Granulosa Cell Specific Loss of Adar in Mice Delays Ovulation, Oocyte Maturation and Leads to Infertility.

Adenosine deaminases acting on RNA-(ADAR) comprise one family of RNA editing enzymes that specifically catalyze adenosine to inosine (A-to-I) editing. A granulosa cell (GC) specific Adar depleted mouse model [Adar flox/flox:Cyp19a1-Cre/+ (gcAdarKO)] was used to evaluate the role of ADAR1 during the periovulatory period. Loss of Adar in GCs led to failure to ovulate at 16 h post-hCG, delayed oocyte germinal vesicle breakdown and severe infertility. RNAseq analysis of GC collected from gcAdarKO and littermate control mice at 0 and 4 h post-hCG following a super-ovulatory dose of eCG (48 h), revealed minimal differences after eCG treatment alone (0 h), consistent with normal folliculogenesis observed histologically and uterine estrogenic responses. In contrast, 300 differential expressed genes (DEGs; >1.5-fold change and FDRP < 0.1) were altered at 4 h post-hCG. Ingenuity pathway analysis identified many downstream targets of estrogen and progesterone pathways, while multiple genes involved in inflammatory responses were upregulated in the gcAdarKO GCs. Temporal expression analysis of GCs at 0, 4, 8, and 12 h post-hCG of Ifi44, Ifit1, Ifit3b, and Oas1g and Ovgp1 confirmed upregulation of these inflammatory and interferon genes and downregulation of Ovgp1 a glycoprotein involved in oocyte zona pellucida stability. Thus, loss of ADAR1 in GCs leads to increased expression of inflammatory and interferon response genes which are temporally linked to ovulation failure, alterations in oocyte developmental progression and infertility.

Ovary-specific depletion of the nuclear receptor Nr5a2 compromises expansion of the cumulus oophorus but not fertilization by intracytoplasmic sperm injection.

The orphan nuclear receptor, liver receptor homolog-1 (aka Nuclear receptor subfamily 5, Group A, Member 2 (Nr5a2)), is widely expressed in mammalian tissues, and its ovarian expression is restricted to granulosa cells of activated follicles. We employed the floxed Nr5a2 (Nr5a2f/f) mutant mouse line and two granulosa-specific Cre lines, Anti-Müllerian hormone receptor- 2 (Amhr2Cre) and transgenic cytochrome P450 family 19 subfamily A polypeptide 1 (tgCyp19Cre), to develop two tissue- and time-specific Nr5a2 depletion models: Nr5a2Amhr2-/- and Nr5a2Cyp19-/-. In the Nr5a2Cyp19-/- ovaries, Nr5a2 was depleted in mural granulosa, but not cumulus cells. We induced follicular development in mutant and wild-type (control, CON) mice with equine chorionic gonadotropin followed 44 h later treatment with human chorionic gonadotropin (hCG) to induce ovulation. Both Nr5a2Amhr2-/- and Nr5a2Cyp19-/- cumulus-oocyte complexes underwent a reduced degree of expansion in vitro relative to wild-type mice. We found downregulation of epiregulin (Ereg), amphiregulin (Areg), betacellulin (Btc) and tumor necrosis factor stimulated gene-6 (Tnfaip6) transcripts in Nr5a2Amhr2-/- and Nr5a2Cyp19-/- ovaries. Tnfaip6 protein abundance, by quantitative immunofluorescence, was likewise substantially reduced in the Nr5a2-depleted model. Transcript abundance for connexin 43 (Gja1) in granulosa cells was lower at 0 h and maximum at 8 h post-hCG in both Nr5a2Amhr2-/- and Nr5a2Cyp19-/- follicles, while Gja1 protein was not different prior to the ovulatory signal, but elevated at 8 h in Nr5a2Amhr2-/- and Nr5a2Cyp19-/- follicles. In both mutant genotypes, oocytes can mature in vivo and resulting embryos were capable of proceeding to blastocyst stagein vitro. We conclude that Nr5a2 is essential for cumulus expansion in granulosa cells throughout follicular development. The disruption of Nr5a2 in follicular somatic cells does not affect the capacity of the oocyte to be fertilized by intracytoplasmic sperm injection.

Reversible infertility in a liver receptor homologue-1 (LRH-1)-knockdown mouse model.

