Context Human embryonic implantation is regulated by neuroendocrine hormones, ovarian steroids, growth factors, and cytokines. Sympathetic innervation of the uterus also may play a role. Objective We tested the hypothesis that cabergoline (Cb), an agonist of type 2 dopamine receptors (DRD2), could influence endometrial decidualization in vitro. Methods Immunohistochemistry confirmed the presence of catecholaminergic neurons in human uterine tissue. DRD2 mRNA and protein expression in endometrial tissue and cells were validated by quantitative RT-PCR, cDNA microarrays, RNA sequencing, and Western blotting. Isolated human endometrial stromal cells (ESC) were subjected to dose-response and time-course experiments in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone, and 0.5 mM dibutyryl cAMP). In some cases, interleukin (IL)-1 beta (0.1 nM) was used as an inflammatory stimulus. Well-characterized in vitro biomarkers were quantified. Results DRD2 were maximally expressed in vivo in the mid-secretory phase of the cycle and upregulated in ESC in response to decidualizing hormones, as were classical (eg, prolactin) and emerging (eg, VEGF and connexin 43) differentiation biomarkers. Cabergoline treatment more than doubled decidual biomarker expression, whereas risperidone, a dopamine receptor antagonist, inhibited ESC differentiation by >50%. Cabergoline induced characteristic decidual morphology changes and blocked detrimental effects of IL-1 beta on decidual cytology. Conclusion Our results support the hypothesis that dopaminergic neurons modulate decidualization in situ. We postulate that dopamine agonists, like Cb, could be developed as therapeutic agents to enhance implantation in couples with inflammation-associated infertility.

Obstetrical complications, often referred to as the "great obstetrical syndromes," are among the most common global causes of mortality and morbidity in young women and their infants. However, treatments for these syndromes are underdeveloped compared with other fields of medicine and are urgently needed. This current paucity of treatments for obstetrical complications is a reflection of the challenges of drug development in pregnancy. The appetite of pharmaceutical companies to invest in research for obstetrical syndromes is generally reduced by concerns for maternal, fetal, and infant safety, poor definition, and high-risk regulatory paths toward product approval. Notably, drug candidates require large investments for development with an unguaranteed return on investment. Furthermore, the discovery of promising drug candidates is hampered by a poor understanding of the pathophysiology of obstetrical syndromes and their uniqueness to human pregnancies. This limits translational extrapolation and de-risking strategies in preclinical studies, as available for other medical areas, compounded with limited fetal safety monitoring to capture early prenatal adverse reactions. In addition, the ethical review committees are reluctant to approve the inclusion of pregnant women in trials, and in the absence of regulatory guidance in obstetrics, clinical development programs are subject to unpredictable regulatory paths. To develop effective and safe drugs for pregnancy complications, substantial commitment, and investment in research for innovative therapies are needed in parallel with the creation of an enabling ethical, legislative, and guidance framework. Solutions are proposed to enable stakeholders to work with a common set of expectations to facilitate progress in this medical discipline. Addressing this significant unmet need to advance maternal and possibly perinatal health requires the involvement of all stakeholders and specifically patients, couples, and clinicians facing pregnancy complications in the dearth of appropriate therapies. This paper focused on the key pharmaceutical research and development challenges to achieve effective and safe treatments for obstetrical syndromes.

DNA damage is a common feature of human spermatozoa associated with an impaired capacity to fertilize the oocyte and an increased mutational load in the offspring. However, the etiology of this damage remains poorly defined. In this study we demonstrate that a major pathway for the induction of DNA damage in mammalian spermatozoa is triggered by exposure to exogenous cell free DNA (cfDNA). Exposure of human and mouse spermatozoa to cfDNA (calf thymus, mouse liver and salmon testes) in vitro induced a dose-dependent increase in sperm DNA damage that could be effectively suppressed by the concomitant presence of DNase. The induction of such damage was not accompanied by any concomitant change in sperm motility or vitality and was not directly associated with the induction of oxidative stress. In vivo the injection of exogenous DNA again precipitated an increase in sperm DNA fragmentation that could be reversed by the prior administration of DNase. Similarly, the induction of a transient unilateral testicular ischemia induced an increase in DNA fragmentation that was evident within 24 h and sustained for at least 14 days via mechanisms that could be completely suppressed by the prior administration of DNase. We conclude that exogenous cfDNA activates a defensive response in human spermatozoa associated with the nuclease-mediated induction of DNA fragmentation, possibly involving the participation of TLR9 and CD4. These novel insights have significant implications for our understanding of DNA fragmentation in the male germ line and open up new pathways for the remediation of this condition.

