OF ENDOCRINOLOGY
PROGRAMME AND ABSTRACTS
ANNUAL SCIENTIFIC MEETING
22
Liggins Institute
The support of
Liggins Institute,
in the organising of this meeting
and Organon / Akzo Nobel
are gratefully acknowledged
AUTHOR INDEX
Aramburo
C
NZ 15
Arnhold
IJP
NZ7
Ashby MA
NZ16
Barrell
GK
NZ9
Bass JJ
NZ20, NZ21
Bava U NZ12,
NZ14
Blair HT
NZ16
Callon KE NZ12,
NZ14
Carvalho
LR
NZ7
Challis J
NZ1
Chaston
J NZ3
Cockrem
J NZ15
Coetsee
CJ
NZ5
Connolly TJ
NZ8
Cornish J NZ11,
NZ12, NZ13, NZ14
Craven AJ
NZ18
Cundy T NZ12,
NZ13
Dattani
M
NZ7
Eayrs K
NZ4
Espiner
EA
NZ9
Evans JJ
NZ8
Fink J
NZ10
Gilmour RS
NZ2
Gluckman
P
NZ1
Grattan
NZ6
Harding J
NZ1
Hawkins P
NZ1
Holloway A
NZ1
Jeanplong
F NZ20,
NZ21
Kambadur
R NZ19,
NZ21
Keelan JA NZ3,
NZ13
Kirk SP
NZ20, NZ21
Ladyman
Lin C
NZ14
Lin J-m
NZ14
Luna M
NZ15
Marvin KW
NZ3
Maxwell L
NZ21
McCowan
SA
NZ12
McLeod B
NZ10
McMahon CD
NZ20, NZ21
Mendonca
BB
NZ7
Middleton-Hardie C NZ13
Mitchell MD
NZ2, NZ3
Naot D NZ12,
NZ13
Nicholls MG
NZ9
Nicholson H
NZ10
Nixon AJ
NZ16, NZ17, NZ18
Oliver M
NZ1
Ormandy
CJ
NZ18
Osepchook
CC NZ20,
NZ21
Pearson AJ
NZ16, NZ17, NZ18
Pitto RP
NZ12
Prickett
TCR
NZ9
Reid IR
NZ12, NZ13, NZ14
Richards AM
NZ9
Sato TA
NZ2, NZ3
Sharma M
NZ19, NZ21
Shelling AN
NZ5
Smith HK
NZ19, NZ21
Soboleva
T
NZ17
Ulrichsen
H
NZ20
Vetharaniam
K
NZ17
Wall DJN
NZ8
Welby M
NZ9
Wildermoth NZ16
Wilkins RJ
NZ18
Woods K
NZ7
Yandle TG NZ9
NZ1
MATERNAL PERICONCEPTIONAL UNDERNUTRITION ALTERS
FETAL HPA DEVELOPMENT AT THE LEVEL OF BOTH THE PITUITARY AND THE ADRENAL GLAND
Frank Bloomfield1,2,
Mark Oliver1, Paul Hawkins1, Alison Holloway2,
Peter Gluckman1, John Challis2 and Jane Harding1
1Liggins Institute,
University of Auckland, Auckland, New Zealand, 2Departments of
Physiology and Obstetrics and Gyncaecology, University
of Toronto, Toronto, Ontario, Canada.
There
are now good epidemiological and experimental data suggesting that alterations
in maternal, and hence fetal, nutrition have important consequences on fetal
development and on the risk of disease in adult life. It has been proposed that corticosteroids may
play a fundamental role in mediating the changes in fetal development that
are seen in response to undernutrition ("fetal programming"). We have recently demonstrated that a moderate
periconceptional nutritional insult (from 60 days
before to 30 days after conception; term = 145 d) to the ewe results in precocious
activation of the fetal hypothalamic-pituitary-adrenal (HPA) axis and non-infectious
preterm birth1.
