The metabolic effects of mirabegron are mediated primarily by ß3 -adrenoceptors.

The ß3 -adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at ß-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [3 H]-2-deoxyglucose uptake, cellular glycolysis, and O2 consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at ß3 -adrenoceptors as they were largely absent in adipocytes derived from ß3 -adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the ß3 -adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the ß3 -adrenoceptor.

Amylin brain circuitry.

Amylin is a peptide hormone that is mainly known to be produced by pancreatic ß-cells in response to a meal but amylin is also produced by brain cells in discrete brain areas albeit in a lesser amount. Amylin receptor (AMY) is composed of the calcitonin core-receptor (CTR) and one of the 3 receptor activity modifying protein (RAMP), thus forming AMY1-3; RAMP enhances amylin binding properties to the CTR. However, amylin receptor agonist such as salmon calcitonin is able to bind CTR alone. Peripheral amylin's main binding site is located in the area postrema (AP) which then propagate the signal to the nucleus of the solitary tract and lateral parabrachial nucleus (LPBN) and it is then transmitted to the forebrain areas such as central amygdala and bed nucleus of the stria terminalis. Amylin's activation of these different brain areas mediates eating and other metabolic pathways controlling energy expenditure and glucose homeostasis. Peripheral amylin can also bind in the arcuate nucleus of the hypothalamus where it acts independently of the AP to activate POMC and NPY neurons. Amylin activation of NPY neurons has been shown to be transmitted to LPBN neurons to act on eating while amylin POMC signaling affects energy expenditure and locomotor activity. While a large amount of experiments have already been conducted, future studies will have to further investigate how amylin is taken up by forebrain areas and deepen our understanding of amylin action on peripheral metabolism.

Unsilencing of native LepRs in hypothalamic SF1 neurons does not rescue obese phenotype in LepR-deficient mice.

Leptin receptor (LepR) signaling in neurons of the ventromedial nucleus of the hypothalamus (VMH), specifically those expressing steroidogenic factor-1 (SF1), have been proposed to play a key role in controlling energy balance. By crossing LepR-silenced (LepRloxTB) mice with those expressing SF1-Cre, we unsilenced native LepR specifically in the VMH and tested whether SF1 neurons in the VMH are critical mediators of leptin's effect on energy homeostasis. LepRloxTB × SF1-Cre [knockout (KO)/Tg+] mice were metabolically phenotyped and compared with littermate controls that either expressed or were deficient in LepRs. Leptin-induced phosphorylated STAT3 was present in the VMH of KO/Tg+ mice and absent in other hypothalamic nuclei. VMH leptin signaling did not ameliorate obesity resulting from LepR deficiency in chow-fed mice. There was no change in food intake or energy expenditure when comparing complete LepR-null mice with KO/Tg+ mice, nor did KO/Tg+ mice show improved glucose tolerance. The presence of functional LepRs in the VMH mildly enhanced sensitivity to the pancreatic hormone amylin. When maintained on a high-fat diet (HFD), there was no reduction in diet-induced obesity in KO/Tg+ mice, but KO/Tg+ mice had improved glucose tolerance after 7 wk on an HFD compared with LepR-null mice. We conclude that LepR signaling in the VMH alone is not sufficient to correct metabolic dysfunction observed in LepR-null mice.

Body weight-dependent and independent improvement in lipid metabolism after Roux-en-Y gastric bypass in ApoE*3Leiden.CETP mice.

