-type contemporaries. In contrast, those homozygous for any of the aforementioned five BMP15 or GDF9 mutations are sterile due to arrested follicular development from the primary (type 2 stage) of growth. The BMP15 and GDF9 mutations are thought to result in reduced levels of mature protein or altered binding to cell-surface receptors. In sheep, GDF9 mRNA is present in germ cells before and after ovarian follicular formation as well as throughout follicular growth. In contrast, BMP15 mRNA is found in oocytes only from the primary stage of growth. Both GDF9 and BMP15 proteins are present in follicular fluid indicating that they are secreted products. In sheep, ALK6 together with related cell-surface receptors such as ALK5 and BMPRII mRNA are present in oocytes at most, if not all, stages of follicular growth. In granulosa cells, BMPRII mRNA is present from the type 1 (primordial) stage of growth, whereas ALK6 and ALK5 are present from the type 2 and 4 stages of growth. Imunisation studies with GDF9 or BMP15 peptides show that both growth factors are essential for ovarian follicular development and normal ovulation and/or corpus luteum formation in sheep. In sheep with mutated ALK6, ovarian follicles undergo precocious maturation leading to 3-7 follicles ovulating at smaller follicular diameters without any increase above wild-types in the ovarian secretion of steroids or inhibins. One important consequence of the mutated ALK6 receptor appears to be a decreased ability of some BMPs to inhibit differentiation of follicular cells. Current findings in sheep suggest that BMP15, GDF9 and ALK6 are targets for new methods of fertility regulation in some mammals.
NZ02
CHARACTERISATION OF THE PSEUDOPREGNANT RAT AS A MODEL OF PREGNANCY-INDUCED LEPTIN RESISTANCE
R.A. Augustine, S.J. Bunn, D.R. Grattan. Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
During pregnancy, food intake increases resulting in increased deposition of adipose tissue, with a consequent increase in plasma leptin. Pregnant rats become resistant to the satiety action of leptin, allowing food intake and body weight to continue increasing. We hypothesised that the hormones of pregnancy may be inducing leptin resistance in the rat. The aim of this experiment was to examine whether pseudopregnancy, induced by a sterile mating, could adequately mimic the hormonal changes of pregnancy, thereby serving as a model to investigate hormonal regulation of leptin sensitivity. Serial blood samples were taken from pseudopregnant and cycling rats via an indwelling jugular cannula at 1000, 1700, 2200 and 0300 hours. Plasma prolactin and leptin concentrations were measured by radioimmunoassay (RIA). Terminal blood samples were collected from diestrous controls and day 3, 6 and 9 pseudopregnant rats, sera removed and progesterone and estradiol measured by RIA. Leptin was administered via an intracerebroventricular cannula in fasted pseudopregnant and cycling rats and food intake measured over 24 hours. Like pregnancy, pseudopregnancy was characterised by twice-daily prolactin surges. These surges have a luteotrophic role, maintaining pregnancy-like, high progesterone levels during pseudopregnancy. Unlike pregnancy, however, there was no significant increase in plasma leptin levels during pseudopregnancy. Furthermore, pseudopregnant rats respond to exogenous leptin by reducing food intake, suggesting that they do not become leptin resistant. These data suggest pseudopregnancy does not completely model the metabolic conditions of pregnancy, and some other aspect of pregnancy, such as placental hormones, must contribute to induction of central leptin resistance seen in the pregnant rat.
NZ03
PREGNANCY INDUCED LEPTIN RESISTANCE IS NOT ASSOCIATED WITH REDUCED ACTIVATION OF THE JAK/STAT3 SIGNALLING PATHWAY
S.R. Ladyman, D.R. Grattan. Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
Pregnancy is characterized by hyperphagia despite elevated concentrations of the satiety hormone, leptin. Furthermore, intracerebroventricular (i.c.v.) leptin administration does not suppress food intake during pregnancy, as it does in non-pregnant rats, indicating a state hypothalamic leptin resistance. Leptin acts in the hypothalamus through signal transduction mechanisms involving the phosphorylation of STAT3 proteins. Previously, we have observed a suppression in the amount of leptin-induced STAT3 activation in the arcuate nucleus during pregnancy, yet no overall change in the number of leptin responsive neurons compared to non-pregnant rats. Arcuate nucleus pro-opiomelanocortin (POMC) neurons produce the anorectic peptide alpha-MSH and are key mediators of the satiety action of leptin. To further investigate the activation of leptin-responsive neurons in the arcuate nucleus during pregnancy, we examined whether the POMC neurons have a reduced response to leptin, which may contribute to the leptin-induced hyperphagia. Pregnant and non-pregnant rats were fasted overnight to suppress endogenous leptin concentrations, then injected i.c.v. with leptin (4 ug) or vehicle. Thirty minutes later rats were anaesthetised and perfused with 2% paraformaldehyde. Brains were then processed for immunohistochemistry. The number of alpha-MSH positive cells colocalised with leptin-induced pSTAT3 in the arcuate nucleus was compared in pregnant and non-pregnant rats. In both groups, leptin induced pSTAT3 expression in approximately 70% of the alpha-MSH neurons, indicating that POMC neurons are responsive to leptin during pregnancy. Therefore it is unlikely that a failure of the leptin signal to activate anorectic POMC neurons mediates the pregnancy-induced leptin resistance and hyperphagia. Impairments in downstream events in leptin signaling cascades, however, can not be ruled out.
NZ04
DIETARY PHYTOESTROGEN EXPOSURE REDUCES FERTILITY OF MALE RATS
A. Glover, H.D. Nicholson, S.J. Assinder
Andrology Research Group of Otago, Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
Phytoestrogens are plant-derived compounds that are particularly abundant in soy-based foods. Exposure to exogenous oestrogenic chemicals has been implicated in declining male fertility. The aim of this study is to deduce whether adult phytoestrogen exposure affects the reproductive function of male rats, and by what mechanisms phytoestrogens may be acting. Six male rats were transferred from a low soy diet (control) to an experimental high soy diet, while 9 males remained on the control diet. On days 3, 6 and 12 all males were mated and litter sizes recorded. A second group of male rats kept on the same dietary regimen were killed after 3, 6 and 12 days on the diets. Real-time PCR was performed to measure mRNA quantities of oxytocin (OT), oxytocin receptor (OTR), oestrogen receptors a (ERa) and _ (ER_), and the androgen receptor (AR). The average litter size following 3 days on the high soy diet was significantly lower than that for rats maintained on the control diet. Litter sizes returned to control levels by day 12. Following 3 days on the high soy diet, ERa and AR mRNA levels increased in the initial segment of the epididymis, while ERa, AR and OTR decreased in the cauda. Short-term exposure to high phytoestrogen levels transiently reduces male fertility, and may involve disruption of hormone receptor expression. The mechanisms by which such disruptions alter fertility are being investigated. The changes in OTR, ERa and AR mRNA levels indicate differential gene regulation between distinct regions of the epididymis.
NZ05
EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS WITHIN TUBEROINFUNDIBULAR DOPAMINERGIC (TIDA) NEURONS DURING DIESTRUS, PREGNANCY AND LACTATION
F.J. Steyn, G.M. Anderson, D.R. Grattan
Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
During late pregnancy, the activity of TIDA neurons (which suppress prolactin secretion) is reduced, resulting in a state of hyperprolactinemia. The reduction in TIDA activity may be mediated by the changes in levels of estrogen and progesterone at this time. The aim of this study was to determine whether ovarian steroid receptors are expressed in TIDA neurons, and whether levels of expression change during late pregnancy and lactation. We perfused animals for brain collection on the morning of diestrus, day 12, 19 and 21 of pregnancy and on day 5 of lactation. Brains were sectioned at 40µm and serial sections containing the arcuate nucleus were stained via dual label peroxidase immunohistochemistry for coexpression of either estrogen receptors (ERa) or progesterone receptors and tyrosine hydroxylase (a marker for TIDA neurons). Both estrogen and progesterone receptors were expressed in TIDA neurons at all times. There was a significant increase in estrogen receptor coexpression during all stages of pregnancy compared to diestrus and lactating animals (mean increase 27%, p<0.05). In contrast, progesterone receptor coexpression was similar across diestrus and pregnancy, however a significant decrease was observed in lactating compared to diestrus (25.9% decrease) animals. Results show that estrogen receptors are increased within TIDA neurons during pregnancy, and thus could mediate direct actions of circulating estrogen at this time. The decrease in progesterone receptor expression observed between pregnancy and lactation is in accordance with prior work. Progesterone receptor expression would be expected to wane during lactation, a time when circulating estrogen levels are low, as its expression is dependent on circulating estrogen levels.