To investigate further the level at which
this activation may have occurred we performed Synacthen
and CRH/AVP stimulation tests in late gestation fetuses in which the ewes
were either fed ad libitum
throughout gestation or were exposed to periconceptional
undernutrition (UN). Hormones were
measured by RIA. The animals were killed
at 131 d gestation and the fetal pituitary glands frozen for in situ hybridisation
(ISH) of mRNA (POMC, PC-1, PC-2, GR) using 45 mer oligonucleotide
probes and sense controls.
Baseline
concentrations of cortisol and ACTH were higher
in UN fetuses (cortisol 2.4 8± 0.45 vs
1.29±0.11 ng/mL, P<0.05, ACTH 46.0 ± 8.8 vs
26.5 ± 1.3 pg/mL, P=0.05). In response to a short Synacthen
test there were no differences between groups in the rise in fetal plasma
ACTH and cortisol concentrations. However in response to a metyrapone challenge only UN fetuses showed a fall in circulating
cortisol concentrations (P<0.05) and a rise in 11-deoxycortisol
and ACTH concentrations (P<0.05). ISH demonstrated no change in POMC mRNA levels
in the pars distalis
(PD), but increased levels in the pars
intermedia (PI).
Similarly, PC-1 mRNA levels were increased in the PI but not the PD. PC-2 levels were not different between groups.
GR mRNA levels in the PD were not different between groups, but
we could not demonstrate GR mRNA in the PI.
These data demonstrate that a nutritional
insult to the ewe around the time of conception has effects on the fetal HPA
axis months later in late gestation. Maturation
of the fetal adrenal gland is advanced, and there are increased levels of
mRNA for POMC and PC-1 in the PI, suggesting that this may be a site of ACTH production.
1.
NZ2
INHIBITION OF PPAR-g RESULTS IN A DECREASE IN
BASAL PROSTAGLANDIN D2 PRODUCTION BY HUMAN CHORIODECIDUAL EXPLANTS
TA Sato1, E.B.E. Berry1,
R.S. Gilmour1 and MD Mitchell2.
1Liggins Institute
and the 2Departments of Pharmacology and Clinical Pharmacology
and Obstetrics & Gynaecology, Faculty of Medical
and Health Sciences, University of Auckland, Auckland, New Zealand.
Enhanced
intrauterine prostaglandin (PG) production has been implicated in the mechanisms
that initiate the onset of both preterm and term labour.
Inflammation and microbial infection have been shown to be strong positive
regulators of PG biosynthesis by gestational tissues (fetal membranes, decidua
and placenta). For this reason, a thorough characterization of the enzymes
involved in PG production is important to better understand the mechanisms
involved in regulating PG biosynthesis. Previous studies conducted in this
laboratory have demonstrated both anti-inflammatory and pro-apoptotic properties
of PPAR-g activation by a naturally occurring ligand, 15-deoxy-D12,14-PGJ2 (15d-PGJ2). PGD2
is the precursor for 15d-PGJ2, and although 15d-PGJ2
has been detected in amniotic fluid and placental-conditioned medium, detailed
knowledge of the extent and regulation of the production of PGD2
by gestational tissues are lacking at this time. Hence, it is uncertain whether
these tissues are able to respond to PPAR-g modulation by endogenous
ligand, and whether PGD2 synthesis is in turn regulated by PPAR-g. These studies were conducted to determine if PPAR-g activity has any influence on the production of PGD2, the precursor
of its ligand, and whether PGD2 is produced by choriodecidual membranes.
Placentas were obtained from women delivered
by Caesarean section at term. The choriodecidua was separated from the amnion
and explant disks were excised (3 disks/well in a 12-well plate) using a 6
mm corkborer. The explants were then cultured and
allowed to equilibrate over night in FCS-containing media. The following day, the media were then replaced
with serum-free media treated with either vehicle control or the specific
and irreversible PPAR-g antagonist, GW9964, in the presence
and absence of bacterial lipopolysaccharide (LPS).
