Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/ß cell ratio and subsequent insulin secretion.

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P = 0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P = 0.001), sustained into adolescence (P = 0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P = 0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P = 0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P = 0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Anti-inflammatory steroid signalling in the human peritoneum.

Peritoneal surface epithelial (PSE) cells participate in adhesion formation following inflammatory injury yet adjacent ovarian SE (OSE) cells regenerate without scarification after ovulation. OSE cells show inflammation-associated expression of 11beta hydroxysteroid dehydrogenase type 1 (11betaHSD1) enzyme, enabling intracrine generation of anti-inflammatory cortisol to minimise tissue damage. We asked if human PSE cells show an 11betaHSD1 response to pro-/anti-inflammatory stimulation and if so, how the 11-oxoreductase activity generated compares with OSE. PSE collected from premenopausal women undergoing surgery for benign gynaecological conditions were used to establish primary PSE cell cultures that were treated for 48 h with interleukin-1alpha (IL-1alpha) with/without anti-inflammatory steroid (cortisol or progesterone). mRNA levels corresponding to the genes of interest (11betaHSD1, 11betaHSD2, cyclooxygenase-2, COX-2) were measured by quantitative RT-PCR. IL-1alpha (0.5 ng/ml) stimulated 11betaHSD1 and COX-2 mRNA levels in PSE cells but 11betaHSD2 was unaffected. Cortisol (1 microM), not progesterone (1 microM), increased 11betaHSD1 mRNA and synergistically enhanced IL-1alpha action. Cortisol suppressed IL-1alpha-stimulated COX-2 more effectively than progesterone. PSE cells had a significantly lower basal 11-oxoreductase enzyme activity than OSE cells; IL-1alpha did not significantly increase the 11-oxoreductase activity in PSE cells but did so in OSE cells. We conclude that PSE cells respond to IL-1alpha and anti-inflammatory steroids in qualitatively similar ways as OSE. However, the enzymatic activity of 11betaHSD1 is lower in PSE and less responsive to IL-1alpha. This could help explain why peritoneal healing often leads to adhesion formation, whereas postovulatory ovarian healing is scar-free.

Maternal nutrient restriction in early pregnancy programs hepatic mRNA expression of growth-related genes and liver size in adult male sheep.

The liver is a major metabolic and endocrine organ of critical importance in the regulation of growth and metabolism. Its function is determined by a complex interaction of nutritionally regulated counter-regulatory hormones. The extent to which hepatic endocrine sensitivity can be programed in utero and whether the resultant adaptations persist into adulthood is unknown and was therefore the subject of this study. Young adult male sheep born to mothers that were fed either a control diet (i.e.100% of total live weight-maintenance requirements) throughout gestation or 50% of that intake (i.e. nutrient restricted (NR)) from 0 to 95 days gestation and thereafter 100% of requirements (taking into account increasing fetal mass) were entered into the study. All mothers gave birth normally at term, the singleton offspring were weaned at 16 weeks, and then reared at pasture until 3 years of age when their livers were sampled. NR offspring were of similar birth and body weights at 3 years of age when they had disproportionately smaller livers than controls. The abundance of mRNA for GH, prolactin, and IGF-II receptors, plus hepatocyte growth factor and suppressor of cytokine signaling-3 were all lower in livers of NR offspring. In contrast, the abundance of the mitochondrial protein voltage-dependent anion channel and the pro-apoptotic factor Bax were up regulated relative to controls. In conclusion, maternal nutrient restriction in early gestation results in adult offspring with smaller livers. This may be mediated by alterations in both hepatic mitogenic and apoptotic factors.

Thyroid hormone signaling in human ovarian surface epithelial cells.