Liver receptor homologue-1 (LRH-1) is an orphan nuclear receptor that has been implicated in steroid hormone biosynthesis and fertility. Herein we describe a transgenic inducible short hairpin (sh) RNA mouse model that was used to study the effect of transient LRH-1 knockdown in vivo. Induction of expression of the shRNA directed against LRH-1 for 2-6 weeks resulted in 80% knockdown of LRH-1 protein in the ovary and complete infertility. Gonadotropin hyperstimulation could not rescue the observed defects in ovulation and corpus luteum formation in LRH-1-knockdown mice. The infertility phenotype was fully reversible because LRH-1-knockdown females became pregnant and delivered normal size litters and healthy pups after cessation of LRH-1 shRNA expression. Timed ovarian microarray analysis showed that, in line with the observed decrease in plasma progesterone levels, key steroid biosynthesis genes, namely Star, Cyp11a1, Hsd3b and Scarb1, were downregulated in LRH-1-knockdown ovaries. In contrast with what has been described previously, no clear effect was observed on oestrogenic activity in LRH-1-knockdown mice. Only Sult1e1 and, surprisingly, Hsd17b7 expression was modulated with potentially opposite effects on oestradiol bioavailability. In conclusion, the fully reversible infertility phenotype of LRH-1-knockdown mice shows the feasibility of an LRH-1 antagonist as new contraceptive therapy with a mechanism of action that most prominently affects cholesterol availability and progesterone production.

Functional transformation of the chromatoid body in mouse spermatids requires testis-specific serine/threonine kinases.

The cytoplasmic chromatoid body (CB) organizes mRNA metabolism and small regulatory RNA pathways, in relation to haploid gene expression, in mammalian round spermatids. However, little is known about functions and fate of the CB at later steps of spermatogenesis, when elongating spermatids undergo chromatin compaction and transcriptional silencing. In mouse elongating spermatids, we detected accumulation of the testis-specific serine/threonine kinases TSSK1 and TSSK2, and the substrate TSKS, in a ring-shaped structure around the base of the flagellum and in a cytoplasmic satellite, both corresponding to structures described to originate from the CB. At later steps of spermatid differentiation, the ring is found at the caudal end of the newly formed mitochondrial sheath. Targeted deletion of the tandemly arranged genes Tssk1 and Tssk2 in mouse resulted in male infertility, with loss of the CB-derived ring structure, and with elongating spermatids possessing a collapsed mitochondrial sheath. These results reveal TSSK1- and TSSK2-dependent functions of a transformed CB in post-meiotic cytodifferentiation of spermatids.

FZD1 regulates cumulus expansion genes and is required for normal female fertility in mice.

WNT4 is required for normal ovarian follicle development and female fertility in mice, but how its signal is transduced remains unknown. Fzd1 encodes a WNT receptor whose expression is markedly induced in both mural granulosa cells and cumulus cells during the preovulatory period, in a manner similar to Wnt4. To study the physiological roles of FZD1 in ovarian physiology and to determine whether it serves as receptor for WNT4, Fzd1-null mice were created by gene targeting. Whereas rare Fzd1(-/-) females were sterile because of uterine fibrosis and ovarian tubulostromal hyperplasia, most were subfertile, producing ≈1 fewer pup per litter on average relative to controls. Unlike WNT4-deficient mice, ovaries from Fzd1(-/-) mice had normal weights, numbers of follicles, steroid hormone production, and WNT4 target gene expression levels. Microarray analyses of granulosa cells from periovulatory follicles revealed few genes whose expression was altered in Fzd1(-/-) mice. However, gene expression analyses of cumulus-oocyte complexes (COCs) revealed a blunted response of both oocyte (Zp3, Dppa3, Nlrp5, and Bmp15) and cumulus (Btc, Ptgs2, Sema3a, Ptx3, Il6, Nts, Alcam, and Cspg2) genes to the ovulatory signal, whereas the expression of these genes was not altered in WNT4-deficient COCs from Wnt4(tm1.1Boer/tm1.1Boer);Tg (CYP19A1-cre)1Jri mice. Despite altered gene expression, cumulus expansion appeared normal in Fzd1(-/-) COCs both in vitro and in vivo. Together, these results indicate that Fzd1 is required for normal female fertility and may act in part to regulate oocyte maturation and cumulus cell function, but it is unlikely to function as the sole ovarian WNT4 receptor.

Sustained release of ancillary amounts of testosterone and alendronate from PLGA coated pericard membranes and implants to improve bone healing.