After 24-h treatment, the media were harvested and PGD2 production
was determined by RIA. Production rates are expressed as pg/mg wet weight/24-hr.
The results (below) are from n=2 placentas, expressed as % of their
respective control (mean ± SEM, * P< 0.5 vs control by ANOVA).
|
PGD2 production |
[GW9964, nM] |
|||
|
|
0.00 |
0.16 |
0.80 |
4.00 |
|
Basal |
100 (3.755) |
45.435 (4.067) |
64.737 (8.244) * |
66.450 (5.038)* |
|
+ LPS (5 mg/mL) |
100 (9.294) |
101.600 (9.896) |
188.706 (19.632) * |
90.810 (11.843) |
These studies demonstrate that PGD2
is present in gestational tissues and that inhibition of PPAR-g
activity results in increased production of PGD2 under basal, but
not LPS-stimulated, conditions. These
results indicate that a positive feed-forward loop may exist in the choriodecidua
through which 15d-PGJ2, derived from PGD2, stimulates
PGD2 production via activation of PPAR-g.
NZ3
Expression and localisation
of bone morphogenic proteins and their receptors in human extra-placental
membranes at term and preterm
Jessica Chaston, Keith W Marvin, Timothy A Sato,
Liggins Institute, University
of Auckland Faculty of Medical and Health Sciences, Private bag 92019,
The transforming growth factor-b
(TGF-b) superfamily comprises a large
number of proteins that share common sequence elements and structural motifs,
which act via binding to homologous type-I and type-II receptors. The bone
morphogenic proteins (BMPs)
represent an important subgroup of the superfamily,
with proven roles in mesoderm formation, neural patterning, skeletal and limb
development, organogenesis, and gametogenesis. The
human placenta and extraplacental membranes are
known to be sources of many TGF-b superfamily members where they
serve various functions in pregnancy. We have explored the hypothesis that
BMPs and their receptors are expressed in the fetal membranes
and may play a role in aspects of membrane function both during, and at the
end of, pregnancy.
Initial data
on mRNA expression of BMP-2, -3, -6, -7, -8, BMP-RIA, -RIB and -RIIA were
obtained from cDNA expression array studies performed using a commercial 385-gene
cytokine array system (R&D Systems, MN) on RNA extracted from amnion and
choriodecidual membranes (n=4 per group) derived from term deliveries (with
and without labour) and preterm deliveries (with
and without intrauterine infection)1.
All genes were expressed ubiquitously, with no significant differences between
groups. BMP-6 expression was very high in amnion and choriodecidua; BMP-2
and 7 were expressed an order of magnitude lower, with BMP-3 and 8 expression
lower still. All 3 BMP receptor mRNAs were present at very low levels in both
tissues. To localise the cellular sites of BMP receptor
expression, immunohistochemistry was employed on frozen sections (5 mM)
of full-thickness membranes from term placentas delivered prior to onset of
labour (n=5). Polyclonal antisera
(R&D Systems) were employed, visualised by immunoperoxidase staining developed using biotin-streptavidin-HRPO
with tyramide amplification. BMP-RIA staining was localised
predominantly to chorionic trophoblasts
at the maternal-fetal interface with some weaker staining throughout the chorion. BMP-RIB staining was less restricted, being widespread
throughout the chorion and decidua, with occasional
staining in the amnion mesenchyme and epithelium.
Unexpectedly, the RII (ligand binding) receptor subunit appeared to be absent
in the majority of the tissues, with only weak staining detected in the amnion
mesenchyme and the apical surface of the epithelium.
BMP-6 immunoreactivity was detected only in the
cytoplasm of the chorionic trophoblast.