NZ06
THE EFFECTS OF 11-KETOTESTOSTERONE ON HYPOPHYSECTOMIZED SHORT-FINNED EELS (ANGUILLA AUSTRALIS)
M. Algie, K.A.N. George, S.L. Divers, P.M. Lokman
Department of Zoology, University of Otago, Dunedin, NZ
The main fish androgen, 11-ketotestosterone (11KT), has been found to play an influential role in the metamorphosis of female anguillid eels. Adult metamorphosis prepares anguillids for a long-distance marine spawning migration. Changes in oocyte, heart, gut, liver and eye size are all observed before the freshwater eels migrate. Previous studies have implicated 11-KT in controlling these metamorphic changes. In this study, hypophysectomy has been used to answer whether or not the action of 11KT is direct. Nine animals were hypophysectomized and 17 sham-operated. Steroid-treated animals received pellets containing 5mg 11-KT per kg body weight, while control animals received placebo pellets. The trial lasted 3 weeks. Hepatosomatic (HeI), gut (GU) and heart (HsI) indices and change in eye index (EI) were calculated at the end of the trial. Plasma 11KT levels in the animals confirmed that the 11KT pellets were effective. 11KT treatment induced significant (p<0.01) increases in HeI, HsI and change in EI, while significantly (p<0.001) decreasing GI. Hypophysectomy, however, did not have a significant effect (p>0.05) on any of the indices. The analysis of changes in gut epithelial cell heights and relative oocyte sizes is ongoing. The preliminary results from this study indicate that the effects of 11KT treatment are direct and not the result of 11KT triggering the increase or decrease of a pituitary factor. This study confirms that hypophysectomy is not required to study the effects of 11KT in female anguillid eels.
NZ07
ADVANCEMENT OF SEASONAL REPRODUCTIVE ACTIVITY IN STOATS BY USE OF ARTIFICIAL LIGHTING
D.N. McLane2, J.A. Duckworth1, S. Scobie1, J.E. Turner1, G.K. Barrell2
1Landcare Research, Lincoln, NZ
2Agriculture and Life Sciences Division, Lincoln University, NZ
To develop reproductive control technologies for wild stoat (Mustela erminea) populations there is a requirement for year-round breeding in captive animals. This study tested the hypothesis that a long-day photoperiod applied to stoats during winter months would stimulate reproduction in these animals. Adult stoats (12 males and 12 females) were captured from the wild during summer and autumn. From 14 May half of the animals were subjected to artificial lighting, which reached 16 h d-1 on 30 June and continued at this daily duration until November. Controls experienced natural changes in daylight. Faecal samples were collected for hormone analysis. Vaginal cytology and physical changes associated with oestrus were monitored in females and scrotal size was monitored in males. Mean faecal oestradiol concentration was highly variable (1Ð20 ng g-1 dry wt) and did not differ between treated and control groups of females. However, in early spring months, there was a high incidence of keratinised vaginal epithelial cells and signs of oestrus in light- treated females that were absent in the controls. Also, light-treated males had higher (P < 0.05) mean faecal testosterone concentrations than controls (e.g. 272 + 32 versus 163 + 31, ng g-1 dry wt respectively, on 4 September) and larger (P < 0.05) scrotal dimensions in August. These results show that stoats are amenable to photoperiodic stimulation of breeding activity and provide some of the first reproductive endocrinology data for this species in NZ
NZ08
EVIDENCE FOR PARACRINE ACTIONS OF OXYTOCIN IN THE RAT ADRENAL GLAND
B. Huang, S.J. Bunn, S.J. Assinder
Department of Anatomy and Structural Biology, University of Otago, Otago School of Medical Sciences, Dunedin, NZ
Oxytocin has a significant role in the physiological adaptation to stress. In response to stress that activates the hypothalamo-pituitary-adrenal (HPA) axis, oxytocin is released in the brain to modulate the stress response. The effects of oxytocin in peripheral stress response have not been examined in detail and the sites of action are unknown. Adrenal glands from male rats were examined for expression of oxytocin and oxytocin receptor by RT-PCR and protein distribution determined by immunolocalisation. Both oxytcoin and oxytocin receptor were shown to be expressed in the adrenal cortex and medulla. In the cortex oxytocin receptor immunoreactivity was greatest in the outer regions of the zona glomerulosa whilst oxytocin immunoreactivity appeared greatest in cells adjacent to this region. In the adrenal medulla oxytocin receptor was identified in chromaffin cells, whilst oxytocin was localised to ganglion cells. In conclusion, this distribution suggests paracrine pathways for oxytocin action within the adrenal of the rat, and that oxytocin may be released from and act upon adrenal cells that secrete stress hormones (corticosterones and catecholamines).
NZ09
PLACENTAL ABC TRANSPORTER EXPRESSION: REGULATION AND IMPLICATIONS FOR TROPHOBLAST CELL SURVIVAL
D.A. Evseenko1, J.W. Paxton2,, J.A. Keelan1,2
1The Liggins Institute, Faculty of Medical and Health Science, University of Auckland, Auckland, NZ
2Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Science, University of Auckland, Auckland, NZ
The human placenta expresses a number of ABC efflux pumps such as P-glycoprotein (Pgp) and multidrug resistance proteins (MRPs) that are believed to facilitate elimination of steroid hormone metabolites, apoptotic mediators, and xenobiotics from both the placenta and fetal circulation. The choriocarcinoma BeWo cell line was used to study the regulation of expression and function of placental ABC transporters by steroid receptor ligands and cytokines. Expression of Pgp, MRP1 and MRP2, as determined by quantitative immunoblotting, was stimulated by ~50% (P<0.05, ANOVA) following 24 h treatment with retinoic acid (5 _M) and lysophosphatidic acid (2 _M), two endogenous nuclear receptor ligands. The PPAR_ agonist rosiglitazone (1 _M) also stimulated Pgp and MRP2 expression. In contrast, TNF-_ (20 ng/ml) inhibited Pgp protein expression by ~50%. The glucocorticoid agonist dexamethasone did not exert any discernable effects on their expression, while progesterone treatment exerted inhibitory effects on Pgp levels which did not reach statistical significance. We hypothesized that Pgp might protect against apoptosis in trophoblast cells through facilitating efflux of ceramides and other apoptotic mediators. Exposure of BeWo cells to TNF-_ (50 ng/ml) for 12 h did not cause apoptosis (measured as activation of caspase-3); however, in the presence of cyclosporine A (10 _M), an inhibitor of Pgp / MRP1 activity, caspase activity was increased by ~40% (P<0.05). We conclude that placental efflux transporters are differentially regulated by various steroid nuclear receptor ligands and cytokines. Exposure to pro-inflammatory cytokines in pathological pregnancies may impair placental function and viability through decreased Pgp/MRP expression, effects which can be modulated by steroid and lipid-derived nuclear receptor ligands.
NZ10
OSTEOPROTEGERIN (OPG) MUTATIONS THAT CAUSE IDIOPATHIC HYPERPHOSPHATSIA IMPAIR OPG PROTEIN SECRETION AND BIOLOGICAL ACTIVITY
D. Naot, C.A. Middleton-Hardie, H. Cundy, I.R. Reid, T. Cundy, J. Cornish
Department of Medicine, The University of Auckland, Auckland, NZ
Familial Idiopathic Hyperphosphatasia (FIH) is a rare genetic bone disease characterised by increased bone turnover. Recently, genetic studies have established that mutations in OPG, a key regulator of bone remodeling, can cause FIH. This study aimed to investigate genotype-phenotype correlation between specific mutations, the function of the mutant proteins and the severity of disease in the families. The patients were grouped into mild, intermediate and severe phenotypes using clinical, biochemical and radiographic data. We produced constructs corresponding to five different mutations: two from patients with intermediate disease (_D182 and F117L), two from patients with severe disease (C65R and C87Y), and a mild C-terminal mutation (CtFS). When expressed in the human osteoblastic cell line SaOS2, the constructs did not effect cell proliferation and measurement of OPG mRNA by real-time PCR demonstrated that all constructs were transcribed with comparable efficiency. However, different levels of OPG protein were secreted into the media: F117L had similar levels to wtOPG; while C65R, C87Y and CtFS all had greatly reduced yields; _D182 had an intermediate secretion level. Functional studies using surface plasmon resonance technology (BIAcore), showed significant variability in the ability of the different mutant proteins to bind the OPG ligand, RANKL. Measuring the activity of the intermediate mutants in an osteoclastogenesis assay showed they were significantly less potent than wtOPG in inhibiting osteoclast formation. The various OPG mutations identified in FIH families and the phenotypic variation of the disease offer a unique opportunity for a structure-function study of OPG. Our investigations suggest that the FIH phenotype results from a combination of impaired intracellular processing and reduced activity of the OPG mutants.