Chronic pain and subfertility are the main symptoms of concern in women with endometriosis. In order to find new therapeutic options to suppress the pain, translational animal models are indispensable. We have developed a new automated, experimental setup, with full consideration for animal wellbeing, to determine whether operant behaviour can reveal abdominal hyperalgesia in rats with surgically-induced endometriosis, in order to assess whether abdominal hyperalgesia affect behavioural parameters. Endometriosis was induced by transplantation of uterine fragments in the abdominal cavity. Control groups consisted of sham-operated rats and non-operated rats. We have developed an operant chamber (Skinnerbox) which includes a barrier. The rat can climb the barrier in order to reach the food pellet, increasing in this way the pressure to the abdomen. We show that endometriosis rats collect significantly less sugar pellets when compared with the control rats after the introduction of the barrier. In the Skinnerbox experiment, we showed that in a positive operant setting, the introduction of a barrier results in a contrast of operant behaviour of endometriosis rats and control groups, perchance as a result of abdominal discomfort/hyperalgesia due to surgically-induced endometriosis. This is a promising start for the further development of a refined animal model to monitor abdominal discomfort/hyperalgesia in rats with surgically-induced endometriosis.

Asthma is a chronic inflammatory disease characterized by recurrent airway obstruction, airway hyperresponsiveness (AHR), airway inflammation, and airway remodeling, which is often associated with allergy and allergic rhinitis. Many patients with asthma are poorly controlled by current drug treatment, particularly a subgroup of patients with difficult-to-treat severe asthma, characterized by chronic symptoms, severe exacerbations, progressive loss of lung function, and resistance to corticosteroids. New therapeutic options are therefore highly warranted. Our patent covers arginase as a new drug target for the treatment of asthma and/or allergic rhinitis, making use of an arginase inhibitor.1 Arginase is the final enzyme of the hepatic urea cycle, converting l-arginine to l-ornithine and urea. Arginase is also expressed in nonhepatic tissues, including the airways. Two isoforms have been identified, arginases 1 and 2, which are encoded by different genes and are differentially expressed in the body.2 Since l-arginine is also substrate for constitutive and inducible nitric oxide synthases (cNOS and iNOS) yielding l-citrulline and NO, one biological function of extrahepatic arginase may be regulating NO levels through competition with NOS for their common substrate2 (Figure 1). Under healthy conditions, NO, derived from cNOS in airway epithelium and inhibitory nonadrenergic-noncholinergic (iNANC) nerves, has a protective role in the airways by inducing bronchodilation as well as inhibiting airway inflammation and mediator release from mast cells. In allergic asthma, arginases can be upregulated by Th2 cytokines (IL-4, IL-13) and TGF-β, causing reduced cNOS-derived NO production and increased production of pro-contractile and pro-inflammatory peroxynitrite (ONOO−) by particularly inflammation-induced iNOS, by reduced bioavailability of l-arginine to these enzymes. Moreover, increased arginase activity increases the production of l-ornithine and its downstream products polyamines and l-proline, which may be involved in airway remodeling by inducing cell proliferation, and enhanced collagen production and fibrosis, respectively2 (Figure 1). Supporting evidence for this mechanism and thus for a role of arginase in the pathophysiology of allergic asthma and, potentially, other allergic disorders like allergic rhinitis was found in a guinea pig model of allergic asthma. Using this model, we discovered that inhalation of the potent specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) considerably reduces the airway sensitivity to inhaled allergen and protects against allergen-induced early and late asthmatic reactions, AHR after these reactions, and airway inflammation. Moreover, ABH acutely reversed AHR after the early and late asthmatic reaction.3 Based on the observed anti-allergic, bronchoprotective, and anti-inflammatory effects of ABH and indications that arginase may be involved in asthma and allergic rhinitis in patients, we claimed the use of an arginase inhibitor in the prophylactic maintenance treatment of patients with asthma and/or allergic rhinitis, by preventing the development of allergen-induced upper and lower airway obstruction and AHR, wherein the arginase inhibitor is administered by topical inhalation.1 In 1996, by performing perfusion experiments in intact airways from allergen-challenged guinea pigs ex vivo, we demonstrated that a deficiency of cNOS-derived NO may contribute to allergen-induced AHR after the early asthmatic reaction.4 The mechanism of this NO deficiency was unknown. In 1997, we presented some of our data on NO deficiency and asthma on a symposium on the pharmacology of NO in