These data
are the first to describe expression and localisation
of BMPs and their receptors in human gestational
membranes, and suggest that these tissues are both sources of,
and targets for, a number of BMPs. The disparate
localisation of type I and II receptors indicates that functional
receptor coupling may not exist (at least in the expected configuration) outside
of the amnion. BMP-RIA may play an as-yet undertermined role at the maternal-fetal interface, perhaps
mediating the effects of BMPs in association with
another type-II receptor. The production profile of BMPs
by these tissues has yet to be determined, although the chorion
appears likely to be an important source, for BMP-6 at least.
1KW Marvin, JA Keelan, RL Eykholt, TA Sato, and MD Mitchell. (2002) Use of cDNA arrays to generate differential expression
profiles for inflammatory genes in human gestational membranes delivered at
term and preterm. Molecular Human Reproduction,
8(4):399-408.
NZ4
A LATERAL FLOW TEST FOR OESTRONE SULPHATE TO DETERMINE PREGNANCY
STATUS IN HORSES
Keith Henderson and Kim Eayrs
Reproductive Technologies, AgResearch Ltd., Wallaceville Animal Research Centre, Upper
Measurement of serum concentrations
of oestrone sulphate (OS)
is an established, accurate means of determining the pregnancy status of horses
from 100 days post-mating through to expected foaling.
Veterinary diagnostic laboratories generally use the relatively complex techniques
of radioimmunoassay or enzymeimmunoassay for measuring
serum OS concentrations. Recently this laboratory described a much simpler
dipstick immunoassay procedure for determining OS concentrations in mare serum
(1). Despite its relative simplicity, the dipstick test still required mixing
of the test serum with antibody coated microspheres
and buffer in a reaction tube before adding the dipstick, and waiting 15 to
20 minutes for the colour change end-point to develop.
To further simplify the test, it has been re-formatted into a lateral flow
test.
A nitrocellulose membrane based lateral flow immunoassay device
was developed using membrane bound 1,3,5, (10)-estratrien-3-ol-17-one conjugated
to bovine serum albumin as the capture antigen, and an OS detection monoclonal
antibody coupled to colloidal gold as the visible detection reagent. The test
was run by adding 0.1 ml of test serum to the sample well of a plastic well
encasing the nitrocellulose membrane. As the serum migrated along the membrane,
a test ‘line’ and control ‘line’ were generated on the membrane within 5 to
10 minutes. The intensity of the test ‘line’ was inversely proportional to
the concentration of OS in the serum sample being tested. The concentration
of the capture antigen and OS monoclonal antibody were optimised so that the working range of the lateral flow assay
would allow pregnancy status of the mare being tested to be determined from
the assay’s visual end point. Serum samples with OS concentrations <10
ng/ml (indicative of non-pregnancy in mares over 100 days post-mating) generated
a test end-point consisting of a visible test ‘line’ and control ‘line’, whereas
serum OS concentrations >50 ng/ml (indicative of pregnancy) generated a
control ‘line’ only. The sensitivity and specificity of the test in diagnosing
pregnancy was 97.1% and 98.7% respectively, based on the analysis of 701 blood
samples obtained from non-pregnant (389) and pregnant (312) mares >100
days post-mating. The lateral flow test devices were stable for at least 12
months when stored at 4oC sealed in aluminium
pouches with desiccant.
It is concluded that this novel, rapid, easy to use lateral flow
immunoassay offers a practical alternative to traditional laboratory based
immunoassays for measuring serum OS concentrations in mares for determining
their pregnancy status. Moreover, this test gives veterinarians the opportunity
to undertake serum OS measurements for mare pregnancy determination themselves,
either mare-side or in their practice laboratory.
(1)
Chrisna
J Coetsee, John T France, Andrew N Shelling
Department of Obstetrics
and Gynaecology, University of Auckland,
Faculty of Medical and Health Sciences, Private Bag 92019, Auckland
Steroid sulphatase deficiency,
more commonly known as placental sulphatase deficiency
or X-linked ichthyosis, is defined as a deficiency
in the enzyme steroid sulphatase (STS).