NZ11
CARDIOTROPHIN-1: CIRCULATING LEVELS, CARDIAC SECRETION AND MOLECULAR FORMS IN CARDIOVASCULAR DISEASE
C.J. Pemberton, S.D. Raudsepp, T.G. Yandle, A.M. Richards
Christchurch Cardioendocrine Research Group, Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, NZ
Cardiotrophin-1 (CT-1) is a interleukin-6 related cytokine reported to play a role in cardiovascular disease (CVD), yet key questions regarding its biochemistry and basic physiology remain unanswered. Accordingly, we developed a sensitive radioimmunoassay (RIA) for CT-1, and used it to study the in vivo and in vitro circulating and tissue levels, cardiac secretion and molecular forms of the cytokine in human and experimental CVD. Plasma levels of CT-1 in healthy humans (915.1±21.6 pmol/L, mean±SEM) were not different from those in patients with myocardial infarction (MI) (887.5±22.6 pmol/L). However, CT-1 levels were reduced in patients with heart failure (734.4±16.4 pmol/L, P<0.01 vs. control/MI), but did not correlate with well described circulating markers of cardiac function or prognosis. Plasma CT-1 levels in 40 week old, male spontaneously hypertensive rats (SHR, 936.7±31.2 pmol/L) were lower than those of Wistar Kyoto controls (WKY, 1295±98.1 pmol/L, P<0.01). However, left ventricle tissue CT-1 protein was significantly higher in SHR animals compared with WKY controls. In isolated hearts, ventricular stretch resulted in significant increases in perfusate CT-1 and BNP in WKY and SHR preparations. High performance liquid chromatography revealed CT-1 in human/rat plasma, isolated rat heart perfusate and rat heart tissue extracts to consist of high molecular weight forms. In conclusion, we provide the first evidence that the heart releases CT-1 in response to in vitro ventricular stretch. However, detailed correlation analysis suggests that plasma measurement of CT-1 is unlikely to have cardiovascular prognostic utility, although cardiac tissue increases in CT-1 protein in CVD may have paracrine importance. The circulating form of CT-1 is complex, which may have implications for its in vivo actions.
NZ12
INFLUENCE OF SEX HORMONES AND ANGIOTENSIN II ON SECRETION OF ADRENOMEDULLIN FROM INDIVIDUAL HUMAN ENDOTHELIAL CELLS
L.J. Pearson, C. Rait, K. Wright, T.G. Yandle, J.J. Evans
Department of Obstetrics and Gynaecology and Department of Medicine, Christchurch School of Medicine and Health Sciences, Christchurch, NZ
Males have a higher incidence of cardiovascular disease than women. The role sex hormones play in the regulation of blood vessel functioning is uncertain. Contractility of vascular tissues is partly influenced by vasoactive substances released from endothelial cells, which line the lumen of blood vessels. Therefore we investigated the effects of angiotensin-II, an established vasoconstrictor, and the sex hormones, oestradiol and testosterone, on the release of adrenomedullin, a peptide with vasodilator activity. We used the cell immunoblot method. This method involves immunohistological staining of proteins secreted from individual endothelial cells on a protein-binding membrane. Our results indicated that angiotensin-II can increase the number of cells that secrete adrenomedullin from human endothelial cells. Testosterone also recruits more cells to the secreting population, but oestradiol had little effect. The combination of testosterone with angiotensin-II also caused an increase in the numbers of secreting cells. Our investigation suggests that the reported vasodilator actions of oestrogens work independently of adrenomedullin secretion from endothelial cells. The interactions of angiotensin-II and adrenomedullin may be important for modulating vascular function. We conclude that there is potential for selective modification by sex steroids of vasoactive peptide secretion.
NZ13
MECHANO-GROWTH FACTOR (MGF) REDUCES ISCHAEMIC INJURY IN ACUTE MYOCARDIAL INFARCTION K.G.
Matthews1, C.D. McMahon1, G.P. Devlin2, P.H. Goldspink4, S.Y. Yang3, J.V. Conaglen2, J.J. Bass1, G. Goldspink3
1AgResearch Ltd., Hamilton, NZ
2Waikato Clinical School, Hamilton, NZ
3Royal Free and University College Medical School, London, UK 4University of Illinois, Chicago, USA
We sought to determine whether the mature domain of IGF-I, or the novel E domain of MGF, a splice-variant of IGF-I, would reduce the severity of damage to the heart after myocardial infarction (MI). MI was induced by injecting 10 _m microspheres into the left circumflex coronary artery of 24 sheep. Ten minutes post-MI, sheep received one of four treatments (n=6 per group), delivered into the infarct-related artery: vehicle, 200 nM mature IGF-I, 200 nM of full MGF or 200 nM MGF E domain. Left ventricular function was assessed by echocardiography before MI and at days 1, 2 and 6 post-MI. Sheep were killed on d 8. To distinguish between viable and Ôat riskÕ areas the myocardium was perfused via the coronary arteries with 0.15% Evans blue dye. Hearts were sectioned transversely into 1 cm slices, then incubated in triphenyl tetrazolium chloride and photographed. The respective areas were assessed using image quantification software. MGF (E and full) reduced the area Ôat-riskÕ by 48% and 63% respectively (P<0.01). Histology revealed that Evans Blue dye was restricted to myocardium with disrupted extracellular spaces. Ejection fraction was reduced by 40% compared to baseline (P<0.001) at d 1 in all sheep but in sheep treated with full MGF improved by 4.5% at d 6 (P<0.05). These data support a role for MGF to protect myocardium in the Ôat-riskÕ region and to improve the mechanical performance of the heart after MI. We conclude that MGF provides greater protection to Ôat-riskÕ myocardium than mature IGF-I.
NZ14
ERYTHROPOIETIN ENHACES CARDIAC CONTRACTILITY AND SECRETION OF VASOACTIVE FACTORS AND PROTECTS AGAINST ISCHEMIA-REPERFUSION INJURY
J. Piuhola1 , C.J. Pemberton1, J.I. Keenan2, M.B. Hampton2 A.M. Richards1
1Christchurch Cardioendocrine Research Group Christchurch School of Medicine and Health Sciences, University of Otago, Dunedin, NZ
2Free Radical Reserch Group, Christchurch School of Medicine and Health Sciences, University of Otago, Dunedin, NZ
Erythropoietin (EPO) is a glycoprotein hormone regulating red blood cell maturation. Recently, the expression of EPO receptor has been verified in various tissues, including heart. In symptomatic heart failure circulating EPO levels are elevated. In this study, the acute cardiac effects of EPO were investigated with isolated perfused rat hearts in vitro. Effect of EPO on ischaemia-reperfusion injury was studied with EPO administered for 30 minutes at reperfusion after 35 minutes global ischemia. At the EPO dose 1 U/ml the developed pressure (DP) increased maximally by 18±2% (mean change from the baseline ± SEM) (P< 0.01 vs. vehicle). Endothelin A and B receptor antagonist bosentan (1 µM) abolished the positive inotropic effect of EPO (P< 0.02 bosentan + EPO vs. EPO alone). B-type natriuretic peptide secretion was elevated by 26 ± 10 % throughout the EPO infusion (P< 0.005). When EPO was administered after ischaemia the DP at 5 minutes after cessation of the infusion was 72 ± 4% of baseline compared to 54 ± 4% in the vehicle treated hearts (P< 0.01). 60 minutes cumulative creatine kinase release after ischemia was attenuated significantly by EPO. Immunohistochemistry revealed that the number of cells with active caspase-3 was 64 ± 7% lower in EPO treated hearts than in vehicle treated hearts (P< 0.05) suggesting that inhibition of apoptosis is involved in the cardioprotective effect. In conclusion, EPO enhances cardiac contractility acutely through endothelin-1 mediated mechanism in isolated rat hearts in pharmacologically relevant doses. EPO alleviates myocardial ischaemia-reperfusion injury, even when administered during the reperfusion only.