Testosterone and alendronate have been identified as two bone healing compounds which, when combined, synergistically stimulate bone regeneration. This study describes the development of a novel ultrasonic spray coating for sustained release of ancillary amounts of testosterone and alendronate encapsulated in PLGA 5004A as a carrier. Due to the low amounts of testosterone and alendronate used, sensitive in vitro assays were developed to determine in vitro release. The ultrasonic spray coating technology was optimized for coating titanium screws and pericardial collagen membranes, with the aim to improve osseo-integration and (guided) bone regeneration, respectively, without interfering with their primary mode of action. In vitro release analysis of collagen membranes and screws showed up to 21 days sustained release of the compounds without a burst release. Subsequent preclinical studies in rat and rabbit models indicated that testosterone and alendronate coated membranes and screws significantly improved bone regeneration in vivo. Coated membranes significantly improved the formation of new bone in a critical size calvarial defect model in rats (by 160% compared to controls). Coated screws implanted in rabbit femoral condyles significantly improved bone implant contact (69% vs 54% in controls), bone mineral density (121%) and bone volume (119%) up to 1.3 mm from the implant. Based on the results obtained, we suggest that implants or membranes enabled with local sustained delivery of ancillary amounts of testosterone and alendronate can be a promising system to stimulate local bone regeneration resulting in improved osseo-integration of implants and improved healing of bone defects and fractures.

Evaluation of Collagen Membranes Coated with Testosterone and Alendronate to Improve Guided Bone Regeneration in Mandibular Bone Defects in Minipigs.

OBJECTIVES: The purpose of the present in vivo study was to evaluate whether pericard collagen membranes coated with ancillary amounts of testosterone and alendronate in a poly-lactic glycolic acid (PLGA) carrier as compared to uncoated membranes will improve early bone regeneration. MATERIAL AND METHODS: In each of 16 minipigs, four standardized mandibular intraosseous defects were made bilaterally. The defects were filled with Bio-Oss® granules and covered with a non-coated or coated membrane. Membranes were spray-coated with 4 layers of PLGA containing testosterone and alendronate resulting in 20, 50 or 125 µg/cm2 of testosterone and 20 µg/cm2 alendronate (F20, F50, F125). Non-coated membranes served as controls (F0). Animals were sacrificed at 6 and 12 weeks after treatment. Qualitative and quantitative histological evaluations of bone regeneration were performed. Differences between groups were assessed by paired Student's t-test. RESULTS: Light microscopical analysis showed new bone formation that was in close contact with the Bio-Oss® surface without an intervening non-mineralized tissue layer. Histomorphometric analysis of newly formed bone showed a significant 20% increase in area in the F125 coated membrane treated defects (40 [SD 10]%) compared to the F0 treated defects after 6 weeks (33 [SD 10]%, P = 0.013). At week 12, the total percentage of new bone was increased compared to week 6, but no increase in newly formed bone compared to F0 was observed. CONCLUSIONS: The data from this in vivo study indicate that F125 collagen membranes coated with testosterone and alendronate resulted in superior bone formation (+24%) when normalized to control sites using uncoated membranes.

Early postnatal lethality and cardiovascular defects in CXCR7-deficient mice.

CXCR7 is a G-protein coupled receptor that was recently deorphanized and shown to have SDF1 and I-TAC as high affinity ligands. Here we describe the characterization of CXCR7-deficient mice that were generated to further investigate the function of this receptor in vivo. Expression analysis using a LacZ reporter knockin revealed that postnatally Cxcr7 was specifically expressed in cardiomyocytes, vascular endothelial cells of the lung and heart, the cerebral cortex and in osteocytes of the bone. Adult tissues revealed high expression in cardiomyocytes and osteocytes. The observation that 70% of the Cxcr7-/- mice died in the first week after birth coincides with expression of Cxcr7 in vascular endothelial cells and in cardiomyocytes. An important role of CXCR7 in the cardiovascular system was further supported by the observation that hearts of the Cxcr7-/- mice were enlarged, showed myocardial degeneration and fibrosis of postnatal origin, and hyperplasia of embryonic origin. Despite high expression in osteocytes no apparent bone phenotype was observed, neither in combination with ovariectomy nor orchidectomy. Thus as CXCR7 does not seem to play an important role in bone our data indicate an important function of CXCR7 in the cardiovascular system during multiple steps of development.

Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development.