Steroid sulphatase cleaves the sulphate
group at the 3ß position of sterols and steroids, with the best-known natural
substrates being dehydroepiandrosterone sulphate
(DHEAS) in the placenta and cholesterol sulphate
in the skin. This deficiency occurs in 1 in 6000 males and is associated with
female carriers. To understand this deficiency it is necessary to have an
understanding of the distribution and function of the enzyme within normal
placenta and epidermis. The deficiency is caused by mutations in the cognate
STS gene. It is thought that 90% of patients with steroid sulphatase
deficiency have total STS gene deletions, and that the 10% of remaining cases
have point mutations or partial deletions, associated in particular with the
3’ end of the gene.
Twenty eight
Our results
showed that most steroid sulphatase deficient cases
are due to total gene deletions. The
most interesting results found were the identification of two novel point
mutations found in three patients, and new partial deletions found in three
of our patients. These results were
used to determine the correlation between biochemical activity and genetic
defects. The results will also improve understanding of the structure-function
relationship of the STS gene.
NZ6
EFFECT OF INTRACEREBROVENTRICULAR LEPTIN ADMINISTRATION ON
FOOD INTAKE IN PREGNANT AND CYCLING RATS
Sharon Ladyman and Dave Grattan
Center for Neuroendocrinology, Department
of Anatomy and Structural Biology,
Leptin is an adipose-derived peptide
hormone that acts in the hypothalamus to regulate the amount of body fat by
decreasing appetite and increasing metabolic rate. Leptin secretion is proportional to the amount
of body fat present, thereby maintaining fat levels within an appropriate
range. During pregnancy the maternal
body undergoes metabolic adaptations to support the growing conceptus and prepare for the subsequent demands of lactation,
resulting in increases in food intake and fat mass. Serum leptin concentrations also increase during
pregnancy, but these rising levels of leptin are apparently unable to prevent
the pregnancy-induced increases in food intake. The aim of this study was to test the hypothesis
that pregnant rats are resistant to the satiety action of leptin.
The effect of intracerebroventricular
administration of leptin on food intake following a 24 hour fast was measured
in diestrus (control) rats and day 14 pregnant rats. Intracerebroventricular cannulae
were surgically implanted into diestrus rats and
day 7 pregnant rats. Following surgery rats were housed individually so daily
food consumption could be measured during a recovery period of approximately
one week. On metestrus and day
13 of pregnancy food was removed from the cages one hour before lights off.
After a twenty four hour fast, leptin (4 µg) or vehicle was injected
into the left lateral ventricle in a volume of 2 µl.
One hour later, at the time of lights off, food was returned and food
consumption was measured 3 hours and 24 hours later.
On the days preceding treatment the pregnant rats (n=13) ate significantly
more than the cycling rats (n=15). Pregnant rats that were allocated to the treatment
group (n=7) did not have significantly different mean body weight or food
intake prior to leptin administration compared to those pregnant rats allocated
to the vehicle group (n=6). This was also the case for the cycling rat groups.
In diestrus rats, leptin treatment (n=8)
resulted in significantly reduced food intake compared to vehicle-treated
diestrus rats (n=7) at both the 3 hour (6.7 ± 0.4 g vs 9.0 ± 0.3 g P<0.05) and 24
hour (17.2 ±
0.6 g vs 24.8 ± 0.9 g P<0.05) time point
following the injection. In the pregnant rats however, there was no difference
in food intake between the leptin-treated or vehicle-treated rats after 3
hours (7.6 ± 0.2 g vs 7.2 ± 0.2 g) or after 24 hours
(23.5 ±
0.5 g vs 23.8±0.5 g). These results indicate that during pregnancy
leptin no longer acts to decrease food intake, thus supporting the hypothesis
that during pregnancy a state of leptin resistance develops in the hypothalamus.