Odense, Denmark. Coincidently, at the same meeting there was the first demonstration that inhibition of arginase by a bona fide arginase inhibitor increased NOS activity in rat alveolar macrophages.5 Although it took almost 10 years to obtain proof of concept,2, 3 it provided an important clue to the underlying mechanism of the allergen-induced NO deficiency and the therapeutic potential of arginase inhibitors in asthma. By using a novel potent and specific arginase inhibitor (Nω-hydroxy-nor-l-arginine), we demonstrated that arginase inhibition reduces guinea pig airway responsiveness in vitro by increasing NO production (see Ref. 2). In ex vivo studies, using a guinea pig model of allergic asthma, we discovered that arginase activity in the airways is increased after allergen challenge, causing AHR after the early asthmatic reaction by reducing the production of neuronal as well as non-neuronal cNOS-derived NO by reduced bioavailability of l-arginine to the enzyme (Ref. 2). Moreover, we found evidence that AHR after the late asthmatic reaction is caused by arginase-induced attenuation of l-arginine availability to particularly iNOS, switching the enzyme to simultaneous production of NO and and, consequently, detrimental ONOO− (Ref. 2). Collectively, these observations paved the way to the proof-of-concept in vivo study presented above.3 Whereas the bronchoprotective effect of ABH was anticipated based on the ex vivo studies, the anti-allergic effect became apparent from the ~30-fold higher allergen dose needed to induce airway obstruction. More recently, we confirmed a role for arginase in airway remodeling by demonstrating that arginase inhibition attenuated airway smooth muscle hyperplasia, airway fibrosis, mucosal gland hypertrophy, and goblet cell hyperplasia following repeated allergen exposure6 (Figure 1). There is growing evidence for an important role of arginase in patients with asthma. Arginase 1 and arginase 2 expression and/or arginase activity are enhanced in asthmatic airways and in serum, and there is an association between arginase expression in bronchial brushings, serum arginase activity, plasma l-arginine, and metabolite concentration and disease severity (lung function and Fe(NO); Refs. 2 and 7). Moreover, ARG1 and ARG2 polymorphisms are associated with asthma, asthma severity (lung function, AHR), and reduced responsiveness to β2-agonists and glucocorticosteroids.8 In addition, enhanced expression of arginases 1 and 2 in nasal mucosa and increased arginase activity in serum have recently been found in patients with allergic rhinitis (Ref. 7). Studies in animal models and in asthmatic patients indicate an important role for both arginase 1 and arginase 2 in the pathophysiology of, particularly severe, asthma and allergic rhinitis. Therefore, arginase inhibitors, having an unique anti-allergic, bronchoprotective, anti-inflammatory, and anti-remodeling profile, may be effective in the treatment of these diseases, possibly guided by the arginine metabolome in blood. Potential drugs are presently under development, some in clinical trials. As no subtype-selective arginase inhibitors are presently available, development of such selective inhibitors to address potential, as yet unknown, differential roles of arginases 1 and 2 may further benefit patients suffering from these diseases. Dr. Meurs reports grants from Lung Foundation Netherlands, grants from N.V. Organon, Oss, The Netherlands, grants from Schering-Plough Research Institute, Oss, The Netherlands, grants from Merck Sharpe and Dohme, Oss, The Netherlands, during the conduct of the study; grants from Netherlands Organisation for Scientific Research (NWO), grants from Carmolex Inc., Pittsburgh, PA, USA, outside the submitted work; In addition, Dr. Meurs has a patent Use of arginase inhibitors in the treatment of asthma and allergic rhinitis licensed to Carmolex Inc., Pittsburgh, PA, USA. Dr. Zaagsma reports grants from Lung Foundation Netherlands, grants from N.V. Organon, Oss, The Netherlands, grants from Schering-Plough Research Institute, Oss, The Netherlands, grants from Merck Sharpe and Dohme, Oss, The Netherlands, during the conduct of the study; In addition, Dr. Zaagsma has a patent Use of arginase inhibitors in the treatment of asthma and allergic rhinitis licensed to Carmolex Inc., Pittsburgh, PA, USA. Dr. Maarsingh reports grants from N.V. Organon, Oss, The Netherlands, grants from Schering-Plough Research Institute, Oss, The Netherlands, personal fees from Schering-Plough, Kenilworth, NJ, USA, grants from Merck Sharpe and Dohme, Oss, The Netherlands, during the conduct of the study; In addition, Dr. Maarsingh has a patent Use of arginase inhibitors in the treatment of asthma and allergic rhinitis licensed to Carmolex Inc., Pittsburgh, PA, USA. At the time of this work Dr. van Duin was an employee of Organon/Schering Plough/Merck MSD and involved in the scientific collaboration with the corresponding author, Dr. Meurs. In addition, Dr. van Duin has a patent Use of arginase inhibitors in the treatment of asthma and allergic rhinitis licensed to Carmolex Inc., Pittsburgh, PA, USA.