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.

Differential functions of the Aurora-B and Aurora-C kinases in mammalian spermatogenesis.

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.

Supercritical carbon dioxide decellularised pericardium: Mechanical and structural characterisation for applications in cardio-thoracic surgery.

INTRODUCTION: Many biomaterials are used in cardio-thoracic surgery with good short-term results. However, calcification, dehiscence, and formation of scar tissue are reported. The aim of this research is to characterise decellularised pericardium after supercritical carbon dioxide (scCO2) processing as an alternative biological material for uses in cardio-thoracic surgery. METHODS: Porcine and bovine pericardium were decellularised using scCO2. Mechanical properties such as tensile strength, elastic modulus, fracture toughness and suture retention strength were determined. Ultrastructure was visualised using Scanning Electron Microscopy. Water uptake and swelling was experimentally determined. Commercially available glutaraldehyde treated bovine pericardium was used as gold standard for comparison. RESULTS: scCO2 decellularised porcine (and bovine pericardium) maintained their tensile strength compared to untreated native pericardium (13.3 ± 2.4MPa vs 14.0 ± 4.1MPa, p = 0.73). Tensile strength of glutaraldehyde treated pericardium was significantly higher compared to untreated pericardium (19.4 ± 7.3MPa vs 10.2 ± 2.2MPa, p = 0.02). Suture retention strength of scCO2 treated pericardium was significantly higher than glutaraldehyde treated pericardium (p = 0.01). We found no anisotropy of scCO2 or glutaraldehyde treated pericardium based on a trouser tear test. Ultrastructure was uncompromised in scCO2 treated pericardium, while glutaraldehyde treated pericardium showed deterioration of extracellular matrix. CONCLUSION: scCO2 processing preserves initial mechanical and structural properties of porcine and bovine pericardium, while glutaraldehyde processing damages the extracellular matrix of bovine pericardium. Decellularisation of tissue using scCO2 might give long-term solutions for cardio-thoracic surgery without compromising initial good mechanical properties.

Impaired nipple development and parturition in LGR7 knockout mice.

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.

Core Binding Factor-ß Knockdown Alters Ovarian Gene Expression and Function in the Mouse.

Core binding factor (CBF) is a heterodimeric transcription factor complex composed of a DNA-binding subunit, one of three runt-related transcription factor (RUNX) factors, and a non-DNA binding subunit, CBFß. CBFß is critical for DNA binding and stability of the CBF transcription factor complex. In the ovary, the LH surge increases the expression of Runx1 and Runx2 in periovulatory follicles, implicating a role for CBFs in the periovulatory process. The present study investigated the functional significance of CBFs (RUNX1/CBFß and RUNX2/CBFß) in the ovary by examining the ovarian phenotype of granulosa cell-specific CBFß knockdown mice; CBFß f/f * Cyp19 cre. The mutant female mice exhibited significant reductions in fertility, with smaller litter sizes, decreased progesterone during gestation, and fewer cumulus oocyte complexes collected after an induced superovulation. RNA sequencing and transcriptome assembly revealed altered expression of more than 200 mRNA transcripts in the granulosa cells of Cbfb knockdown mice after human chorionic gonadotropin stimulation in vitro. Among the affected transcripts are known regulators of ovulation and luteinization including Sfrp4, Sgk1, Lhcgr, Prlr, Wnt4, and Edn2 as well as many genes not yet characterized in the ovary. Cbfß knockdown mice also exhibited decreased expression of key genes within the corpora lutea and morphological changes in the ovarian structure, including the presence of large antral follicles well into the luteal phase. Overall, these data suggest a role for CBFs as significant regulators of gene expression, ovulatory processes, and luteal development in the ovary.

Localization of LGR7 gene expression in adult mouse brain using LGR7 knock-out/LacZ knock-in mice: correlation with LGR7 mRNA distribution.

Knowledge of the distribution of the relaxin receptor, LGR7, in the brain provides a basis for studies of the physiologic actions of relaxin. LGR7 knock-out (KO) mice were produced by the in-frame replacement of LGR7 exon 10 and 11 with a LacZ-reporter cassette (knock-in [KI]), and in this study we used LGR7-KO/LacZ-KI mice to determine the regional/cellular distribution of LGR7 gene expression in adult mouse brain by assessing beta-galactosidase activity in perfusion-fixed sections. High densities of beta-galactosidase-positive neurons were detected in anterior olfactory and claustrum/endopiriform nuclei, deep layers of cortex (particularly somatosensory), and the subiculum. Low to moderate densities were detected in olfactory bulb (periglomerular layer), cingulate cortex, subfornical organ, hippocampal CA2/dentate hilus, amygdala, hypothalamus, and thalamus. This LGR7/LacZ expression appears to recapitulate that of native LGR7 in wild-type mice and provides a model to further investigate the phenotype of LGR7-responsive neurons in the brain and to help reveal functions associated with central relaxin signaling.

