Chromosomal missegregation and aberrant embryo development in repro57 female mice with Rnf212 homozygous mutation.

In brief: Repro57 mice, bearing an Rnf212 gene mutation, exhibit infertility in both homozygous mutant males and females, revealing arrested spermatogenesis in males and investigating unclear mechanisms in females. The study highlights aneuploidy and altered kinetochore patterns in repro57 homozygous mutant oocytes, which impact later stages of embryo development. Abstract: Repro57 mice, induced with N-ethyl-N-nitrosourea and harboring a mutation in the Rnf212 gene, exhibit infertility in both homozygous mutant males and females. Rnf212 plays a crucial role in recombination and crossover designation. In male repro57 homozygous mutants, spermatocytes often degenerate during late prophase, and mature spermatozoa are absent in the seminiferous epithelium, indicating arrested spermatogenesis as the cause of infertility. Despite reports of infertility in Rnf212-knockout female mice, the specific mechanisms underlying infertility in female repro57 homozygous mutants remain elusive. This study investigates the chromosomal and kinetochore patterns of mature oocytes and their developmental potential following in vitro fertilization in female repro57 homozygous mutant mice. While all wild-type oocytes progress to metaphase II and exhibit euploidy, all repro57 homozygous mutant mouse oocytes display aneuploidy. Additionally, kinetochore distances in repro57 homozygous mutant oocytes exceed those observed in wild-type counterparts. Although no significant differences are noted in fertilization and early embryo development rates between wild-type and repro57 homozygous mutant mice, embryos derived from repro57 homozygous mutants exhibit significantly lower morula and blastocyst rates, accompanied by frequent cytokinesis failure and vacuole formation. These findings suggest that the premature segregation of sister chromatids in repro57 homozygous mutant mice adversely impacts the later stages of embryo development.

Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.

Serum amino acid profiles in clinically normal Noma horses.

Plasma or serum amino acids are used to evaluate nutritional status and metabolic disorders. In this study, we aimed to set reference values of serum amino acid concentrations in the Noma horse, a Japanese native horse. Thirty-one horses were classified into six age groups: neonatal foal (0-4 days), foal (0.5-1 years), youth (5 years), middle age (10 years), old (15 years), and extra-old (>20 years). Horses >5 years of age were analyzed together as the adult group. In the adult horses, there were no significant differences among the serum amino acid concentrations of each age group. The foal group had higher concentrations of alanine, aspartic acid, glutamic acid, α-aminoadipic acid, and 3-methyl-histidine than the adult group. The neonatal foal group had higher serum concentrations of phenylalanine, lysine, alanine, proline, aspartic acid, glutamic acid, ß-alanine, and ß-amino-iso-butyric acid and lower tryptophan concentrations and Fischer's ratios than the adult group. The neonatal foal group had higher ß-amino-iso-butyric acid concentrations and lower tryptophan and 3-methyl-histidine concentrations than the foal group. Therefore, reference values might be set separately in neonatal foals, foals, and adult horses. The data for the serum amino acid concentrations can be used for health care through physiological and pathological evaluations in Noma horses.

Genetic characterization of Kushum horses in Kazakhstan based on haplotypes of mtDNA and Y chromosome, and genes associated with important traits of the horses.

The Kushum is a relatively new breed of horses in Kazakhstan that was established in the middle of the 20th century through a cross between mares of Kazakhstan local horses and stallions of Thoroughbred, Trotter, and Russian Don breeds to supply military horses. To reveal the genetic characteristics of this breed, we investigated haplotypes of mitochondrial DNA (mtDNA) and single-nucleotide polymorphisms of the Y chromosome, as well as genotypes of five functional genes associated with coat color, body composition, and locomotion traits. We detected 10 mtDNA haplotypes that fell into 8 of the 17 major haplogroups of horse mtDNA, indicating a unique haplotype composition with high genetic diversity. We also found two Y-chromosomal haplotypes in Kushum horses, which likely originated from Trotter and/or Don breeds. The findings regarding the mtDNA and Y-chromosomal haplotypes are concordant with the documented maternal and paternal origins of the Kushum horses. The allele frequencies of ASIP, MC1R, and MATP associated with coat color were consistent with the coat color variations of Kushum horses. The allele frequencies of MSTN associated with endurance performance and those of DMRT3 associated with gait suggested that the observed allele frequencies of these genes were the result of selective breeding for these traits. As a result of this study, we were able to obtain useful information for a better understanding of the origin and breeding history of the Kushum horse breed using molecular markers.