CONTEXT: Ovarian surface epithelial (OSE) cells express multiple nuclear hormone receptor genes, including those encoding thyroid hormone and estrogen receptors (TR and ER, respectively). Ovarian cancer is hormone-dependent, and epidemiological evidence links hyperthyroidism, inflammation of the ovarian surface, and increased risk of ovarian cancer. OBJECTIVE: The objective of this study was to assess T3 action on human OSE cells in vitro, asking 1) is there evidence for (pre)receptor control, 2) is T3 inflammatory, and 3) does T3 affect ER expression? DESIGN: Immunohistochemical analysis of fixed human ovaries and in vitro analysis of human OSE primary cell cultures were performed. PATIENTS: Twelve women aged 29-50 yr (median, 41 yr) undergoing elective gynecological surgery for nonmalignant conditions were studied. RESULTS: Messenger RNA transcripts for TRalpha1, TRalpha2, TRbeta1, and T3 activating deiodinase 2 and inactivating deiodinase 3 were present in primary OSE cell cultures by RT-PCR. TRalpha and TRbeta proteins were also localized to intact OSE by immunohistochemistry. Treatment of OSE cell cultures for 24 h with T3 caused dose-dependent mRNA expression of inflammation-associated genes: cyclooxygenase-2, matrix metalloproteinase-9, and 11betahydroxysteroid dehydrogenase type 1, determined by quantitative RT-PCR. Finally, treatment with T3 dose dependently stimulated ERalpha mRNA expression without affecting ERbeta1 or ERbeta2. CONCLUSION: The ovarian surface is a potential T3 target. T3 exerts direct inflammatory effects on OSE cell function in vitro. OSE cell responses to T3 include increased expression of ERalpha mRNA, which encodes the ER isoform most strongly associated with ovarian cancer. This could help explain suggested epidemiological links between hyperthyroidism and ovarian cancer.

cAMP response element-binding (CREB) signalling and ovarian surface epithelial cell survival.

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREB(S133A)) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREB(WT)) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREB(S133A) markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.

Effects of maternal undernutrition during early pregnancy on apoptosis regulators in the ovine fetal ovary.

This study aimed to determine whether reduced fetal ovary folliculogenesis in ewes undernourished during early/midpregnancy is associated with altered ovarian cell proliferation and/or the expression of apoptosis-regulating genes. Groups of ewes (n = 11-19) were fed either 100% (high; H) or 50% (low; L) of metabolisable energy requirements for live-weight maintenance during selected windows of gestation. All animals were killed at days 50, 65 or 110 of gestation. Between mating and slaughter, control animals were fed the H ration, while animals of other subgroups were fed the L ration from (a) mating to slaughter at 50, 65 or 110 days; (b) 0 to 30 days; (c) 31 to 50 or 65 days; or (d), in the day 110 slaughter group only, from 66 to 110 days. Bouin's-fixed fetal ovaries were examined for (a) Ki67 immunoexpression (proliferation) and (b) Bax and Mcl-1 (apoptosis-regulating genes) expression by in situ hybridisation (day 110) and immunohistochemistry (days 50, 65 and 110). At day 50, maternal nutrition had no effect on Ki67, predominant in germ cells, or Bax and Mcl-1, predominant in the oocytes. Restricted maternal food intake from 0 to 30 days significantly reduced staining for Ki67 in germ cells at day 65 (P < 0.05) but increased staining in granulosa cells at day 110 (P < 0.05). In animals fed the L ration for 110 days, primordial follicle Bax and Mcl-1 were significantly increased (Bax: P < 0.01; Mcl-1: P < 0.05). Granulosa cell Bax was also increased (P < 0.05). When the L ration was fed from 66 to 110 days, granulosa cell Bax (P < 0.05) and primordial follicle Mcl-1 (P < 0.01) were also significantly increased. In the fetal ovarian vasculature, animals underfed for 0-110 days had significantly elevated perivascular Mcl-1 (P < 0.001) and endothelial Bax expression (P < 0.05). Moreover, at day 110, endothelial Mcl-1 was increased by underfeeding from 0 to 30 days (P < 0.05). These data indicate that maternal undernutrition alters proliferation and the expression of apoptosis-regulating genes in the developing fetal ovary. The precise mechanism depends on the window of maternal food restriction.

Inflammation-associated gene expression is altered between normal human ovarian surface epithelial cells and cell lines derived from ovarian adenocarcinomas.