NZ15
REACTIVE OXYGEN SPECIES (ROS) MAY INITIATE CACHEXIA
S.J. Falconer, J.M. Oldham, F. Jeanplong, K.G. Matthews, C.D. McMahon
AgResearch Ltd., Hamilton, NZ
Cachexia is a progressive and uncontrolled wasting of skeletal muscle. The primary cause is an increase in proteolysis associated with upregulation of the ubiquitin-proteasome system, but this system alone cannot degrade intact muscle fibres in vitro and requires the calcium-calpain system to disrupt sarcomeres. While these pathways are upregulated during muscle wasting conditions, it remains unclear how these systems are activated. In the current investigation, we sought to identify candidate genes that may initiate cancer induced cachexia. Three male rats were injected with the AH130 tumour and killed at day 7 along with three saline injected controls. Muscles had lost 20% of their mass at 7d. RNA was extracted, labeled with Cy3 and Cy5 fluorescent dyes and hybridized on 10,000 oligomicroarray slides (MWG Ltd). Of the 10,000 genes on the oligoarrays, 160 decreased and 320 increased differentially. As expected, 30 genes associated with the ubiquitin-proteasome pathway were upregulated, in particular, atrogin, a muscle specific ubiquitin ligase, was upregulated 6.5 fold. Four genes associated with the calcium-calpain system were also increased. Interestingly, several genes that code for proteins that scavenge reactive oxidative species (ROS) (metalliothioneins (3), thioredoxins (5), glutathione (2)) were upregulated. One in particular, metallothionein 2 was upregulated by 150 fold. Interestingly, there was no change in expression of myostatin, suggesting that it does not play a role in the regulation of muscle wasting in this type of cancer. Confirmation of gene expression was performed using real-time PCR (Roche, Lightcycler). We interpret these data to mean that generation of ROS may be the principle mechanism regulating degradation of muscle fibres. Furthermore, the ubiquitin-proteasome pathway may be secondarily activated to eliminate liberated proteins, rather than to generate them.
NZ16
NUCLEAR EXPRESSION OF §C-ACTIVIN IN OVARIAN CYSTS FROM MICE WITH VARYING OVULATION NUMBER
M.J. Richardson, C.R. BeaugiŽ, H.J. McQuillan, J.S. Fleming
Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
Incessant ovulation in mice increases ovarian surface epithelium (OSE) invagination and cyst formation [1]. Some cysts arise from dilatation of rete ovarii (RO) tubules at the ovarian hilus [3]. In rats, §C-activin immunoreactivity is found in RO epithelia, but not OSE [2]. We determined §C-activin expression in ovaries from mice with a range of lifetime ovulation numbers (OV#). We hypothesised expression of §C-activin in cyst epithelia would reflect the cellular origin of the cyst. Incessant ovulation was induced by housing Swiss Webster mice (n = 10 per treatment) until 9 months old, in screen-divided cages [1]. Group-housed or breeding females were used as age-matched controls with lower OV#. §C-activin expression was observed by immunohistochemistry with an affinity-purified goat polyclonal antibody (Santa Cruz) and DAB visualisation. Cysts were observed in all groups and their location classified as hilar (13/18), or intra-ovarian (5/18). Strong cytoplasmic §C-activin staining of all RO epithelia was observed, whereas mouse OSE stained weakly in some invaginations and the OSE-mesothelial transition. Intra-ovarian inclusion cysts with pseudostratification, papillae, apical nuclei or Òsignet ringÓ cells showed strong nuclear §C-activin immunoreactivity. Conversely all hilar cysts showed weak and patchy §C-activin nuclear immunoreactivity. These data suggest strong §C-activin cyst immunostaining does not indicate an RO origin. The strong nuclear immunoreactivity observed in cystic structures showing a higher degree of metaplasia warrants further investigation. (1) Clow OL, Hurst PR & Fleming JS Molecular & Cellular Endocrinology 191: 105-111 (2002). (2) Gold EJ, OÕBryan MK, Mellor SL, Cranfield M, Risbridger GP, Groome NP & Fleming JS Molecular & Cellular Endocrinology 222: 60-69 (2004). (3) Long GG. Toxicologic Pathology 30: 592-598 (2002).
NZ17
CAN A MODEL DESCRIBING FOLLICLE GROWTH TAILOR INDIVIDUAL GONADRPHIN TREATMENTS TO WOMEN PRESENTING FOR ART?
A.J. Peterson1 , T.K. Soboleva1, J. Peek2, A.B. Pleasants1
1AgResearch, Ruakura Reseach Cenre, NZ
2Fertility Associates, Auckland, NZ
A dynamical model to describe ovarian follicle development following their commitment has been developed [1] and successfully applied to both monoovulatory and polyovulatory mammals [1,2]. The model allows the possibility for incorporation of the effect of exogenous gonadotrophin treatment and therefore is able to predict the ovarian response to FSH stimulation based on scan data of ovarian follicles before stimulation and the stimulation protocol. Tailoring gonadotrophin treatments to individuals can be improved by setting model parameters according to the estradiol profiles in the cycle that precedes treatment. Recently the model had been modified to match clinical observations of ovarian follicles development following FSH stimulation of individual women. An application of the modified model to a urinary estradiol profiles in 20 patients of Fertility Associates indicated that it provides a good quantitative description of follicle growth and ovulation in women. The computer simulations of follicle response to withdrawal of stimulation mimicked the phenomenon known as ÔcoastingÕ in human IVF suggesting that the model may be useful for management of ovarian hyperstimulation syndrome. (1) TK Soboleva, AJ Peterson, AB Pleasants, KP McNatty, FM Rhodes. Animal Reproduction Science, 2000, 58, 45-57. (2) T.K. Soboleva, A.B. Pleasants, B.T.T.M. van Rens, T. van der Lende and A.J. Peterson. Journal of Animal Science, 2004, 82, 2329-2332.
NZ18
GONADAL STEROID HORMONES DURING EARLY DEVELOPMENT IN THE BRUSHTAIL POSSUM
D.C. Eckery, G. Shuttleworth, B. Mester, B.P. Thomson
AgResearch, Wallaceville Animal Research Centre, Upper Hutt, NZ
Steroid hormones are known to play important roles in early gonadal development and are responsible for the expression of sexual phenotype. Relatively little is known about these processes in marsupials. The aim of this study was to determine the steroid hormone content of gonads (ovaries and testes) collected during early development in the brushtail possum. Gonads were collected and weighed from pouch young at days 1, 20, 60 and >75 after birth. Steroids were extracted and measured using specific radioimmunoassays for progesterone, androstenedione, testosterone and oestradiol. The bodyweights of male and female pouch young increased with age at similar rates. While gonadal weights also increased with age, testicular weight increased more dramatically than ovarian weight. Concentrations of progesterone were low around the time of birth, but steadily increased with age in both male and female pouch young. Levels of progesterone were much higher in testes than ovaries. In testes, androstenedione and testosterone concentrations showed similar patterns to progesterone. Androstenedione was largely undetectable in ovaries, whereas, testosterone was detectable at low levels at all ages. Oestradiol was undetectable in testes at any age, but present in ovaries at days 20 and >75. These data provide evidence that steroids are produced by the gonads of pouch young during development. Differences in the levels and patterns of steroid production suggest differing roles in males and females.
NZ19
EXPRESSION OF mRNA ENCODING STEROIDOGENIC RECEPTORS IN THE DEVELOPING BRUSHTAIL POSSUM OVARY
L.J. Haydon, D.C. Eckery, B.P. Thomson, J.L. Juengel AgResearch, Wallaceville Animal Research Centre, Upper Hutt, NZ
While it is known that the developing ovary of several species synthesizes steroids, the cellular types in the developing ovary responsive to these steroids are not well characterized. Therefore, expression of mRNAs encoding oestrogen receptor (ER_, ER__, progesterone receptor (PR) and androgen receptor (AR) was determined in ovaries (n > 3 per group) of brushtail possums collected 1-2, 10-21, 36-63, 110-131 and 155+ days following birth using in situ hybridization. The ER (either ER__and/or ER__ mRNA was expressed in the ovary from birth (around the time of morphological sexual differentiation). Initially, expression (ER_ and ER_) was observed in cells of the blastema and in cells migrating into the ovary from the mesonephros. Expression was also observed in the medullary cord cells from day 20. Some cells of the surface epithelium expressed ER (ER_ followed by ER_) by day 40. Expression of ER in primordial and primary follicles was limited to ER_ in the oocyte. The mRNA for PR was expressed by day 20. The medullary cords expressed PR mRNA but oogonia did not. Primordial and primary follicles did not express PR mRNA although granulosa cells of some secondary follicles did. Signal for AR mRNA expression prior to day 40 was very faint; thereafter, variable expression was observed in the medullary cords peaking between days 60-120. While AR mRNA was not detected in oogonia, oocytes of primary and larger follicles did express AR mRNA. Thus, the expression of mRNAs encoding the steroidogenic receptors in the developing ovary, particularly in the medullary cords and oocytes, supports a role for steroids in the process of ovarian and follicular formation.