NZ7
THE FIRST HUMAN MUTATION
(I26T) IN THE REPRESSOR DOMAIN (EH-1) OF THE TRANSCRIPTION
FACTOR HESX1 IS ASSOCIATED WITH
EVOLVING COMBINED PITUITARY HORMONE DEFICIENCY (CPHD) WITHOUT MIDLINE DEFECT.
Luciani R. Carvalho.1; Kathryn Woods.2; Mehul
Dattani 2; Berenice
B Mendonca1, Ivo JPArnhold
1.
1.Laboratório
de Hormônios e Genética Molecular LIM/42, Disciplina de Endocrinologia HCFMUSP;
Sao Paulo, Brazil. 2.Center for
Paediatric Endocrinology, BEM Unit, Inst of Child Health,
HESX1 is a paired-like
homeobox gene implicated in forebrain and pituitary
embryogenesis. The first human mutation (R160C) was identified in
2 siblings with septo-optic dysplasia
(SOD) and an ectopic posterior pituitary lobe (PPL).
Subsequent 4 heterozygous mutations associated with mild phenotype were described.
We screened 78 patients with GH deficiency (GHD), isolated or associated with
other hormonal deficiencies and/or midline defects. We identified a novel
homozygous missense mutation (T77C) leading to a
non-conservative substitution (I26T). The patient was born to consanguineous parents and had -4.7 SD height at age 5 yrs;
pituitary stimulation tests revealed GHD, prepubertal
LH/FSH and normal cortisol, TSH, and PRL responses.
At 14 yr LH and FSH deficiencies were documented. At 21 yr, she had low cortisol and T4 levels indicating ACTH and TSH deficiencies.
MRI revealed a hypoplastic anterior pituitary, thin
pituitary stalk, and ectopic PPL. The normal parents and one sister were heterozygous
for I26T, as well as 1 of 100 normal controls. Polymorphic markers using gene
scan analysis showed a founder effect of this mutation in HESX1. I26 lies in a highly conserved region
of the HESX1 N-terminal domain (eh-1;
aa 21-27), implicated in
transcriptional repression. HESX1 fused to heterologous DNA-binding domain of GAL4 repressed transcription
in a construction containing GAL4-binding sites upstream of the SV40 promoter
driving luciferase reporter gene in transient transfections
in COS-7 cells. HESX1 (I26T) led to impaired repression when compared
with wild-type. Both HESX1 or HESX1
I26T co- trasnfected in COS 7 cells together
with TLE1/Gro fused to VP16 and reporter plasmid containing P3 site
upstream E4 promoter driving luciferase gene showed
an impaired ability to recruit TLE1, groucho mammalian
homologue corepressor. EMSA
with recombinant HESX1 I26T protein bound to a consensus dimeric
P3 site in a similar manner to the wild-type protein. In conclusion, we describe
the first human mutation in the repressor domain of HESX1 (I26T) in a patient
born to consanguineous parents with evolving CPDH, ectopic
PPL whose functional study indicates that the repressor domain has an important
role in pituitary cell differentiation through recruitment of TLE1 correpressor.
NZ8
DELINEATING INTERACTIVE PROCESSES IN THE PITUITARY AND BLOOD
VESSELS
John J Evans, T John Connolly and David
J N Wall
Department of Obstetrics and Gynaecology,
Christchurch School of Medicine and Health Sciences, University of Otago,
Christchurch, and Biomathematics Research Centre, Department of Mathematics
and Statistics, University of Canterbury, Christchurch.
Although biological processes
are necessarily studied as distinct activities, physiologically each compound
acts in concert with many others. The
description of physiology as delineated processes that act in a co-ordinated manner is a challenge.
The pituitary produces a large surge of LH in the female ovulatory cycle as a necessary precursor to ovulation. Gonadotrophin-releasing
hormone (GnRH) is recognised
as a prime factor in forming of the surge.
The action of GnRH has been extensively studied
but there continues to remain debate about a number of details of its action.