Hybrid Sterility with Meiotic Metaphase Arrest in Intersubspecific Mouse Crosses.

Although organisms belonging to different species and subspecies sometimes produce fertile offspring, a hallmark of the speciation process is reproductive isolation, characterized by hybrid sterility (HS) due to failure in gametogenesis. In mammals, HS is usually exhibited by males, the heterogametic sex. The phenotypic manifestations of HS are complex. The most frequently observed are abnormalities in both autosomal and sex chromosome interactions that are linked to meiotic prophase arrest or postmeiotic spermiogenesis aberrations and lead to defective or absent gametes. The aim of this study was to determine the HS phenotypes in intersubspecific F1 mice produced by matings between Mus musculus molossinus-derived strains and diverse Mus musculus domesticus-inbred laboratory mouse strains. Most of these crosses produced fertile F1 offspring. However, when female BALB/cJ (domesticus) mice were mated to male JF1/MsJ (molossinus) mice, the (BALBdomxJF1mol)F1 males were sterile, whereas the (JF1molxBALBdom)F1 males produced by the reciprocal crossings were fertile; thus the sterility phenotype was asymmetric. The sterile (BALBdomxJF1mol) F1 males exhibited a high rate of meiotic metaphase arrest with misaligned chromosomes, probably related to a high frequency of XY dissociation. Intriguingly, in the sterile (BALBdomxJF1mol)F1 males we observed aberrant allele-specific expression of several meiotic genes, that play critical roles in important meiotic events including chromosome pairing. Together, these observations of an asymmetrical HS phenotype in intersubspecific F1 males, probably owing to meiotic defects in the meiotic behavior of the XY chromosomes pair and possibly also transcriptional misregulation of meiotic genes, provide new models and directions for understanding speciation mechanisms in mammals.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Liver transplantation: New treatment for mucopolysaccharidosis type VI in rats.

BACKGROUND: Mucopolysaccharidosis (MPS) VI is a rare, autosomal recessive congenital metabolic disorder caused by deficient activity of the lysosomal metabolic enzyme, N-acetylgalactosamine 4-sulfatase. Enzyme replacement therapy (ERT) is the current treatment for MPS VI, although it involves limited compliance to the therapy and high cost. The aim of this study was to develop a new method of treatment by conducting an orthotopic liver transplantation (LTx) using an animal model of human MPS VI, and to evaluate and examine its effectiveness for treating MPS VI. METHODS: LTx was carried out from normal unaffected to affected MPS VI rats (MPR), which were then killed after LTx, and tissues from the heart, spleen, and knee joint, as well as serum, collected for biological and morphologic evaluation. RESULTS: Liver-transplanted (LTx) MPR had the same level of N-acetylgalactosamine 4-sulfatase activity in the liver and lungs as normal unaffected MPR, and the urinary secretion of mucopolysaccharides/glycosaminoglycan (GAG) in LTx MPR was significantly decreased. Furthermore, on histopathology, the spleens of LTx MPR showed elimination of vacuole cells. In the knee joints, growth plates became thinner, and on radiography the facial and cranial bones of LTx MPR were morphologically normal. CONCLUSIONS: LTx from normal to affected MPR was effective for symptoms of MPS and accumulation of GAG, suggesting that LTx could be a promising alternative approach for MPS VI.

Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice.

Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi protein MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi protein MIWI2 (PIWIL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain protein in mediating complex formation with other participants during secondary piRNA biogenesis.

Generation of Evc2/Limbin global and conditional KO mice and its roles during mineralized tissue formation.

Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis 53:612-626, 2015. © 2015 Wiley Periodicals, Inc.

A missense mutation in Rev7 disrupts formation of Polζ, impairing mouse development and repair of genotoxic agent-induced DNA lesions.

Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ζ (Polζ), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Polζ. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive γH2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Polζ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.

Brain natriuretic peptide is able to stimulate cardiac progenitor cell proliferation and differentiation in murine hearts after birth.

Brain natriuretic peptide (BNP) contributes to heart formation during embryogenesis. After birth, despite a high number of studies aimed at understanding by which mechanism(s) BNP reduces myocardial ischemic injury in animal models, the actual role of this peptide in the heart remains elusive. In this study, we asked whether BNP treatment could modulate the proliferation of endogenous cardiac progenitor cells (CPCs) and/or their differentiation into cardiomyocytes. CPCs expressed the NPR-A and NPR-B receptors in neonatal and adult hearts, suggesting their ability to respond to BNP stimulation. BNP injection into neonatal and adult unmanipulated mice increased the number of newly formed cardiomyocytes (neonatal: +23 %, p = 0.009 and adult: +68 %, p = 0.0005) and the number of proliferating CPCs (neonatal: +142 %, p = 0.002 and adult: +134 %, p = 0.04). In vitro, BNP stimulated CPC proliferation via NPR-A and CPC differentiation into cardiomyocytes via NPR-B. Finally, as BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction. In pathological conditions, BNP treatment was cardioprotective by increasing heart contractility and reducing cardiac remodelling. At the cellular level, BNP stimulates CPC proliferation in the non-infarcted area of the infarcted hearts. In the infarcted area, BNP modulates the fate of the endogenous CPCs but also of the infiltrating CD45(+) cells. These results support for the first time a key role for BNP in controlling the progenitor cell proliferation and differentiation after birth. The administration of BNP might, therefore, be a useful component of therapeutic approaches aimed at inducing heart regeneration.

An ENU-induced mutation in the mouse Rnf212 gene is associated with male meiotic failure and infertility.

The ENU-induced repro57 mutation was identified in an unbiased screen for the discovery of novel genes for fertility. Male repro57 homozygous mice are infertile and exhibit significantly reduced testis weight compared with WT mice. Histological examination of mutant testes revealed that spermatocytes degenerated during late prophase, and no mature spermatozoa were found in the seminiferous epithelium, suggesting that infertility is caused by the arrest of spermatogenesis at late meiotic prophase. Consistent with this hypothesis, the number of foci with MLH1, a protein essential for crossing over, is greatly reduced in repro57 mutant spermatocytes, which also lack chiasmata between homologs and exhibit premature dissociation of XY chromosomes. In repro57 mutant mice, we identified a mutation in the Rnf212 gene, encoding Ring finger protein 212. The overall phenotype of repro57 mice is consistent with the recently reported phenotype of the Rnf212 knockout mice; slight differences may be due to genetic background effects. Thus, the repro57 nonsense mutation provides a new allele of the mouse Rnf212 gene.

A mutation in the nuclear pore complex gene Tmem48 causes gametogenesis defects in skeletal fusions with sterility (sks) mice.