NZ20
MODELLING PEPTIDE INTERACTIONS AND THE INVOLVEMENT OF cAMP IN THE REGULATION OF LUTEINISING HORMONE
T.J. Connolly, J.J. Evans, D.J.N. Wall
Laboratory for Cell and Protein Regulation, Department of O&G, Christchurch School of Medicine and Health Sciences and Biomathematics Research Centre, University of Canterbury, Christchurch, NZ
Pulsatile gonadotrophin-releasing hormone (GnRH) is considered to be the predominant regulator of the preovulatory surge of luteinising hormone (LH), which is a prerequisite of normal reproductive function in female mammals. However it is now believed that a community of peptides participates in the full production of the physiological LH surge. By appropriate mathematical analysis details of the profile of release of LH in response to GnRH and other peptides can be discerned, and hence the components of the physiological response, derived from experimental studies using in vivo protocols, can be examined. The response of pituitary tissue to a time-restricted bolus of GnRH was examined using perifusion systems in which consecutive sampling of perfusate allowed construction of a time course of LH release. The actual response to GnRH was dependent on a variety of circumstances as exemplified by the phenomenon of GnRH self-priming, and synergistic responses to peptides such as oxytocin and neuropeptide Y in association with GnRH. These actions involve the production of cyclic AMP with subsequent de novo protein synthesis. Mathematical modelling was performed utilising non-linear differential equations, and takes account of both concentrations and temporal factors. The model includes production of cAMP with resulting activation of transcription factors and incorporates a time delay to account for effects of protein translation. Our model integrates data from experiments which investigate isolated processes in order to describe the dynamic alterations of physiological environments.
NZ21
EFFECT OF CHANGING THE RATIO OF ANDROGEN:OESTROGEN IN NORMAL AND MALIGNANT EPITHELIAL PROSTATE CELLS
K.J. King, H.D. Nicholson, S.J. Assinder
Andrology Research Group of Otago, Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin, NZ
Both benign and malignant disease of the prostate present a significant health issue, although aetiology of prostate disease is not well understood. Tissue culture has been used as a model system to understand progression of prostate disease, with most systems employing cells derived from prostate cancer tumours. Studies involving cells derived from normal or benign tissue have been infrequent. The aim of this study was to investigate if altered ratios of androgen:oestrogen affected growth of normal epithelial prostate cells in vitro and compare with the effects on the prostate cancer cell line LNCaP. Cells were cultured in the absence of dihydrotestosterone (DHT) or oestradiol (E2) (control); constant E2 (5pmol.L-1) and varying concentrations of DHT (0.1-100 nmol.L-1); constant DHT (10 nmol.L-1) and varying concentrations of E2 (0.05-50pmol.L-1) . These treatments were repeated in a third experimental group in which PrEC were grown with normal stromal prostate cells (PrSC) in a co-culture system (n ³ 10). When grown in isolation, no changes in normal epithelial cell proliferation was determined. LNCaP cell proliferation was increased with both constant androgen with increasing oestrogen, and constant oestrogen with increasing androgen. When in co-culture, PrEC cell proliferation was increased by both constant oestrogen with increasing androgen, and constant androgen with increasing oestrogen. In conclusion, there is an alteration in the sensitivity to gonadal steroids in malignant prostate cancer cells. Furthermore, these results suggest that the increased epithelial cell growth seen in benign prostatic hyperplasia is a result of an indirect action of steroids mediated by stromal cells.
NZ22
INTERFERON-_ STIMULATION OF BOVINE ADRENAL MEDULLARY CHROMAFFIN CELLS RESULTS IN PHOSPHORYLATION AND NUCLEAR TRANSLOCATION OF STAT-1, 2 AND -3 PROTEINS
S.J. Bunn, S. Douglas
Centre for Neuroendocrinology and Department of Anatomy and Structural Biology University of Otago, Dunedin, NZ
The chromaffin cells of the adrenal medulla play an important role in the physiological adaptation to stress. This is achieved through the release of catecholamines and a range of peptides, which have diverse actions throughout the body. It is our working hypothesis that microbiological infection and tissue damage represent significant physiological stressors and therefore chromaffin cells may be required to respond to immune system signals. We have demonstrated that bovine chromaffin cells do indeed respond to interferon-_ (IFN-_), an important mediator in immune system signalling. Incubation of chromaffin cells with IFN-_ resulted in a time- and concentration-dependent phosphorylation and nuclear translocation of selected STAT (signal transducers and activators of transcription) proteins. STAT-1 phosphorylation, measured by immunoblotting, was evident with IFN-_ concentrations as low as 0.01 nM. STAT-2 and STAT-3 phosphorylation occurred over a similar concentration range, although to a lesser extent than STAT-1. IFN-__mediated STAT-1 phosphorylation was relatively rapid, seen after a 10 min incubation and appeared to be maximal after 30 mins. Interestingly, despite evidence of a decline in the magnitude response by 120 min the phosphorylation of STAT-1 was still elevated 24 hrs after the initial stimulus. Immunocytochemistry confirmed that in addition to phosphorylation IFN-_ resulted in a nuclear translocation of STAT-1, -2 and -3. Translocation of STAT-1 and STAT-2 occurred in the majority of chromaffin cells, whereas STAT-3 translocation was limited to a small subpopulation. These data provide evidence that adrenal chomaffin cells are capable of a rapid but sustained response to IFN-_. The cellular consequences of this interaction remain to be determined.
NZ23
DO SUPPRESSORS OF CYTOKINE SIGNALLING (SOCS) PROTEINS CAUSE HYPERPROLACTINAEMIA DURING LATE PREGNANCY AND LACTATION?
G.M. Anderson, D.R. Grattan
Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago, NZ
Prolactin secretion is regulated by negative feedback. Prolactin activates tuberoinfundibular (TIDA) neurons within the arcuate nucleus to release dopamine, which then suppresses further pituitary prolactin release. Activation of TIDA neurons by prolactin involves the JAK/STAT5b signalling pathway, which in other systems is negatively regulated by the SOCS family of protein. We hypothesized that increased SOCS expression might be responsible for the loss of TIDA neuronal responsiveness to prolactin (and consequent hyperprolactinaemia) that is known to occur during late pregnancy and lactation. On gestational (G) days 17, 20 and 22 and day 7 of lactation (L), rats (n=6-10) were decapitated and the brains rapidly frozen for real-time RT-PCR analysis of SOCS mRNA (SOCS-1, -2 and -3 and CIS) in microdissected arcuate nuclei. A further 2 groups of rats (G22 and L7) were pretreated (8 h before decapitation) to reduce prolactin levels by administering bromocriptine or removing pups, respectively. Serum prolactin concentration from trunk blood was low (<10 ng/ml) on G17 and G20 but high (>200 ng/ml) on G22 and L7 (P<0.001). Both bromocriptine and pup removal cause a return to basal prolactin levels (P<0.001). Levels of mRNA for all 4 SOCS proteins were low-undetectable on G17 and G20, however SOCS-1 and SOCS-3 levels increased 5-8-fold on G22 and L7 (P<0.01). This effect was mostly reversed by bromocriptine and pup removal (P<0.05 for SOCS-1; P=0.07 and 0.09 respectively for SOCS-3). These results support the idea that SOCS-1 and SOCS-3 contribute to the reduction in TIDA neuronal activity that permits hyperprolactinaemia in late pregnancy and lactation. This effect appears to be largely dependent on prolactin.