Preterm labor occurs in nearly 10% of U.S. pregnancies and is the leading cause of neonatal morbidity and mortality worldwide. Prostaglandins, specifically of the E and F series, play a key role in myometrial contractility and have been recognized as activators of parturition. As such, modulation of select prostaglandin signaling pathways provides a potential approach to pharmaceutical intervention of preterm labor. Prostaglandin E2 is the most abundantly produced prostanoid and functions through four subtypes of G protein coupled receptors (EP1, EP2, EP3, and EP4). It is generally accepted that EP1 and EP3 serve as pro‐contractility receptors while EP2 and EP4 are relaxatory receptors that promote myometrial quiescence. Prostaglandin F2α (PGF2α) functions through the FP receptor and plays an essential role in uterine contractility during preterm or term labor and delivery. The dynamicity and complexity of prostaglandin pharmacology is an important factor to consider during assay development for the testing of molecules which may not be selective to one receptor subtype.Translational ex vivo assays that enhance throughput of drug candidate screening and decrease the use of experimental animals are increasingly desirable. The objective of this research was to determine the relevance of employing either mesenteric resistance artery or uterine artery segments as surrogate tissues to the uterus in the evaluation of prostaglandin‐induced smooth muscle contraction. The relative expression of EP1, EP2, EP3, EP4, and FP receptor mRNA was determined by RT‐qPCR and compared between the uterine body, uterine artery, and mesenteric artery in the rat. Additionally, the efficacy of PGF2α in eliciting contraction was measured by isometric wire myography and compared between the uterine and mesenteric artery.The results of this study demonstrate that rat mesenteric artery may have utility as a model in the screening of prostaglandin receptor‐targeting molecules. Together, these findings will contribute to the identification of suitable and efficient ex vivo assays by which new pharmacological approaches for the treatment of preterm labor can be evaluated.Support or Funding InformationFerring Research Institute, San Diego, CAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.