Ovulation is believed to contribute to the development of ovarian cancers that derive from the ovarian surface epithelium (OSE). The process of ovulation is synonymous with inflammation and inflammatory cytokines such as interleukin-1alpha (IL-1alpha) have recently been shown to induce both inflammatory and anti-inflammatory responses in human OSE (HOSE) cells. In this study we directly compared levels of IL-1alpha-induced gene expression by analysing the levels of 11beta-hydroxysteroid dehydrogenase (11betaHSD) types 1 (11betaHSD-1) and 2 (11betaHSD-2), cyclooxygenase-2 (COX-2), IL-1 receptor (IL-1R) and glucocorticoid receptor alpha (GRalpha) mRNA between normal HOSE cells and cell lines derived from poorly differentiated (SKOV-3, BG-1, PEO-4) and well-differentiated (PEO-14) ovarian adenocarcinoma. In HOSE cell cultures, and to a lesser extent PEO-14 cells, the basal mRNA levels of COX-2 and 11betaHSD-1 were relatively high and further shown to be induced in response to IL-1alpha (for HOSE cells; >20-fold, P<0.05 and PEO-14 cells; >3fold, P<0.05). However, whereas HOSE cells expressed a low level of 11betaHSD-2 mRNA that was only mildly responsive to IL-1alpha (1.3-fold, P<0.001), all cell lines exhibited a higher basal level of 11betaHSD-2 mRNA that was in some cases further stimulated in PEO-4 cells (five-fold; P<0.05) or suppressed in SKOV-3 cells (two-fold; P<0.01) in response to IL-1alpha. All cells tested expressed IL-1R and, with the exception of BG-1, GRalpha. These results indicate that cell lines derived from ovarian cancers have lost the ability to respond normally to inflammatory cytokines such as IL-1alpha. The finding that normal OSE cells, in contrast to cell lines derived from patients with ovarian adenocarcinoma, abundantly express 11betaHSD-1 mRNA but are essentially devoid of 11betaHSD-2 mRNA supports the concept that the pattern of 11betaHSD isoform gene expression is a defining feature of neoplastic cellular transformation, which might have particular relevance to the ovary.

Impact of maternal undernutrition and fetal number on glucocorticoid, growth hormone and insulin-like growth factor receptor mRNA abundance in the ovine fetal kidney.

Epidemiological and animal studies strongly indicate that the environment experienced in utero determines, in part, an individual's likelihood of developing cardiovascular disease in later life. This risk has been further linked to impaired kidney function, as a result of compromised development during fetal life. The present study therefore examined the influence of maternal nutrient restriction (NR), targeted at specific periods of kidney development during early to mid gestation, on the mRNA abundance of receptors for glucocorticoid (GCR), growth hormone (GHR) and insulin-like growth factors-I (IGF-IR) and -II (IGF-IIR), and the IGF-I and -II ligands. This was undertaken in both singleton and twin fetuses. At conception ewes were randomly allocated to either an adequately fed control group or one of four nutrient-restricted groups that were fed half the control amount from 0 to 30, 31 to 65, 66 to 110 or 0 to 110 days gestation. At 110 days gestation all ewes were humanely euthanased and fetal kidneys and surrounding adipose tissue sampled. There was no effect of NR or fetal number on kidney weight, shape or nephron number, but the surrounding fat mass was increased in singleton fetuses exposed to NR for 110 days. An increase in kidney mRNA abundance with NR only occurred in singleton fetuses where IGF-IR mRNA was enhanced with NR from 66-110 days gestation. In twin fetuses, NR had no effect on mRNA abundance. However, for all genes examined mRNA expression was lower in the kidneys of twin compared with singleton fetuses following NR, and the magnitude of the effect was dependent on the timing of NR. In conclusion, the abundance of mRNA for receptors which regulate fetal kidney development are lower in twin animals compared with singletons following periods of nutrient deficiency. This may impact on later kidney development and function.

Steroid signalling in human ovarian surface epithelial cells: the response to interleukin-1alpha determined by microarray analysis.