A mathematical modelling method was applied to perifusion
data. The model was consistent with
cyclic AMP being an important component of the latter phase of the LH response
to GnRH. Further,
the model suggested that the cAMP activated by a
pulse of GnRH was important to the GnRH self-priming phenomenon exhibited by gonadotrophs in response to subsequent pulses of GnRH. Complicating
the understanding of the formation of the physiological LH surge are observations
that peptides other than GnRH also contribute to
LH regulation. The involvement of cAMP
with the synergistic response of gonadotrophs to oxytocin plus GnRH was added to the model. The mathematical description of the activity
of the community of peptides (including NPY, galanin,
substance P, and endothelin) that modulate LH may
predict targets for pharmacological interventions.
Blood vessels have recently been observed to contain a number
of peptides with vasoactive peptides which participate
in the tonic regulation and pathophysiological alterations
of blood pressure. Investigations have
revealed that the peptides are involved in paracrine interactions between
the endothelial and smooth muscle layers of the vascular tissues. Additionally our studies indicated that paracrine/autocrine processes take place within the endothelial
cells. We conclude that an understanding
of the network of vasoactive peptides (which include
adrenomedullin, endothelin-1 and C-type natriuretic
peptide) will require models that accept the introduction of new peptides
as they are discovered.
Models that describe the dynamic alterations of physiological
environments are required to integrate data from experiments which investigate
isolated processes.
NZ9
Identification
of Amino-Terminal pro-C-Type Natriuretic Peptide in Human, Sheep and Deer
Plasma
Timothy C.R. Pricketta, Timothy G. Yandlea,
Graham K. Barrellb, Martin Wellbyb, M Gary Nichollsa,
Eric A. Espinera and
A. Mark Richardsa.
a Department
of Medicine,
Atrial natriuretic
peptide (ANP), brain natriuretic peptide (BNP) and
C-type natriuretic peptide (CNP) comprise a family
of structurally related peptides that play an important role in the control
of blood pressure, renal function and volume homeostasis. CNP is secreted
by endothelial cells lining blood vessels, influencing vascular tone and remodeling.
CNP is also present in the brain, pituitary and pineal glands, kidney and
at low concentrations in the heart. Like the other members of the natriuretic peptide family, CNP is synthesized as a precursor
(proCNP) which is cleaved to release the biologically
active portion (carboxy-terminal). There are two
biologically active molecular forms of CNP which have distinct tissue distributions,
CNP-22 and CNP-53. However in contrast to other natriuretic peptides, CNP forms so far identified have been
virtually undetectable in blood. By analogy with ANP and BNP we hypothesize
that amino-terminal fragments of proCNP would circulate
at detectable levels. Further we postulate that the length and identity of
the amino-terminal proCNP (NT-proCNP) peptides produced would be determined by the processing
sites for CNP-22 and CNP-53 production.
A specific radioimmunoassay (RIA) was established based on antisera to
the synthetic peptide proCNP(1-15). Plasma NT-proCNP concentrations in 21
normal adults ranged from 15-32 pM and were 15-45 times greater than corresponding
CNP levels. Subjecting sheep pituitary
(known to contain predominantly CNP-53) or deer pituitary extracts to size
exclusion HPLC coupled to RIA revealed
the presence of an immunoreactive peptide with a molecular weight (Mr
approximately 5 kDa) similar to NT-proCNP(1-50), the fragment predicted
from processing of proCNP to CNP-53. Extracts of human, sheep and deer venous
plasma separated by size exclusion HPLC also contained a major 5 kDa immunoreactive peak. There was however little evidence of NT-proCNP(1-81),
a theoretical fragment resulting from a single cleavage of proCNP to yield
CNP-22 the major bioactive form found in blood. It
is possible that NT-proCNP(1-81) is further processed to NT-proCNP(1-50) or
that CNP-53 is processed to CNP-22.