BACKGROUND:In women, different events of folliculogenesis can be measured and evaluated using ultrasound (US) technology. The availability of a non-invasive translational non-human primate model to study these processes would represent a major contribution to further advance R&D efforts toward novel therapies in assisted reproduction.METHODS:In our study, follicular growth and ovulation was measured in six cyclic Cynomolgus monkeys using abdominal Doppler US.RESULTS:The mean follicular diameter on cycle day -6 (cycle day 0=day of ovulation) was 3.7mm that increased to 6.8mm on cycle day -1. After ovulation, the mean diameter decreased to 4.6mm, confirming ovulation. The mean percentage of follicular size reduction after ovulation was 31%.CONCLUSION:Ultrasonography in combination with color-flow Doppler imaging was shown to be a useful, non-invasive translational method to measure ovarian follicular growth and occurrence and timing of follicular rupture in Cynomolgus monkeys.

Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B–SLLP1 as a pair of novel sperm–egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.

RATIONALE In a guinea pig model of allergic asthma, using perfused tracheal preparations ex vivo, we demonstrated that L-arginine limitation due to increased arginase activity underlies a deficiency of bronchodilating nitric oxide (NO) and airway hyperresponsiveness (AHR) after the allergen-induced early and late asthmatic reaction. OBJECTIVES Using the same animal model, we investigated the acute effects of the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) and of L-arginine on AHR after the early and late reaction in vivo. In addition, we investigated the protection of allergen-induced asthmatic reactions, AHR, and airway inflammation by pretreatment with the drug. METHODS Airway responsiveness to inhaled histamine was measured in permanently instrumented, freely moving guinea pigs sensitized to ovalbumin at 24 hours before allergen challenge and after the allergen-induced early and late asthmatic reactions by assessing histamine PC(100) (provocative concentration causing a 100% increase of pleural pressure) values. MEASUREMENTS AND MAIN RESULTS Inhaled ABH acutely reversed AHR to histamine after the early reaction from 4.77 +/- 0.56-fold to 2.04 +/- 0.34-fold (P < 0.001), and a tendency to inhibition was observed after the late reaction (from 1.95 +/- 0.56-fold to 1.56 +/- 0.47-fold, P < 0.10). Quantitatively similar results were obtained with inhaled l-arginine. Remarkably, after pretreatment with ABH a 33-fold higher dose of allergen was needed to induce airway obstruction (P < 0.01). Consequently, ABH inhalation 0.5 hour before and 8 hours after allergen challenge protected against the allergen-induced early and late asthmatic reactions, AHR and inflammatory cell infiltration. CONCLUSIONS Inhalation of ABH or l-arginine acutely reverses allergen-induced AHR after the early and late asthmatic reaction, presumably by attenuating arginase-induced substrate deficiency to NO synthase in the airways. Moreover, ABH considerably reduces the airway sensitivity to inhaled allergen and protects against allergen-induced bronchial obstructive reactions, AHR, and airway inflammation. This is the first in vivo study indicating that arginase inhibitors may have therapeutic potential in allergic asthma.

The Aurora kinases are cell cycle-regulatory serine-threonine kinases that have been implicated in the function of the centrosomes, kinetechores, chromosome dynamics, and cytokinesis. In comparison with other tissues, there are high levels of expression of Aurora-B and -C in testis. What their respective roles in mammalian spermatogenesis are is an open question. Here we describe the expression and distribution patterns of the three kinases in mouse testis using in situ hybridization and immunohistochemistry. Importantly, the localization of Aurora-B is tightly regulated during spermatogenesis, whereas Aurora-C expression appears to be testis specific. To address the function of Aurora-B in spermatogenesis, we have generated transgenic mice using a pachytene-stage-specific promoter driving the expression of either wild-type Aurora-B or an inactive form of the kinase. Expression of the inactive Aurora-B results in abnormal spermatocytes, increased apoptosis, spermatogenic arrest, and subfertility defects. The function of Aurora-C may also be targeted in the Aurora-B transgenic mutants. To address the function of Aurora-C in testis, we generated Aurora-C knockout mice by homologous recombination. Remarkably, Aurora-C null mice were viable, yet the males had compromised fertility. Aurora-C mutant sperm display abnormalities that included heterogenous chromatin condensation, loose acrosomes, and blunted heads. These findings indicate that Aurora-B and Aurora-C serve specialized functions in mammalian spermatogenesis.