The human ovarian surface epithelium (HOSE) is a common site of gynaecological disease including endometriosis and ovarian cancer, probably due to serial injury-repair events associated with successive ovulations. To comprehend the importance of steroid signalling in the regulation of the HOSE, we used a custom microarray to catalogue the expression of over 250 genes involved in the synthesis and reception of steroid hormones, sterols and retinoids. The array included a subset of non-steroidogenic genes commonly involved in pro-/anti-inflammatory signalling. HOSE cells donated by five patients undergoing surgery for non-malignant gynaecological conditions were cultured for 48 h in the presence and absence of 500 pg/ml interleukin-1alpha (IL-1alpha). Total RNA was reverse-transcribed into biotin-labelled cDNA, which was hybridised to the array and visualised by gold-particle resonance light scattering and charge-coupled device (CCD) camera detection. Results for selected genes were verified by quantitative reverse-transcription PCR. In five out of five cases, untreated HOSE cells expressed genes encoding enzymes required for de novo biosynthesis of cholesterol from acetate and subsequent formation of C21-pregnane and C19-androstane steroids. Consistent with the inability of HOSE cells to synthesise glucocorticoids, oestrogens or 5alpha-reduced androgens de novo, CYP21, CYP19 and 5alpha-reductase were not detected. The only steroidogenic gene significantly up-regulated by IL-1alpha was 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1). Other cytokine-induced genes were IL-6, IL-8, nuclear factor kappaB (NFkappaB) inhibitor alpha, metallothionein-IIA and lysyl oxidase: inflammation-associated genes that respond to glucocorticoids. The only steroidogenic gene significantly suppressed by IL-1alpha was 3betaHSD1. Other genes suppressed by IL-1alpha were aldehyde dehydrogenase (ALDH) 1, ALDH 10, gonadotrophin hormone-releasing hormone receptor, peroxisome proliferation-activated receptor-binding protein (PPAR-bp) and nuclear receptor subfamily 2 group F member 2. These results define a steroidogenic phenotype of cultured HOSE cells and provide a limited expression profile for genes with associated signalling functions. IL-1alpha co-ordinately induces 11betaHSD1 and a panel of glucocorticoid-regulated, inflammation-associated genes in HOSE cells, providing further evidence that cortisol generated by 11betaHSD1 could participate in the local resolution of inflammation associated with ovulation.

Anti-inflammatory and proliferative responses in human and ovine ovarian surface epithelial cells.

The majority of ovarian cancers (>90%) are believed to derive from the ovarian surface epithelium (OSE); a single layer covering the entire surface of the ovary. At ovulation, the OSE cell layer undergoes an inflammatory response, involving cell death and growth, in order to overcome ovarian surface rupture. Abnormalities during these processes are believed to contribute to the development of tumours. Using primary cultures of OSE cells, we have compared anti-inflammatory and proliferative responses directly between human and ovine OSE cells to further establish the use of ovine OSE cells as a suitable model system for the study of human OSE cells. In order to compare effects of inflammatory stimulation, expression and activity of 11betahydroxysteroid dehydrogenase (11betaHSD) type 1 was measured in OSE cells in response to interleukin (IL)-1alpha. As previously identified in human OSE cells, treatment of ovine OSE cells with IL-1alpha stimulated a concomitant increase of 11betaHSD type 1 mRNA (31-fold; P <0.05) and oxoreductase activity, indicating an increased production of anti-inflammatory cortisol. To compare the growth of human and ovine OSE cells, OSE cell number was measured in response to treatment with gonadotropins or growth factors. In the presence of FSH, LH or human chorionic gonadotropin (hCG), ovine and human OSE cell growth was similarly stimulated >1.2-fold (P <0.05). In the presence of connective tissue growth factor (CTGF) and more significantly insulin growth factor I (IGF-I), human and ovine OSE cell growth was also similarly stimulated >1.2-fold (P <0.05) and >1.5-fold (P <0.01), respectively. The induction of both human and ovine OSE cell growth by IGF-I or hCG was further shown to be dependent on activation of the MAP kinase/extracellular-signal-regulated kinase (ERK) pathway. Stimulation of ovine OSE cell growth by hepatocyte growth factor (HGF) was similarly shown to be ERK-dependent; however, for human OSE cells, HGF only mildly stimulated ERK phosphorylation and failed to stimulate OSE cell growth. The demonstration that human and ovine OSE cells share similarities at the level of cell signalling, gene expression and cellular growth supports the use of ovine OSE cells as a suitable model for the study of human OSE cells.

Programming of adult cardiovascular function after early maternal undernutrition in sheep.