We
have established that amino-terminal proCNP peptides circulate in humans and
other species, and that NT-proCNP(1-50)
is the major form present in human, sheep and deer plasma and pituitary extracts.
NZ10
SEASONAL CHANGES IN PROSTATIC MESOTOCIN AND ANDROGENS
IN THE BRUSHTAIL POSSUM (TRICHOSURUS
VULPECULA)
Jo Fink, Bernie McLeod* and Helen
Nicholson
Department of Anatomy and Structural Biology,
This study investigates
the changes in androgen levels that occur in relation to the seasonal changes
of prostate size in the brushtail possum. The presence of an oxytocin-like
hormone in the prostate was also investigated, as oxytocin
has been implicated in prostate growth and the regulation of androgens in
eutherian mammals.
Male possums were sacrificed throughout the year during the breeding
and nonbreeding periods, and blood samples and prostate
tissue collected. Prostates were divided
into cranial (Cr) and caudal (Ca) areas and either fixed in 10% neutral buffered
formalin for immunocytochemistry of oxytocin and
mesotocin, or frozen in preparation for measurement
by radioimmunoassay of oxytocin and mesotocin, and the androgens testosterone and dihydrotestosterone (DHT).
Significant changes (P<0.05)
in prostate weight occurred throughout the year, with the largest prostate
weights occurring in the breeding season months of March (26.52 ± 8.25
g), April (19.61 ± 6.42 g) and May (23.03 ± 6.62 g), and lowest in the nonbreeding month of January (7.54 ± 1.48 g). Mesotocin immunoreactivity, but not oxytocin
was identified in the prostate. The hormone
was immunolocalised to all the epithelial cells
of the glandular acini. The concentration of mesotocin
in the prostate was significantly higher (P<0.01)
in the breeding season (Cr 124.75 ± 11.43 pg/g, Ca 187.25 ± 31.84 pg/g) when
prostate weights were largest, than in the nonbreeding
season (Cr 100 ± 11.04 pg/g, Ca 122.38 ± 16.32 pg/g) when prostate weights
were lowest. Testosterone and DHT levels
also changed in relation to the breeding periods.
Plasma testosterone was significantly higher in January (4.93 ± 0.71
ng/mL, P<0.01) than in the breeding
months of April (2.09 ± 0.49 ng/mL) and May (2.2 ± 0.45 ng/mL). Plasma DHT was lowest (P<0.05) in March (7.54 ± 1.4 ng/mL), increasing in concentration
throughout the year, and peaking in January (13.31 ± 3.91 ng/mL), before the
onset of the breeding period. In the
cranial prostate, testosterone concentrations were significantly higher in
January (7.83 ± 1.77 ng/g, P<0.05)
than in the breeding month of March (4.63 ± 0.21 ng/g). DHT in the cranial prostate was also significantly
higher (P<0.01) in January (33.86
± 5.06 ng/g), than in March (18.72 ± 0.61 ng/g), and the remainder of the
year.
The seasonal increases in prostate weight
in the months of March, April and May correspond to the time that mating occurs.
Androgen levels begin to increase several months before the onset of
the breeding season, returning to lower levels when mating has ceased.
The concentration of prostatic mesotocin
closely mimics changes in prostate weight, supporting an involvement for the
hormone in prostate growth in the marsupial in addition to the eutherian.
Jillian Cornish
Department of Medicine,
A healthy skeleton is maintained by
the continuous
renewal of bone and this requires that a highly controlled and complexly regulated
balance between bone formation and resorption occurs.
Circulating hormones are important in contributing to the fine control necessary
to achieve and maintain balance, but recent insights indicate that locally
generated cytokines, growth factors and peptides are crucial in influencing
these processes. These paracrine influences are not exclusive of endocrine
effects, because there are many interactions between the circulating hormones
and locally generated factors, the understanding of which would provide a
better appreciation of the cellular and molecular basis of bone remodelling, and could therefore be valuable in approaches
to new therapies.
The B