BACKGROUND/OBJECTIVES: The incidence of obesity and metabolic syndrome (MetS) has rapidly increased worldwide. Roux-en-Y gastric bypass (RYGB) achieves long-term weight loss and improves MetS-associated comorbidities. Using a mouse model with a humanized lipoprotein metabolism, we elucidated whether improvements in lipid and glucose metabolism after RYGB surgery are body weight loss-dependent or not. SUBJECTS/METHODS: Male ApoE*3Leiden.CETP (ApoE3L.CETP) mice fed Western type diet for 6 weeks underwent RYGB or Sham surgery. Sham groups were either fed ad libitum or were body weight-matched (BWm) to the RYGB mice to discriminate surgical effects from body weight loss-associated effects. Before and after surgery, plasma was collected to assess the metabolic profile, and glucose tolerance and insulin sensitivity were tested. Twenty days after surgery, mice were sacrificed, and liver was collected to assess metabolic, histological and global gene expression changes after surgery. RESULTS: RYGB induced a marked reduction in body weight, which was also achieved by severe food restriction in BWm mice, and total fat mass compared to Sham ad libitum mice (Sham AL). Total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C) and ceramide were strongly reduced 20 days after surgery in RYGB compared to BWm mice. Glucose tolerance and insulin sensitivity improved 13 days after surgery similarly in RYGB and BWm mice. Liver histology confirmed lipid reduction in RYGB and BWm mice while the transcriptomics data indicated altered genes expression in lipid metabolism. CONCLUSIONS: RYGB surgery improves glucose metabolism and greatly ameliorates lipid metabolism in part in a body weight-dependent manner. Given that ApoE3L.CETP mice were extensively studied to describe the MetS, and given that RYGB improved ceramide after surgery, our data confirmed the usefulness of ApoE3L.CETP mice after RYGB in deciphering the metabolic improvements to treat the MetS.

Phenotypical heterogeneity in responder and nonresponder male ApoE*3Leiden.CETP mice.

The metabolic syndrome (MetS) is a major health issue worldwide and is associated with obesity, insulin resistance, and hypercholesterolemia. Several animal models were used to describe the MetS; however, many of them do not mimic well the MetS pathophysiology in humans. The ApoE*3Leiden.CETP mouse model overcomes part of this limitation, since they have a humanised lipoprotein metabolism and a heterogeneous response to MetS, similar to humans. The reported heterogeneity among them and their common classification refer to responder (R) and nonresponder (NR) mice; R mice show increased body weight, cholesterol, and triglycerides levels, whereas NR mice do not show this expected phenotype when fed a Western type diet. To define better the differences between R and NR mice, we focused on feeding behavior, body weight gain, glucose tolerance, and lipid parameters, and on an extensive pathological examination along with liver histology analysis. Our data confirmed that R mice resemble the pathological features of the human MetS: obesity, dysplipidemia, and glucose intolerance. NR mice do not develop the full dysmetabolic phenotype because of a severe inflammatory hepatic condition, which may heavily affect liver function. We conclude that R and NR mice are metabolically different and that NR mice have indications of severely impaired liver function. Hence, it is critical to identify and separate the respective mice to decrease data heterogeneity. Clinical chemistry and histological analysis should be used to confirm retrospectively the animals' classification. Moreover, we point out that NR mice may not be an appropriate control for studies involving ApoE*3Leiden.CETP R mice. NEW & NOTEWORTHY When compared with some other animal models, ApoE*3Leiden.CETP mice are better models to describe the metabolic syndrome. However, there is phenotypic heterogeneity between "responder" and "nonresponder" mice, the latter showing some evidence of hepatic pathology. A full phenotypic characterization and eventually postmortem analysis of the liver are warranted.

Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus.

Amylin phosphorylates ERK (p-ERK) in the area postrema to reduce eating and synergizes with leptin to phosphorylate STAT3 in the arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei to reduce food intake and body weight. The current studies assessed potential amylin and amylin-leptin ARC/VMN interactions on ERK signaling and their roles in postnatal hypothalamic pathway development. In amylin knockout mice, the density of agouti-related protein (AgRP)-immunoreactive (IR) fibers in the hypothalamic paraventricular nucleus (PVN) was increased, while the density of α-melanocyte-stimulating hormone (αMSH) fibers was decreased. In mice deficient of the amylin receptor components RAMP1/3, both AgRP and αMSH-IR fiber densities were decreased, while only αMSH-IR fiber density was decreased in rats injected neonatally in the ARC/VMN with an adeno-associated virus short hairpin RNA against the amylin core receptor. Amylin induced p-ERK in ARC neurons, 60% of which was present in POMC-expressing neurons, with none in NPY neurons. An amylin-leptin interaction was shown by an additive effect on ARC ERK signaling in neonatal rats and a 44% decrease in amylin-induced p-ERK in the ARC of leptin receptor-deficient and of ob/ob mice. Together, these results suggest that amylin directly acts, through a p-ERK-mediated process, on POMC neurons to enhance ARC-PVN αMSH pathway development.