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC−/− spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC−/− spermatozoa and are undetectable after sperm purification. HCO3− is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca2+ entry into sAC−/− spermatozoa. Moreover, the delayed HCO3−-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC−/− spermatozoa. However, sAC−/− sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC−/− sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC−/− spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.

To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility. sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive.

LGR7 is a G-protein coupled receptor with structural homology to the gonadotrophin and thyrotrophin receptors. Recently, LGR7 was deorphanized, and it was shown that relaxin is the ligand for LGR7. To further study the function of this receptor, mice deficient for LGR7 were generated by replacing part of the transmembrane-encoding region with a LacZ reporter cassette. Here we show that LGR7 is expressed in various tissues, including the uterus, heart, brain, and testis. Fertility studies using female LGR7-/- mice showed normal fertility and litter size. However, some females were incapable of delivering their pups, and several pups were found dead. Moreover, all offspring died within 24 to 48 h after delivery because female LGR7-/- mice were unable to feed their offspring due to impaired nipple development. In some male LGR7-/- mice, spermatogenesis was impaired, leading to azoospermia and a reduction in fertility. Interestingly, these phenomena were absent in mutant mice at older ages or in later generations. Taken together, results from LGR7 knockout mice indicate an essential role for the LGR7 receptor in nipple development during pregnancy. Moreover, a defect in parturition was observed, suggesting a role for LGR7 in the process of cervical ripening.

Glycoprotein hormone receptors, including LH receptor, FSH receptor, and TSH receptor, belong to the large G protein-coupled receptor (GPCR) superfamily but are unique in having a large ectodomain important for ligand binding. In addition to two recently isolated mammalian LGRs (leucine-rich repeat-containing, G protein-coupled receptors), LGR4 and LGR5, we further identified two new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors. Phylogenetic analysis showed that there are three LGR subgroups: the known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup represented by LGR7. LGR6 has a subgroup-specific hinge region after leucine-rich repeats whereas LGR7, like snail LGR, contains a low density lipoprotein (LDL) receptor cysteine-rich motif at the N terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed in multiple tissues. Although the putative ligands for LGR6 and LGR7 are unknown, studies on single amino acid mutants of LGR7, with a design based on known LH and TSH receptor gain-of-function mutations, indicated that the action of LGR7 is likely mediated by the protein kinase A but not the phospholipase C pathway. Thus, mutagenesis of conserved residues to allow constitutive receptor activation is a novel approach for the characterization of signaling pathways of selective orphan GPCRs. The present study also defines the existence of three subclasses of leucine-rich repeat-containing, G protein-coupled receptors in the human genome and allows future studies on the physiological importance of this expanding subgroup of GPCR.

Transgenic mice with deletion of the GDF-9 (growth differentiation factor-9) gene are characterized by the arrest of ovarian follicle development at the primary stage. Based on the hypothesis that GDF-9 is important for early follicle development, we isolated rat GDF-9 complementary DNA (cDNA) and generated recombinant GDF-9 protein to study its physiological role. Using bacteria-derived GDF-9-glutathione S-transferase (GST) fusion protein, specific antibodies to the mature form of GDF-9 was generated. Immunohistochemical staining of ovarian sections indicated the localization of GDF-9 protein in the oocyte of primary, secondary and preantral follicles, whereas immunoblotting demonstrated the secretion of GDF-9 by mammalian cells transfected with GDF-9 cDNAs. Recombinant GDF-9 was shown to be an N-glycosylated protein capable of stimulating early follicle development. Growth of preantral follicles isolated from immature rats was enhanced by treatment with either GDF-9 or FSH whereas the combined treatment showed an additive effect. In addition, treatment with GDF-9, like forskolin, also stimulated inhibin-alpha content in explants of neonatal ovaries. In contrast, the stimulatory effects of GDF-9 were not mimicked by amino-terminal tagged GDF-9 that was apparently not bioactive. Thus, the present study demonstrates the important role of GDF-9 in early follicle growth and differentiation. The availability of recombinant bioactive GDF-9 allows future studies on the physiological role of GDF-9 in ovarian development in vivo.