The orphan nuclear receptor Nr5a2 is essential for luteinization in the female mouse ovary.

In the ovary, the follicular granulosa cells express the nuclear receptor Nr5a2 (nuclear receptor subfamily 5 group A member 2), also known as liver receptor homolog-1, and after ovulation, Nr5a2 expression persists in the corpus luteum. Previous studies demonstrated that Nr5a2 is required for both ovulation and luteal steroid synthesis. Our objectives were to analyze the temporal sequence in the regulatory effects of Nr5a2 in the ovary, with focus on its contribution to luteal function. We developed a female mouse model of granulosa-specific targeted disruption from the formation of the antral follicles forward (genotype Nr5a2(Cyp19-/-)). Mice lacking Nr5a2 in granulosa cells of antral follicles are infertile. Although their cumulus cells undergo expansion after gonadotropin stimulation, ovulation is disrupted in those mice, at least in part, due to the down-regulation of the progesterone receptor (Pgr) gene. The depletion of Nr5a2 in antral follicles permits formation of luteal-like structures but not functional corpora lutea, as evidenced by reduced progesterone levels and failure to support pseudopregnancy. Progesterone synthesis is affected by depletion of Nr5a2 due to, among others, defects in the transport of cholesterol, evidenced by down-regulation of Scarb1, Ldlr, and Star. Comparison of this mouse line with the models in which Nr5a2 is depleted from the primary follicle forward (genotype Nr5a2(Amhr2-/-)) and after the ovulatory signal (genotype Nr5a2(Pgr-/-)) demonstrates that Nr5a2 differentially regulates female fertility across the trajectory of follicular development.

Ovulation involves the luteinizing hormone-dependent activation of G(q/11) in granulosa cells.

The LH receptor (LHR) activates several families of heterotrimeric G proteins, but only the activation of Gs and subsequent generation of cAMP are universally accepted as important mediators of LH actions. To examine the involvement of the Gq/11 family on the actions of LH, we crossed Cyp19Cre and Gαq(f/f);Gα11(-/-) mice to generate mice with a granulosa cell-specific deletion of Gαq in the context of a global deletion of Gα11. Granulosa cells from Gαq(f/f);Gα11(-/-);Cre(+) mice have barely detectable levels of Gαq/11, have a normal complement of LHR, and respond to LHR activation with a transient increase in cAMP accumulation, but they fail to respond with increased inositol phosphate accumulation, an index of the activation of Gαq/11. The LHR-provoked resumption of meiosis, cumulus expansion, and luteinization are normal. However, the Gαq(f/f);Gα11(-/-);Cre(+) mice display severe subfertility because many of the oocytes destined for ovulation become entrapped in preovulatory follicles or corpora lutea. Because follicular rupture is known to be dependent on the expression of the progesterone receptor (Pgr), we examined the LHR-induced expression of Pgr and 4 of its target genes (Adamts-1, Ctsl1, Edn2, and Prkg2). These actions of the LHR were impaired in the ovaries of the Gαq(f/f);Gα11(-/-);Cre(+) mice. We conclude that the defect in follicular rupture is secondary to the failure of the LHR to fully induce the expression of the Pgr. This is the first conclusive evidence for the physiological importance of the activation of Gq/11 by the LHR and for the involvement of Gαq/11 in ovulation.

Loss of GATA-6 and GATA-4 in granulosa cells blocks folliculogenesis, ovulation, and follicle stimulating hormone receptor expression leading to female infertility.