Ghrelin receptor inverse agonists as a novel therapeutic approach against obesity-related metabolic disease.

AIMS: Ghrelin is implicated in the control of energy balance and glucose homeostasis. The ghrelin receptor exhibits ligand-independent constitutive activity, which can be pharmacologically exploited to induce inverse ghrelin actions. Because ghrelin receptor inverse agonists (GHSR-IA) might be effective for the treatment of obesity-related metabolic disease, we tested 2 novel synthetic compounds GHSR-IA1 and GHSR-IA2. MATERIALS AND METHODS: In functional cell assays, electrophysiogical and immunohistochemical experiments, we demonstrated inverse agonist activity for GHSR-IA1 and GHSR-IA2. We used healthy mice, Zucker diabetic fatty (ZDF) rats and diet-induced obese (DIO) mice to explore effects on food intake (FI), body weight (BW), conditioned taste aversion (CTA), oral glucose tolerance (OGT), pancreatic islet morphology, hepatic steatosis (HS), and blood lipids. RESULTS: Both compounds acutely reduced FI in mice without inducing CTA. Chronic GHSR-IA1 increased metabolic rate in chow-fed mice, suppressed FI, and improved OGT in ZDF rats. Moreover, the progression of islet hyperplasia to fibrosis in ZDF rats slowed down. GHSR-IA2 reduced FI and BW in DIO mice, and reduced fasting and stimulated glucose levels compared with pair-fed and vehicle-treated mice. GHSR-IA2-treated DIO mice showed decreased blood lipids. GHSR-IA1 treatment markedly decreased HS in DIO mice. CONCLUSIONS: Our study demonstrates therapeutic actions of novel ghrelin receptor inverse agonists, suggesting a potential to treat obesity-related metabolic disorders including diabetes mellitus.

The area postrema (AP) and the parabrachial nucleus (PBN) are important sites for salmon calcitonin (sCT) to decrease evoked phasic dopamine release in the nucleus accumbens (NAc).

The pancreatic hormone amylin and its agonist salmon calcitonin (sCT) act via the area postrema (AP) and the lateral parabrachial nucleus (PBN) to reduce food intake. Investigations of amylin and sCT signaling in the ventral tegmental area (VTA) and nucleus accumbens (NAc) suggest that the eating inhibitory effect of amylin is, in part, mediated through the mesolimbic 'reward' pathway. Indeed, administration of the sCT directly to the VTA decreased phasic dopamine release (DA) in the NAc. However, it is not known if peripheral amylin modulates the mesolimbic system directly or whether this occurs via the AP and PBN. To determine whether and how peripheral amylin or sCT affect mesolimbic reward circuitry we utilized fast scan cyclic voltammetry under anesthesia to measure phasic DA release in the NAc evoked by electrical stimulation of the VTA in intact, AP lesioned and bilaterally PBN lesioned rats. Amylin (50µg/kg i.p.) did not change phasic DA responses compared to saline control rats. However, sCT (50µg/kg i.p.) decreased evoked DA release to VTA-stimulation over 1h compared to saline treated control rats. Further investigations determined that AP and bilateral PBN lesions abolished the ability of sCT to suppress evoked phasic DA responses to VTA-stimulation. These findings implicate the AP and the PBN as important sites for peripheral sCT to decrease evoked DA release in the NAc and suggest that these nuclei may influence hedonic and motivational processes to modulate food intake.

RYGB progressively increases avidity for a low-energy, artificially sweetened diet in female rats.