NZ24
PROLACTIN ALTERS STAT5A BUT NOT STAT5B SIGNALLING IN GNRH NEURONS
M.A. Fenwick, F.Y. Ma, D.R. Grattan, G.M. Anderson
Centre for Neuroendocrinology, and Department of Anatomy and Structural Biology, University of Otago, NZ
Hyperprolactinaemia, caused by anti-psychotic drugs, pathology or physiological states such as lactation, results in infertility partly due to suppression of gonadotrophin-releasing hormone (GnRH) secretion from the hypothalamus. We have previously shown that the negative feedback mechanism that regulates prolactin secretion utilises the transcription factor STAT5b to activate tuberoinfundibular dopaminergic (TIDA) neurons of the arcuate nucleus. This project aimed to determine whether the same signalling mechanism occurs in GnRH neurons. Normally cycling (diestrous) and lactating rats (n= 4-6 each) received prolactin (500 µg sc) or vehicle 40 minutes before perfusion with 4% paraformaldehyde. Thick (40 µm) cryosections were taken through the preoptic area of the hypothalamus and immunolabelled for GnRH and either STAT5a or STAT5b, and imaged by confocal microscopy. Results showed that after normalisation against background staining levels, a greater proportion of GnRH neurons expressed STAT5a than STAT5b irrespective of treatment (75% vs. 49% respectively; P<0.001). There was no evidence for nuclear translocation, or any change in the proportion of GnRH neurons expressing STAT5b in any of the treatment groups in response to prolactin. These data suggest that if prolactin exerts direct effects on GnRH neurons, the signal transduction pathway is different to that used by TIDA neurons. In contrast, prolactin caused an increase in the proportion of GnRH neurons expressing STAT5a (P<0.05) along with a small but significant increase in nuclear translocation (P<0.01 vs. vehicle treated) in diestrous rats. This may be evidence for a direct action of prolactin on GnRH neurons, but to be conclusive we will also need to demonstrate that these neurons express prolactin receptors.
NZ25
MATERNAL NUTRITION DURING PREGNANCY DETERMINES RESPONSIVENESS TO PERIPHERAL LEPTIN TREATMENT IN OFFSPRING.
S.O. Krechowec1, M.H. Vickers1, A. Gertler2, B.H. Breier1
1Liggins Institute, University of Auckland, Auckland, NZ
2Hebrew University of Jerusalem, Jerusalem, Israel
Our previous research has shown that offspring of mothers undernourished during pregnancy are predisposed to the development of obesity as adults. We hypothesize that the development of leptin resistance provides a mechanism linking adverse prenatal influences with postnatal obesity. In the present study, we examine leptin sensitivity in adult offspring from a rodent model of maternal undernutrition. Virgin Wistar rats were time-mated and assigned to receive chow either ad-libitum (AD) or at 30% of ad-libitum intake (UN) throughout pregnancy. From weaning until completion of the study offspring were maintained on either an ad-libitum chow diet (C), a regime of caloric restriction (70% of control (CR)) or a high fat diet (HF) ad-libitum. At 142±5 days, female AD and UN offspring were weight-matched and placed into treatment groups receiving either saline or recombinant-rat leptin (2.5ug/g/day) for 14 days by twice-daily subcutaneous injection. Leptin treatment caused a significant weight loss in all animals. On a control diet UN offspring demonstrated a resistance to the weight reducing effects of leptin, losing significantly less weight than matched AD offspring (ADC -5.2% vs UNC -2.7% BW, P<0.05). Offspring on HF nutrition demonstrated a similar level of leptin resistance with both AD and UN offspring showing reduced weight loss in response to treatment (ADHF -2.6% Vs UNHF -2.7% BW). While peripheral leptin treatment significantly reduced food intake in all offspring on chow and high fat diets, UN offspring lost significantly less body weight independent of postnatal nutrition, suggesting leptin resistance. This is strong evidence for our hypothesis that leptin sensitivity may be set during fetal development and may contribute to weight gain and obesity during postnatal life.
NZ26
LYSOPHOSPHOLIPIDS: LOCALISATION OF RECEPTORS AND ACTIVITIES IN HUMAN PLACENTA AND BEWO CHORIOCARCINOMA CELL
S E. Okuda, J.A. Keelan
Liggins Institute, Faculty of Medical and Health Sciences, University of Auckland, Auckland, NZ
Lysophospholipids (LPLs), such as lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), sphingosylphosphorylcholine (SPC), and lysophosphatidylcholine (LPC), are bioactive lipid mediators that act as high affinity ligands for a family of G-protein coupled receptors, exerting a wide variety of mitogenic, immunological, and regulatory effects in different cell types. Although there is some evidence suggesting that LPLs are involved in various aspects of pregnancy, little is known about the properties and functions of these molecules in the pregnant uterus. In this study, we characterised the localisation and expression of LPL receptors in the term human placenta and human choriocarcinoma (BeWo) cells using immunohistochemistry and RT-PCR. S1P2 receptors were localised immunohistochemically to the syncytium of term villous placenta, while LPA1 receptors were localized in the endothelium of the term villous placenta, with weak staining of the syncytium. RT-PCR analysis of LPL receptor expression indicated that S1P1 and S1P2 receptors are expressed in BeWo cells while LPA and S1P receptors are expressed in term amnion, choriodecidua and villous placenta. Using the BeWo model, dose response studies revealed that LPA (0.1-10 mM) exerts significant and dose-dependent anti-apoptotic effects (P<0.05; ANOVA), whereas S1P, SPC and LPC were not protective. S1P and SPC (10 mM) were both shown to enhance the production of the proinflammatory cytokine IL-6 in BeWo cells by ~60% and ~200%, respectively, while LPA was inhibitory, significantly reducing IL-6 production by ~50% in the presence of IL-1b stimulation (P<0.05). Production of TNF-a was stimulated by S1P to ~200% of control (P<0.05). These results collectively suggest that the placenta is a target for LPL actions, with viability and immuno-endocrine activity potentially being responsive to LPL modulation.
NZ27
EFFECTS OF CHRONIC PULSATILE GROWTH HORMONE (GH) INFUSION TO THE GROWTH-RESTRICTED FETAL SHEEP ON mRNA LEVELS OF IGF-I AND OF THE GH AND IGF TYPE 1 RECEPTORS
H.H. Phua, F.H. Bloomfield, M.K. Bauer, J.E. Harding
The Liggins Institute, University of Auckland, Auckland, NZ
The accepted dogma is that in fetal life IGF-I, the major fetal growth factor, is not regulated by GH. However, chronic pulsatile infusion of GH to growth-restricted (IUGR), but not normally-grown, fetal sheep elevated fetal circulating IGF-1 levels. Fetal growth was not increased, and kidney weight was decreased1. To test the hypothesis that IUGR induced hepatic GH receptor (GHR) expression but that IGF receptor (IGF-1R) was downregulated in response to GH, we measured mRNA levels of GHR, IGF-I and IGF-1R in fetal and placenta; tissues from three groups of fetuses: controls, and IUGR fetuses treated for 10 d with either saline or GH (n = 5 per group). mRNA levels were measured relative to 18s mRNA using real-time relative quantitative RT-PCR. Compared to controls, hepatic GHR mRNA levels were reduced 3-fold and placental GHR mRNA increased 2-fold in saline fetuses. Hepatic GHR mRNA levels were similar in GH fetuses, but placental and kidney GHR mRNA levels were increased compared with saline and control fetuses. Placental IGF-I mRNA levels were increased in saline, but not GH, treated fetuses; otherwise IGF-I mRNA levels were not different among groups. IGF-1R mRNA levels were increased in placenta in saline- and GH-treated fetuses, and were decreased in kidney in GH-treated fetuses. Increased circulating IGF-1 levels in GH-treated IUGR fetuses cannot be explained by induction of the hepatic GHR, or by increased IGF-I mRNA expression, and may therefore be due to decreased clearance. The placental somatotrophic axis is regulated differently from that in the liver in IUGR. (1) Bauer et al. Journal of Endocrinology 2003;177:83-92.