The prenatal nutritional environment influences the subsequent risk of hypertension in adulthood. Animal studies have used, generally, the rat as a model species to illustrate the association between maternal nutrient intake and blood pressure in the resulting adult offspring. No study to date has shown programming of adult cardiovascular function in the sheep through maternal dietary intervention. We therefore fed pregnant sheep to either 100% recommended intake from day 0 of gestation to term [ approximately 147 days gestational age (dGA); controls n = 8] or to 50% recommended intake from day 0 to 95 dGA and thereafter to 100% intake (NR; n = 9). Sheep lambed naturally, offspring were weaned at 16 wk, and the male offspring were reared on pasture until 3 yr of age. At this time, cardiovascular catheters were inserted under halothane anesthesia and sheep were allowed 2-4 days recovery. Basal cardiovascular status and pressor responses to infusion of norepinephrine, angiotensin II, and captopril were then assessed alongside basal plasma concentrations of glucose, cortisol, and leptin. NR sheep were of similar birth weight to controls but at 3 yr of age had higher blood pressure before, but not after, feeding. Peripheral sensitivity to vasoconstrictor infusion was similar between dietary groups, although a reflex bradycardia was not apparent in NR sheep during norepinephrine infusion. Circulating leptin correlated well with fat mass and increased more after vasoconstrictor infusion in NR sheep. In conclusion, early NR has been shown to program aspects of cardiovascular control and adipocyte function in adult sheep.

Environmental influences on the fetus and neonate--timing, mechanisms of action and effects on subsequent adult function.

Environmental influences on fetal and neonatal development can affect neural, reproductive, immune and cardiovascular function in adult humans and animals. The effects can be exerted at many different stages of development from before conception to after birth. Effects may even be exerted during a preceding generation. Some known and some possible mechanisms are reviewed. Systems likely to be affected include the brain, hypothalamus, pituitary and adrenal glands and the gonads. The effects may be exerted through altered gene expression at any stage of development or through changes in organ structure or physiology.

Undernutrition of ewe lambs in utero and in early post-natal life does not affect hypothalamic-pituitary function in adulthood.

The effect of undernutrition in utero, during late gestation (from day 100), and early neonatal life on hypothalamic-pituitary function was investigated in female lambs born to ewes fed rations calculated to provide either 100% (high; H) or 70% (low; L) of the energy requirements to sustain a twin pregnancy. Following parturition in early spring, ewes and lambs were maintained on pasture with sward heights of 6 cm (H) or 4 cm (L) until week 8 of lactation and then sward heights of 5 cm (H) or 3 cm (L) until weaning at week 14. Mean lamb birth weights were 18% lower in L than H animals (P<0.05) and mean liveweights were 23% lower in the L animals (P<0.001) at weaning at 14 weeks of age. Liveweight differences were not significant at, or after, 26 weeks of age. There were no significant differences between pre-pubertal H and L animals, either before (26 weeks) or after ovariectomy (31 weeks), with respect to hypothalamic or pituitary activity, as measured by LH pulse frequency, pulse amplitude or mean plasma LH and FSH concentrations and the responses to GnRH injection as measured by LH peak amplitude, respectively. Similarly there were no differences in any of these variables in pubertal animals at 18 months of age. At 31 weeks of age, H animals had significantly lower pituitary GnRH receptor binding (P<0.01) and lower ERalpha mRNA content (P<0.05) than L lambs. There were no differences with treatment in the abundance of mRNA for LHbeta, FSHbeta or GnRH-receptor at 31 weeks of age or in pubertal animals aged 18 months, when there were no significant differences with treatment in GnRH receptor binding or ERalpha mRNA expression. It is concluded that effects on lifetime reproductive function of female sheep of undernutrition during late gestation and early neonatal life are unlikely to be expressed through permanent changes in hypothalamic-pituitary function and are therefore attributable to effects exerted directly on the ovary.

Non-genomic steroid receptors in the bovine ovary.

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [3H]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins (M(r), 55 and 60kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.

Effect of maternal undernutrition on fetal testicular steroidogenesis during the CNS androgen-responsive period in male sheep fetuses.