Weight re-gain within 2 y after Roux-en-Y gastric bypass (RYGB) is significantly associated with increased intake of and cravings for sweet foods. Here we describe a novel model of this late increase in sweet appetite. Ovariectomized RYGB and Sham-operated rats, with or without estradiol treatment, were maintained on Ensure liquid diet and offered a low-energy, artificially sweetened diet (ASD) 2 h/d. First, we tested rats more than six months after RYGB. ASD meals were larger in RYGB than Sham rats, whereas Ensure meals were smaller. General physical activity increased during ASD meals in RYGB rats, but not during Ensure meals. Second, new rats were adapted to ASD before surgery, and were then offered ASD again during 4-10 wk following surgery. Estradiol-treated RYGB rats lost the most weight and progressively increased ASD intake to >20 g/2 h in wk 9-10 vs. ∼3 g/2 h in Sham rats. Finally, the same rats were then treated with leptin or saline for 8 d. Leptin did not affect body weight, Ensure intake, or activity during meals, but slightly reduced ASD intake in estradiol-treated RYGB rats. Food-anticipatory activity was increased in estradiol-treated RYGB rats during the saline-injection tests. Because increased meal-related physical activity together with larger meals is evidence of hunger in rats, these data suggest that (1) RYGB can increase hunger for a low-energy sweet food in rats and (2) low leptin levels contribute to this hunger, but are not its only cause. This provides a unique rat model for the increased avidity for sweets that is significantly associated with weight recidivism late after RYGB.

Glicentin-related pancreatic polypeptide inhibits glucose-stimulated insulin secretion from the isolated pancreas of adult male rats.

Peptides derived from the glucagon gene Gcg, for example, glucagon and glucagon-like peptide 1 (GLP-1), act as physiological regulators of fuel metabolism and are thus of major interest in the pathogenesis of diseases, such as type-2 diabetes and obesity, and their therapeutic management. Glicentin-related pancreatic polypeptide (GRPP) is a further, 30 amino acid Gcg-derived peptide identified in human, mouse, rat, and pig. However, the potential glucoregulatory function of this peptide is largely unknown. Here, we synthesized rat GRPP (rGRPP) and a closely related peptide, rat GRPP-like peptide (rGRPP-LP), and investigated their actions in the liver and pancreas of adult male rats by employing isolated-perfused organ preparations. Rat GRPP and rGRPP-LP did not affect glucose output from the liver, but both elicited potent inhibition of glucose-stimulated insulin secretion (GSIS) from the rat pancreas. This action is unlikely to be mediated by glucagon or GLP-1 receptors, as rGRPP and rGRPP-LP did not stimulate cyclic adenosine monophosphate (cAMP) production from the glucagon or GLP-1 receptors, nor did they antagonize glucagon- or GLP-1-stimulated cAMP-production at either receptor. GRPP and GRPP-LP may be novel regulators of insulin secretion, acting through an as-yet undefined receptor.

Synthesis of the IGF-II-like hormone vesiculin using regioselective formation of disulfide bonds.

Diabetes mellitus, characterised by hyperglycemia and altered ß-cell function, is an increasingly common disorder affecting millions of individuals world-wide. While therapeutic regimens exist to manage the condition, diabetic individuals remain prone to complications that are detrimental to both their length and quality of life. An improved understanding of the disease which may then enable development of new treatments is therefore a desirable goal. Vesiculin, a novel IGF-II-like protein was recently isolated from the secretory granules of murine ß-cells, and preliminary studies indicate it is capable of signalling via the insulin receptor (IR)/insulin-like growth factor receptor 1(IGF1R) family giving it the potential to elicit both metabolic and mitogenic responses in the beta-cell. In order to facilitate further studies on this new member of the insulin-family of hormones, we undertook a chemical synthesis of the protein using regioselective disulfide bond formation.

A simple and rapid method for identifying and semi-quantifying peptide hormones in isolated pancreatic islets by direct-tissue matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

We describe a new, simple, robust and efficient method based on direct-tissue matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that enables consistent semi-quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet-endocrine cells: (α-cells) glucagon, glicentin-related polypeptide/GRPP; (ß-cells) insulin I, insulin II, C-peptide I, C-peptide II, amylin; (δ-cells) somatostatin-14; and (PP-cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non-ß-cell-hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non-ß-cells. It was also effective in characterizing hormonal phenotype in hemizygous human-amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide-hormone discovery by identifying a prominent new Gcg-gene-derived peptide (theoretical monoisotopic molecular weight 3263.5 Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide-ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies.

Mice lacking the neuropeptide alpha-calcitonin gene-related peptide are protected against diet-induced obesity.