NZ28
A FORM OF OBESITY INDEPENDENT OF INSULIN RESISTANCE IS DETERMINED BY MATERNAL NUTRITION DURING PREGNANCY
N.M. Thompson1, M.H. Vickers1, S.O. Krechowec1, J. Miles1, R. Shankar2, B.H. Breier1
1Liggins Institute, University of Auckland, Auckland, NZ
2Indiana University and Purdue University, Indianapolis, USA
Our previous work has shown that offspring of pregnant rats undernourished during pregnancy develop obesity. Effects on insulin sensitivity in adult life are unknown. Virgin Wistar rats were time-mated and assigned to receive chow either ad-libitum (AD) or at 30% of ad-libitum intake (UN) throughout pregnancy. From weaning until completion of the study offspring were maintained on either ad-libitum chow diet (C), a regime of caloric restriction (70% of C (CR)) or high fat diet (HF) ad-libitum. At 263±2 days of age hyperinsulinaemic-euglycemic clamps were performed under halothane anaesthesia (n=6 / group). A parallel cohort of animals was used for endocrine measurements. Adult UN offspring were shorter (P<0.001) (nose tail (mm) ADC 494±5, ADCR 472±4, ADHF 495±8, UNC 475±4.781, UNCR 458±3, UNHF 479±4), obese (P<0.05) (% supra renal fat pads ADC 2.7±0.2, ADCR 1.1±0.1, ADHF 5.7±0.3, UNC 3.4±0.4, UNCR 1.6±0.1, UNHF 6.1±0.3), hyperinsulinaemic (P<0.001) (plasma insulin (µg/L) ADC 0.7±0.1, ADCR 0.4±0.1, ADHF 0.9±0.1, UNC 3.5±1.0, UNCR 1.1±0.3, UNHF 3.8±0.7) and hyperglycaemic (P<0.001) (plasma glucose (mmol/L) ADC 8.6±0.3, ADCR 7.2±0.3, ADHF 7.6±0.2 UNC 9.7±0.3, UNCR 7.6±0.2, UNHF9.0±0.3). HF animals showed decreased (P<0.001) sensitivity to insulin compared with C (glucose infusion rate (mg/kg/min) ADC 21.5±0.9, ADCR 29.0±1.9, ADHF 17.0±0.6, UNC 23.1±0.5, UNCR 30.5±1.6, UNHF 17.5±1.0). In contrast, caloric restriction throughout postnatal life increased insulin sensitivity (P<0.001). The level of maternal nutrition during foetal development had no independent effect on insulin sensitivity in adult offspring. Our data suggest that the aetiology of obesity induced by maternal undernutrition during pregnancy may be independent of the insulin resistant state observed in other forms of obesity (e.g. diet-induced obesity).
NZ29
CELL PROLIFERATION IN THE EPITHELIAL COMPARTMENTS OF MOUSE OVARIES OF VARYING AGE AND OVULATION NUMBER, MEASURED BY BROMODEOXYURIDINE INCORPORATION
C.R. BeaugiŽ, H.J. McQuillan, J.S. Fleming
Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
Cell division was studied in mice of various ages and total lifetime ovulation number (OV#) to investigate the known association of these factors with epithelial ovarian cancer risk. We have shown incessant ovulation induces inclusion cyst formation in mouse ovaries [1]. Since some of these cysts appear to be dilated rete ovarii (RO) tubules [2], we aimed to compare rates of cell division in RO, with those in cysts and ovarian surface epithelium (OSE). Incessant ovulation was induced by housing Swiss Webster mice (n = 10 at each age) until 3, 6, 9 and 12 months old, in screen-divided cages [1]. Group-housed or breeding females were used as age-matched controls with lower OV#. Animals were injected with 3 x 30 mg bromodeoxyuridine (BrdU) per g body weight i.p. at 2 hour intervals and ovaries collected 2 hours later. Incorporated nuclear BrdU was determined by immunohistochemistry with DAB visualisation. Inclusion cysts were observed in ovaries from mice of all ages and treatments. The percentage of BrdU-stained cells was greater in cysts than in OSE and lowest in RO in all treatments and ages. In all epithelial compartments, BrdU incorporation declined with age. BrdU-stained cells in cyst epithelia were mainly cuboidal and often grouped, whereas papillae or Òsignet ringÓ cells were less frequently stained. We conclude that cell proliferation is increased in ovarian cyst epithelium, compared with the OSE and RO, but that the rate decreases with age in all ovarian epithelia. 1. Clow OL, Hurst PR, Fleming JS Molecular & Cellular Endocrinology 191: 105-111 (2002). 2. Long GG. Toxicologic Pathology 30: 592-598 (2002).
NZ30
PHARMACOGENOMICS AND INFERTILITY TREATMENT IN POLYCYSTIC OVARIAN SYNDROME
A.J. Umbers, N. Johnson, J. Falkiner, A.N. Shelling
Department of Obstetrics and Gynaecology, University of Auckland, NZ
Polycystic ovarian syndrome (PCOS) is a highly prevalent and heterogeneous disease, affecting 5-10% of women of reproductive age. Patients suffer significant reproductive and metabolic morbidity. In addition to infertility, patients often experience recurrent miscarriage, menstrual disturbances, insulin resistance, acne, obesity and hirsutism. Although the aetiology remains unknown, a strong genetic component is recognised. Many candidate genes have been screened but no genetic defect has yet been identified as the primary cause of the disease. Insulin resistance exacerbates the symptoms of PCOS. Recently an insulin sensitising agent, metformin, has been used to treat the metabolic and endocrine abnormalities of this disease. Metformin induces ovulation, returning fertility to some patients. Only 60% of women respond to metformin. Currently no parameters exist to accurately predict which patients will be responsive to treatment. In collaboration with the National WomenÕs Hospital PCOSmic clinical trial of metformin, pharmacogenomics has been employed to predict therapeutic response based on individual genetic variation in PCOS patients. Known polymorphisms in genes hypothesised to coordinate the therapeutic properties of metformin, are undergoing screening. Genetic variation in the insulin receptor, insulin receptor substrate-2, sex hormone binding globulin and P45017a hydroxylase genes have been screened using restriction fragment length polymorphisms and denaturing high pressure liquid chromatography. The use of genetic analysis to determine which patients will respond positively to metformin will lead to targeted and more effective treatment of infertility in PCOS patients.
NZ31
A NEW BREED OF FOX HUNTING: BREAST CANCER AND THE FORKHEAD DYNASTY
R.B. Sprague, K.J. Woad, W.J. Smale, A.N. Shelling
Department of Obstetrics and Gynaecology, University of Auckland, NZ
The FOX (forkhead box) family of transcription factors have been implicated in numerous developmental and signalling pathways and there is a recent and rising body of literature implicating them in various cancers. In hormone-dependent tumours such as breast cancer, FOX transcription factors can mediate the oestrogenic stimulus through direct interaction with the oestrogen receptor, and hence may regulate response to anti-oestrogens such as tamoxifen. This research aims to evaluate the potential for involvement of the FOX genes in the pathology of breast cancer and the mechanisms of acquired tamoxifen and aromatase inhibitor resistance. The presence of known or potentially novel FOX genes is currently being investigated in an oestrogen-dependent breast cancer cell line (MCF-7) via the amplification of the conserved forkhead domain within the FOX genes from cDNA using degenerate primers. The PCR products have been cloned into plasmid vectors and preliminary sequence analysis indicates that FOXA1 is the most widely expressed in MCF-7. The remaining clones will be analysed by a combination of restriction enzyme analysis and DNA sequencing to determine the relative abundance of specific FOX gene expression in MCF-7. The expression levels of the identified FOX genes in MCF-7 will be analysed using real time RT-PCR and the change in FOX expression levels in MCF-7 following exposure to tamoxifen and aromatase inhibitors may also be examined. The potential exists for the FOX genes to augment the existing compilation of diagnostic clinical markers, thus adding to the current endeavours towards individualised treatment regimes. This dynasty also holds promise towards increasing the understanding of resistance mechanisms adopted by neoplasms to escape pharmacological assault.
NZ32
BOYS, BARBEQUES, BAKED BEANS AND BABIES E. Podivinsky, B.M. Thompson
ESR Ltd., Christchurch Science Centre, Christchurch, NZ
Cause and effect studies have implicated environmental estrogen mimics (xenoestrogens) in a raft of biological effects. Xenoestrogens may be from natural sources (the phytoestrogens) or may be synthetic. Both have structures that are analogous to endogenous estrogens and so fit and activate the cellular estrogen receptor. The molecular mechanisms by which estrogen mimics may affect human endocrine pathways are not known and there is little data to identify long-term physiological responses that may be associated with an individualÕs exposure to these factors. A study of dietary exposure data has suggested that men, and particularly young men, may be at risk from environmental estrogen mimics. We are interested in whether the natural phytoestrogens and the synthetic xenoestrogens have similar molecular modes of action and whether exposure is linked to molecular responses that can be correlated with individual disease susceptibilities. In particular, susceptibility to disease conditions associated with male sexual dysfunction eg: lowered sperm count, cryptorchidism, testicular and prostate cancer.