Single GATA-6 (G6(gcko)), GATA-4 (G4(gcko)), and double GATA-4/6 (G4/6(gcko)) granulosa cell-specific knockout mice were generated to further investigate the role of GATA transcription factors in ovarian function in vivo. No reproductive defects were found in G6(gcko) animals. G4(gcko) animals were subfertile as indicated by the reduced number of pups per litter and the release of significantly fewer oocytes at ovulation. In marked contrast, G4/6(gcko) females fail to ovulate and are infertile. Furthermore, G4/6(gcko) females had irregular estrous cycles, which correlate with the abnormal ovarian histology found in unstimulated adult G4/6(gcko) females showing lack of follicular development and increased follicular atresia. Moreover, treatment with exogenous gonadotropins did not rescue folliculogenesis or ovulation in double-knockout G4/6(gcko) mice. In addition, ovary weight and estradiol levels were significantly reduced in G4(gcko) and G4/6(gcko) animals when compared with control and G6(gcko) mice. Aromatase, P450scc, and LH receptor expression was significantly lower in G4(gcko) and G4/6(gcko) mice when compared with control animals. Most prominently, FSH receptor (FSHR) protein was undetectable in granulosa cells of G4(gcko) and G4/6(gcko). Accordingly, gel shift and reporter assays revealed that GATA-4 binds and stimulates the activity of the FSHR promoter. These results demonstrate that GATA-4 and GATA-6 are needed for normal ovarian function. Our data are consistent with a role for GATA-4 in the regulation of the FSHR gene and provide a possible molecular mechanism to explain the fertility defects observed in animals with deficient GATA expression in the ovary.

Inhibitors of cathepsin K: a patent review (2004 - 2010).

INTRODUCTION: Cathepsin K is a lysosomal cysteine protease involved in osteoclast-mediated bone resorption. Inhibition of cathepsin K represents a potentially attractive therapeutic approach for treating diseases characterized by excessive bone resorption, such as osteoporosis. AREAS COVERED: The present review provides an overview of low molecular weight cathepsin K inhibitors published in the patent literature from July 2004 to 2010. Different chemotypes are surveyed and listed according to electrophilic warhead type. Relevant information from original research articles in peer-reviewed journals and clinical investigations is also described. EXPERT OPINION: Between 2004 and 2010, more than 50 patent applications have appeared, underlining the continued interest in small molecule cathepsin K inhibition for therapeutic intervention. Most compounds claimed are peptide-derived inhibitors displaying a reversible binding nitrile or ketone warhead. The success of these compounds in the clinic will be determined by the selectivity that can be achieved against other off-target cathepsin. In this respect, eliminating lysosomotropic characteristics may prove to be crucial in the design of selective cathepsin K inhibitors. During the review period, ONO-5334 and odanacatib have progressed to Phase II and Phase III clinical trials, respectively. The results of these studies are eagerly awaited and may determine the future of these agents as disease-modifying therapeutics.

Gender-specific increase of bone mass by CART peptide treatment is ovary-dependent.

Cocaine- and amphetamine-regulated transcript (CART) has emerged as a neurotransmitter and hormone that has been implicated in many processes including food intake, maintenance of body weight, and reward, but also in the regulation of bone mass. CART-deficient mice are characterized by an osteoporotic phenotype, whereas female transgenic mice overexpressing CART display an increase in bone mass. Here we describe experiments that show that peripheral subcutaneous sustained release of different CART peptide isoforms for a period up to 60 days increased bone mass by 80% in intact mice. CART peptides increased trabecular bone mass, but not cortical bone mass, and the increase was caused by reduced osteoclast activity in combination with normal osteoblast activity. The observed effect on bone was gender-specific, because male mice did not respond to treatment with CART peptides. In addition, male transgenic CART overexpressing mice did not display increased bone mass. Ovariectomy (OVX) completely abolished the increase of bone mass by CART peptides, both in CART peptide-treated wild-type mice and in CART transgenic mice. The effect of CART peptide treatment on trabecular bone was not mediated by 17ß-estradiol (E(2)) because supplementation of OVX mice with E(2) could not rescue the effect of CART peptides on bone. Together, these results indicate that sustained release of CART peptides increases bone mass in a gender-specific way via a yet unknown mechanism that requires the presence of the ovary.

Lrp5 functions in bone to regulate bone mass.

The human skeleton is affected by mutations in low-density lipoprotein receptor-related protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans. We found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also induced an Lrp5 mutation in cells that form the appendicular skeleton but not in cells that form the axial skeleton; we observed that bone properties were altered in the limb but not in the spine. These data indicate that Lrp5 signaling functions locally, and they suggest that increasing LRP5 signaling in mature bone cells may be a strategy for treating human disorders associated with low bone mass, such as osteoporosis.