Alpha-calcitonin gene-related peptide (alphaCGRP) is a neuropeptide that is expressed in motor and sensory neurons. It is a powerful vasodilator and has been implicated in diverse metabolic roles. However, its precise physiological function remains unclear. In this study, we investigated the role of alphaCGRP in lipid metabolism by chronically challenging alphaCGRP-specific knockout (alphaCGRP(-/-)) and control mice with high-fat diet regimens. At the start of the study, both animal groups displayed similar body weights, serum lipid markers, and insulin sensitivity. However, alphaCGRP(-/-) mice displayed higher core temperatures, increased energy expenditures, and a relative daytime (nonactive) depression in respiratory quotients, which indicated increased beta-oxidation. In response to fat feeding, alphaCGRP(-/-) mice were comparatively protected against diet-induced obesity with an attenuated body weight gain and an overall reduction in adiposity across all the three diets examined. AlphaCGRP(-/-) mice also displayed improved glucose handling and insulin sensitivity, lower im and hepatic lipid accumulation, and improved overall metabolic health. These findings define a new role for alphaCGRP as a mediator of energy metabolism and opens up therapeutic opportunities to target CGRP action in obesity.

Effects of active immunization against growth differentiation factor 9 and/or bone morphogenetic protein 15 on ovarian function in cattle.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15 in vivo on ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to <25% of that in the controls. In conclusion, immunization against GDF9 and BMP15, alone or together, altered follicular development and ovulation rate in cattle. Thus, as has been observed in sheep, both GDF9 and BMP15 appear to be key regulators of normal follicular development and ovulation rate in cattle.

Evidence that alpha-calcitonin gene-related peptide is a neurohormone that controls systemic lipid availability and utilization.

Alpha-calcitonin gene-related peptide (alphaCGRP) is released mainly from sensory and motor nerves in response to physiological stimuli. Despite well-documented pharmacological effects, its primary physiological role has thus far remained obscure. Increased lipid content, particularly in skeletal muscle and liver, is strongly implicated in the pathogenesis of insulin resistance, but the physiological regulation of organ lipid is imperfectly understood. Here we report our systematic investigations of the effects of alphaCGRP on in vitro and in vivo indices of lipid metabolism. In rodents, levels of alphaCGRP similar to those in the blood markedly stimulated fatty acid beta-oxidation and evoked concomitant mobilization of muscle lipid via receptor-mediated activation of muscle lipolysis. alphaCGRP exerted potent in vivo effects on lipid metabolism in muscle, liver, and the blood via receptor-mediated pathways. Studies with receptor antagonists were consistent with tonic regulation of lipid metabolism by an endogenous CGRP agonist. These data reveal that alphaCGRP is a newly recognized regulator of lipid availability and utilization in key tissues and that it may elevate the availability of intramyocellular free fatty acids to meet muscle energy requirements generated by contraction by evoking their release from endogenous triglyceride.

The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate.

The aims of these studies were to determine the abilities of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions in which dimerization of the protein or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = 10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = 10) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents.

Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants.

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on (3)H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19-40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes.

Immunization of ewes against growth differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. The aims of the present study were to determine the effects of a short-term immunization regimen against BMP15 and GDF9 on ovulation rate, fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, and the ability of immunized ewes to carry a pregnancy to term. Ewes were given a primary and booster immunization against keyhole limpet hemocyanin (KLH; control, n = 50), a GDF9-specific peptide conjugated to KLH (GDF9, n = 30), or a BMP15-specific peptide conjugated to KLH (BMP15, n = 30). The estrous cycles of all ewes were synchronized, and ewes were joined with fertile rams approximately 14 days after the booster immunization. The number of corpora lutea was determined by laparoscopy 3-4 days following mating. Subsequently, about one-half of the ewes in each group underwent an embryo transfer procedure 4-6 days following mating, with the embryos being transferred to synchronized, nonimmunized recipients. The remaining ewes were allowed to carry their pregnancies to term. Short-term immunization against either BMP15 or GDF9 peptides resulted in an increase in ovulation rate with no apparent detrimental affects on fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, or the ability of the immunized ewes to carry a pregnancy to term. Therefore, regulation of BMP15, GDF9, or both is potentially a new technique to enhance fecundity in some mammals.