NZ33
TESTICULAR OXYTOCIN RECEPTOR EXPRESSION IS INCREASED IN OESTROGEN RECEPTOR ALPHA KNOCKOUT MICE
M. Gould, H.D. Nicholson
Department of Anatomy and Structural Biology, University of Otago, Dunedin, NZ
In the male, oxytocin acts on the testis to modulate androgen synthesis and regulate contractility. There is evidence that oestrogen can affect oxytocin action by altering oxytocin receptor expression. However, the mechanism of this action is not clear. The study uses knockout mice to investigate whether the _ or __oestrogen receptor is involved in regulating oxytocin receptor expression in the mouse testis. Adult _ (_ERKO) and _ oestrogen receptor knockout (_ERKO) and wild type mice were killed (n=8). The testes were removed and either frozen or processed for immunohistochemistry. Trunk blood was collected for testosterone measurement. Using an antibody raised to the 3rd intracellular loop of the oxytocin receptor Western blot analysis and immunohistochemistry were used to identify and localise the oxytocin receptor in the testis. No difference in body or testis weight was observed between the animals. Plasma testosterone concentrations were significantly elevated in the _ERKO (P < 0.001) but not _ERKO mice. Western analysis revealed a single band of ~ 60 kDa in the testes of all mice. No difference in the density of this band was seen between _ERKO and wild type mice. The density of this band was significantly increased in _ERKO mice. Immunohistochemistry localised the oxytocin receptor predominantly to the Leydig cells. The density of immunoreactivity appeared more intense in the _ERKO mice supporting the Western analysis data. These findings suggest that in the male, oestrogen receptor _ is important in the regulation of the oxytocin receptor. Whether this is a direct effect or mediated by testosterone awaits further investigation.
NZ34
PROTEOMIC ANALYSIS OF CHANGES IN STRUCTURE AND VIABILITY OF FETAL MEMBRANES OVERLAYING THE CERVICAL OS PRIOR TO MEMBRANE RUPTURE AT TERM
J.A. Keelan1, B. Nijmeijer1, E. Parry2, P. Stone2
1Liggins Institute and Department of Pharmacology, University of Auckland, Auckland, NZ
2Obstetrics and Gynaecology, University of Auckland, Auckland, NZ
Timely ripening of the cervix and rupture of the fetal membranes is central to the success of parturition. Despite much research, it is still unclear what factors regulate and control these processes. The region of the gestational membranes overlying the cervical os (zone of altered morphology - ZAM) exhibits increased rates of apoptosis and marked histological changes prior to labour compared to fundal regions, reflecting preparative changes required for rupture prior to parturition. To attempt to clarify and define the nature and regulation of these changes with a view to identifying predictive/diagnostic markers of imminent rupture, we have conducted preliminary proteomic analysis of cervical and fundal regions of intact fetal membranes at term prior to rupture and delivery in conjunction with immunoblotting studies of proteins known to be involved in regulation of apoptosis. The apoptotic regulatory proteins survivin, RAIDD, XIAP, SODD and cFLIP were present in membranes at unchanged levels regardless of anatomical location. A ZAM marker, alpha-smooth muscle actin, was abundant by immunoblotting in cervical but not fundal samples. The 2D electrophoretic pattern of proteins was markedly different between cervical and fundal regions with over 40 differentially expressed proteins detected. Of 8 proteins identified to date, fibrinogen precursors were found only in fundal tissue, while tissue transglutaminase, heat shock 27 kDa protein and beta actin were abundant only in cervical tissue. These results point to dramatic changes in protein composition of membranes at the site of rupture, suggesting proteomics may be useful in identifying predictive indicators of premature membrane rupture.
NZ35
EFFECTS OF LEPTIN ON EEL (ANGUILLA AUSTRALIS) PREVITELLOGENIC OOCYTE GROWTH IN VITRO
S.L. Divers, A.R. McLean, P.M. Lokman
Department of Zoology, University of Otago, Dunedin, NZ
There is little understanding to date of the communication between the body nutrient pool and the regulation of the reproductive axis in teleost fish. There is evidence from mammals suggesting that the effect of leptin on reproductive function may relate to its site of expression. The aim of this study was to investigate the in vitro effect of leptin on eel oocyte growth. Fragments of ovary were isolated from five previtellogenic eels and incubated (sixteen days) with dose response (0,1,10,100,1000 ng/ml) treatments of recombinant mouse leptin. Incubations (0-1000nM) with 11-ketotestosterone (11-KT) were used as a positive control. Lastly, we investigated the impact of leptin on the stimulatory effects of 11-KT with a combined 11-KT/leptin treatment. Our preliminary data indicate that leptin had a slight inhibitory effect on eel oocyte diameter. In contrast, 11-KT had a strong positive effect on eel oocyte diameter, but this effect was somewhat suppressed when leptin was added to the cultures. The effect of 11-KT is in agreement with earlier observations (1), and indicate that the cultures were viable during the experimental period. Our present observations on leptin suggest that it has limited effects on previtellogenic oocyte growth in vitro, and may indicate that leptin exerts its effects elsewhere on the teleost hypothalamo-pituitary-gonad axis. (1) Lokman PM, George KAN, & Young G (2003) Fish Physiology and Biochemistry 28: 283-285.
NZ36
11-KETOTESTOSTERONE INDUCES IN VITRO GROWTH OF PREVITELLOGENIC OOCYTES OF EEL (ANGUILLA AUSTRALIS)
P.M. Lokman1, K.A.N. George1, G. Young2
1Department of Zoology, University of Otago, Dunedin, NZ
2Department of Biological Sciences, University of Idaho, USA
11-Ketotestosterone (11-KT), considered a male-specific androgen in teleost fish, has recently been implicated in stimulating lipid accumulation in previtellogenic oocytes of fresh-water eels. To address whether or not these effects were direct or indirect, eel ovarian tissue was isolated and maintained in vitro for 18 days in the presence or absence of 11-KT at 0-3.3 _M. Previtellogenic oocyte diameters were significantly greater, by 10-20% on average, if ovarian explants were incubated in the presence of 11-KT when compared to control incubations. This increase was reflected in visible increases in nuclear size. No other histological changes were noticeable. Similarly, no clear differences in ultrastructure were observed between control and 11-KT-treated ovarian explants. These results confirm that 11-KT has direct effects on the previtellogenic ovary of the eel, but the mechanisms by which lipid accumulation in fish oocytes is regulated remains unclear.
NZ37
IDENTIFYING THE MECHANISM OF A NOVEL GENETIC MUTATION AFFECTING FECUNDITY IN SHEEP
E.S. Feary1,3, J.L. Juengel1, P. Smith1, B.J. Mcleod2, P.A. Farguhar2, G.H. Davis2, K.P. McNatty1.
1Wallaceville Animal Research Centre, Upper Hutt, NZ
2AgResearch Invermay Agriculture Centre, Dunedin, NZ
3Victoria University, Wellington, NZ
The objective of this study was to examine ovarian function in a line of Coopworth sheep called Woodlands which have ovulation rates about o.4 higher than wild-type sheep. These animals have a novel, imprinted, X-linked fecundity gene called FecX2W where Fec = Fecundity, X = X chromosome, 2 = 2nd mutation identified on X chromosome and W = Woodlands. Standard methods of morphometric and histological analysis showed that the ovaries of 4 week-old lambs with the natural mutation are approximately 6 times heavier and have 10 times more antral follicles than wild-type ovaries. The cortical and ovarian volumes were larger than those in the wild-type animals. No differences were observed in the mean numbers of 1, 1a, 2, 3 and 4 follicles between the genotypes. Moreover, no differences were observed between genotypes in follicle or oocyte diameters for any follicle type. Using established in-situ hybridization techniques, the genes bone protein 15, growth differentiating factor 9, estrogen receptor a and b, inhibin a, inhibin/activin bA and bB, follicle stimulating hormone receptor, bone morphogenic protein receptor I and II, showed no observable differences of expression patterns between genotypes. Thus, the Woodlands mutation FecX2W not only affects ovulation rate in adults but also appears associated with a polycystic ovary phenotype in lambs. However, the pathways that the mutation is utilising to affect follicular development has not yet been identified.