The aim of this study was to determine the effects of maternal undernutrition, applied during physiologically relevant stages of development of the reproductive system, on reproductive development in male sheep fetuses. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of metabolizable energy requirements for live weight maintenance during selected 'windows', bounded by days 0, 30, 50, 65 and 110 after mating. Ewes of control groups (HH (Expts 1 and 2) and HHH (Expt 3)) were fed the H ration from mating until they were killed at day 50 (Expt 1), day 65 (Expt 2) or day 110 (Expt 3) of gestation, whereas ewes of other groups were fed the L ration for the periods days 0-30 of gestation (LH and LHH), days 31-50 or days 31-65 of gestation (HL and HLH), days 65-110 of gestation (HHL), or day 0 to day 50, day 65 or day 110 of gestation (LL and LLL) when the animals were killed. At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass or fetal testicular mass, but there was increased expression of mRNA for steroidogenic acute regulatory protein (StAR) in the testes of LL animals (P < 0.05) compared with HH controls. Compared with HH animals, the mean plasma testosterone concentrations of LL fetuses tended to be higher, but this result did not reach significance. At day 65 of gestation there were no significant differences between treatments in mean fetal masses, testicular masses, mean plasma testosterone concentrations or StAR mRNA content. At day 110 of gestation, fetal masses in the LLL group were lower (P < 0.01) than those of control fetuses, although no differences in testicular size or fetal plasma testosterone concentrations were recorded. It is concluded that the effects of undernutrition on reproductive development of male sheep fetuses are dependent on the timing of the period of undernutrition.

The effects of undernutrition, in utero, on reproductive function in adult male and female sheep.

The aim of this study was to determine the effects of maternal undernutrition during pregnancy on adult reproductive function in male and female offspring. Groups of ewes were fed rations providing either 100% (High, H) or 50% (Low, L) of estimated metabolisable energy (ME) requirements for pregnancy, from mating until day 95 of gestation, and thereafter were conventionally managed. At 20 months of age, LH and FSH profiles, and LH responses to exogenous GnRH were measured in male and female offspring and, in males, testicular responses to exogenous LH (as measured by testosterone concentrations) were also measured. Undernutrition had no effect on the mean birth weights of lambs of either sex, or on testicular size in male animals at either 6 weeks or 20 months of age. L males exhibited significantly higher FSH concentrations than H males (P < 0.05) but there were no differences with treatment in FSH profiles in females, basal LH profiles or gonadotrophin responses to GnRH in offspring of either sex, and no difference in basal testosterone concentrations or in the testosterone response to exogenous LH administration in males. Semen quality at 20 months of age was unaffected by pre-natal undernutrition but ovulation rate was significantly reduced in L compared to H female offspring (P < 0.05). It is concluded that pre-natal undernutrition had no effect on male reproductive development and adult function, but reduced ovulation rate in female progeny. This effect was not associated with a change in gonadotrophin profiles or pituitary responsiveness.

Maternal undernutrition alters triiodothyronine concentrations and pituitary response to GnRH in fetal sheep.

The aims of this study were to determine which hormones may have a role in the expression of maternal undernutrition effects on reproductive function, in both the developing fetus and the adult offspring. This was undertaken by measuring the effects of long-term maternal undernutrition on metabolic hormone profiles and pituitary responses to single doses of GnRH and GH-releasing factor (GRF) in fetal sheep. From mating, groups of ewes were fed rations providing either 100% (HIGH) or 50% (LOW) of estimated metabolisable energy requirements for pregnancy throughout the experiment until slaughter at approximately 119 days of gestation. Fetal and maternal blood samples were collected from 113 until 119 days of gestation, via carotid and jugular catheters respectively, and assayed for insulin, IGF-I, GH, thyroxine and triiodothyronine (T(3)). Undernutrition had no effects on fetal weight, fetal gonad weight of either sex, fetal insulin or IGF-I concentrations. Male LOW fetuses exhibited a significantly attenuated response (P<0.05) to a bolus challenge of GnRH compared with HIGH fetuses. Basal fetal GH concentrations and the response to exogenous GRF were similar in both treatment groups, although LOW fetuses exhibited more secretory episodes (P<0.01). Mean T(3) concentrations were significantly lower in both the maternal (P<0.01) and fetal (P<0.05) plasma of LOW animals compared with HIGH animals. It is concluded that pituitary function was altered in fetal males and could influence male reproductive development. On the other hand, in female sheep, fetal gonadal abnormalities and reductions in reproductive capacity in adult life which are associated with fetal undernutrition are unlikely to be attributable to altered pituitary function. Additionally, these studies raise the possibility that thyroid hormones may have a role in the expression of maternal undernutrition effects on fetal development.

Effect of maternal undernutrition during pregnancy on early ovarian development and subsequent follicular development in sheep fetuses.