Skeletal fusions with sterility (sks) is an autosomal recessive mutation of mouse that results in male and female sterility because of defects in gametogenesis. The mutants also have skeletal malformations with fused vertebrae and ribs. We examined testicular phenotypes of sks/sks mice to investigate the defects in spermatogenesis. Histological and immunocytochemical analyses and expression analyses of the marker genes demonstrated that spermatogenesis is arrested at mid to late pachytene stage of meiotic prophase with defective synapsis of the homologous chromosomes. Next, we determined the precise chromosomal localization of the sks locus on a 0.3-Mb region of mouse chromosome 4 by linkage analysis. By sequencing the positional candidate genes in this region and whole exome sequencing, we found a GG to TT nucleotide substitution in exon 6 of the Tmem48 gene that encodes a putative transmembrane protein with six transmembrane domains. The nucleotide substitution causes aberrant splicing, which deletes exon 6 of the Tmem48 transcript. Specific expression of TMEM48 was observed in germ cells of males and females. Furthermore, the phenotypes of the sks mutant were completely rescued by the transgenesis of a genomic fragment containing the wild-type Tmem48 gene. These findings indicate that the Tmem48 mutation is responsible for the gametogenesis defects and skeletal malformations in the sks mice. The TMEM48 protein is a nuclear membrane protein comprising the nuclear pore complex; its exact function in the nuclear pore complex is still unknown. Our finding suggested that the nuclear pore complex plays an important role in mammalian gametogenesis and skeletal development.

C-type natriuretic peptide specifically acts on the pylorus and large intestine in mouse gastrointestinal tract.

C-type natriuretic peptide (CNP) exerts its main biological effects by binding to natriuretic peptide receptor B (NPR-B), a membrane-bound guanylyl cyclase receptor that produces cyclic guanosine monophosphate (cGMP). CNP is known to cause gastrointestinal (GI) smooth muscle relaxation. Experimental evidence suggests a connection between CNP signaling and GI function, with reactive regions in the GI tract possibly affecting transit; however, this relation has not yet been conclusively shown. Here, we show that CNP plays important region-specific roles in the GI tract of mice. We found that treatment with CNP (1 or 2 mg/kg) increased transient cGMP production in the pylorus, colon, and rectum, with the higher dose (2 mg/kg) enhancing gastric emptying in mice; this increase in cGMP levels was however absent in NPR-B-deficient short-limbed dwarfism (SLW) mouse. Furthermore, we found that NPR-B is highly expressed in the pylorus, colon, and rectum, being localized to nerve fibers and to the nuclei and cytoplasm of smooth muscle cells of the GI tract and blood vessels. Our in vivo findings showed that NPR-B-mediated cGMP production after CNP administration specifically acted on the pylorus, colon, and rectum and contributed to gastric emptying. CNP may thus be a potential therapeutic agent for GI motility/transit disorders such as ileus and pyloric stenosis.

Mutant phenotype analysis suggests potential roles for C-type natriuretic peptide receptor (NPR-B) in male mouse fertility.

BACKGROUND: C-type natriuretic peptide (CNP) signaling through its receptor natriuretic peptide receptor B (NPR-B) is a key molecule for mammalian reproduction, and known to play important roles in female fertility. However, the function of these peptides in mouse male reproduction remains largely unknown. To determine the role of CNP/NPR-B signaling in male reproduction we investigated phenotype of Npr2-deficient short-limbed-dwarfism (Npr2(slw/slw)) mice, which have been shown to have gastrointestinal (GI) abnormalities. FINDINGS: In homozygous Npr2(slw/slw) mice, spermatogenesis is developmentally delayed at both 2 and 4 weeks of age, with vacuolation and degenerating apoptotic germ cells being observed at 3 weeks age. However, the adult Npr2(slw/slw) mice exhibited apparently normal spermatogenesis, albeit with some aberrant spermatids, suggesting that developmental delay was overcome. In addition, the adult Npr2(slw/slw) mice showed abnormal penile morphology (paraphimosis). CONCLUSIONS: The potential role of CNP signaling via the NPR-B receptor in male fertility appears to be mediated not through germ-cell development, but may be through maintenance of normal penile function.

t-SNARE Syntaxin2 (STX2) is implicated in intracellular transport of sulfoglycolipids during meiotic prophase in mouse spermatogenesis.