Gonad development in female sheep fetuses is thought to occur in a number of key stages. The aim of this study was to determine the effects of maternal undernutrition, applied at one or more of these critical stages, on fetal ovarian development. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of energy requirements for live weight maintenance during selected 'windows' during gestation. Control ewes (HH and HHH) were fed the H ration from mating until they were killed at days 50, 65 (HH) or 110 (HHH) of gestation, whereas ewes of other groups were fed the L ration for the periods between day 0 and day 30 of gestation (LH and LHH), day 31 and day 50 or 65 of gestation (HL and HLH), day 65 and day 110 of gestation (HHL) or day 0 of gestation until the animals were killed (LL and LLL). At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass but compared with HH animals, mean fetal ovarian mass was significantly lower in HL (P < 0.05) and LL (P < 0.001) animals. At day 65 of gestation, there were significantly fewer germ cells (P < 0.05) at the resting, diplotene stage of initial meiosis in LL animals than there were in HH animals, indicating delayed germ cell maturation and onset of meiosis. Qualitative assessment of proliferative cell nuclear antigen immunostaining indicated that, at day 50 of gestation, staining was located predominantly in the germ cells, whereas by day 65 of gestation, staining was confined predominantly to somatic cells. Undernutrition in each one of these windows was associated with delayed ovarian follicular development (P < 0.05-0.001) as measured by development of the granulosa cell layer at day 110 of gestation. This study demonstrates that undernutrition before and during folliculogenesis can delay fetal follicular development.

Synthesis and characterization of 111In-DTPA-N-TIMP-2: a radiopharmaceutical for imaging matrix metalloproteinase expression.

The matrix metalloproteinases (MMPs) are enzymes involved in the turnover of the extracellular matrix. Their overexpression in tumors is implicated in the metastatic process and may provide a target for diagnostic tumor imaging by using a radiolabeled inhibitor. MMPs are inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs). Thus, TIMPs are potential targeting molecules which could be used as vehicles for selective radionuclide delivery by virtue of their binding to MMPs. The aim of this work was to produce a radiopharmaceutical with which to evaluate this potential. The 127 amino acid N-terminal domain of recombinant human TIMP-2 (N-TIMP-2) was conjugated with the bifunctional chelator diethylenetriamine pentaacetic acid (DTPA). Singly modified DTPA-N-TIMP-2 conjugate (identified by electrospray ionization mass spectrometry) was isolated by anion-exchange chromatography. The primary site of DTPA modification on N-TIMP-2 was mapped to lysine-116, which is distant from the site of MMP interaction. The conjugate was radiolabeled with indium-111 to give 111In-DTPA-N-TIMP-2 with a specific activity of at least 4 MBq/microg and a radiochemical yield and purity of >95%, by incubation with 111InCl3, without need for postlabeling purification. The product was sterile, pyrogen-free, and stable in serum over 48 h and retained full inhibitory activity in a fluorimetric binding assay. With these attributes, 111In-DTPA-N-TIMP-2 is a suitable radiopharmaceutical for in vivo biological and clinical investigation of the potential benefits of imaging MMP expression.

Effects of nutrition and environmental factors on the fetal programming of the reproductive axis.

Research from a wide range of scientific disciplines has shown that the reproductive performance of animals in adult life is determined, in part, by a variety of extraneous influences acting at different stages of development from before conception until after birth. These effects are probably mediated through changes in the hypothalamic-pituitary and gonadal axes but the physiological system that is affected depends on the stage of development at which the influence is applied. The physiological mechanisms through which environmental influences are transmitted to the target organs are, in many cases, complex and poorly understood. Gonadotrophins seem to play a pivotal role in the development of the fetal testis, although effects of environmental influences on GnRH secretion have yet to be demonstrated. Other studies have shown that, at earlier stages of fetal development, the normal ontogeny of gonadal development and function can be disrupted by undernutrition or the influence of endocrine-disrupting compounds. Specifically, in female fetuses, the onset of meiosis is delayed, whereas, in male fetuses, testosterone synthesis is increased as a result of enhanced testicular steroidogenic enzyme activity. Although reproductive performance is clearly influenced by prenatal factors, much further work is required to identify the relationships between developmental abnormalities and adult reproductive function. Work is also required to elucidate further the critical windows in development and the mechanisms by which environmental factors affect the reproductive organs of developing offspring.