Syntaxin2 (STX2), also known as epimorphin, is a member of the SNARE family of proteins, with expression in various types of cells. We previously identified an ENU-induced mutation, repro34, in the mouse Stx2 gene. The Stx2(repro34) mutation causes male-restricted infertility due to syncytial multinucleation of spermatogenic cells during meiotic prophase. A similar phenotype is also observed in mice with targeted inactivation of Stx2, as well as in mice lacking enzymes involved in sulfoglycolipid synthesis. Herein we analyzed expression and subcellular localization of STX2 and sulfoglycolipids in spermatogenesis. The STX2 protein localizes to the cytoplasm of germ cells at the late pachytene stage. It is found in a distinct subcellular pattern, presumably in the Golgi apparatus of pachytene/diplotene spermatocytes. Sulfoglycolipids are produced in the Golgi apparatus and transported to the plasma membrane. In Stx2(repro34) mutants, sulfoglycolipids are aberrantly localized in both pachytene/diplotene spermatocytes and in multinucleated germ cells. These results suggest that STX2 plays roles in transport and/or subcellular distribution of sulfoglycolipids. STX2 function in the Golgi apparatus and sulfoglycolipids may be essential for maintenance of the constriction between neighboring developing spermatocytes, which ensures ultimate individualization of germ cells in later stages of spermatogenesis.

Detection of bovine leukemia virus in beef cattle kept in the Central Coast Regions of Vietnam.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leucosis. Our previous study showed the BLV existence in cattle kept in the Red River Delta Region of Vietnam. However, no positive samples were identified in beef cattle. Besides, information related to the BLV circulation in the remained parts of Vietnam is limited. Therefore, we tested the existence of BLV in 48 beef cattle kept in the Central Coast Regions. Nested PCR targeting the BLV-env-gp51 confirmed the prevalence of 14.6% in investigated regions. Phylogenetic analysis suggested the co-existence of genotypes 1 and 10. The close relationship between strains found in Vietnam, Thailand, Myanmar, and China was revealed suggesting the possibility of BLV transmission through the movement of live cattle.

NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation during follicular development in the mouse ovary.

Natriuretic peptide type C (NPPC) and its high affinity receptor, natriuretic peptide receptor 2 (NPR2), have been assumed to be involved in female reproduction and have recently been shown to play an essential role in maintaining meiotic arrest of oocytes. However, the overall role of NPPC/NPR2 signaling in female reproduction and ovarian function is still less clear. Here we report the defects observed in oocytes and follicles of mice homozygous for Nppc(lbab) or Npr2(cn), mutant alleles of Nppc or Npr2 respectively to clarify the exact consequences of lack of NPPC/NPR2 signaling in female reproductive systems. We found that: i) Npr2(cn)/Npr2(cn) female mice ovulated a comparable number of oocytes as normal mice but never produced a litter; ii) all ovulated oocytes of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice exhibited abnormalities, such as fragmented or degenerated ooplasm and never developed to the two-cell stage after fertilization; iii) histological examination of the ovaries of Npr2(cn)/Npr2(cn) and Nppc(lbab)/Nppc(lbab) mice showed that oocytes in antral follicles prematurely resumed meiosis and that immediately before ovulation, oocytes showed disorganized chromosomes or fragmented ooplasm; and iv) ovulated oocytes and oocytes in the periovulatory follicles of the mutant mice were devoid of cumulus cells. These findings demonstrate that NPPC/NPR2 signaling is essential for oocyte meiotic arrest and cumulus oophorus formation, which affects female fertility through the production of oocytes with developmental capacity.

CNP/NPR2 signaling maintains oocyte meiotic arrest in early antral follicles and is suppressed by EGFR-mediated signaling in preovulatory follicles.

Oocyte meiosis is arrested at prophase I by factors secreted from surrounding somatic cells after oocytes acquire meiotic competence at an early antral stage, and meiosis resumes in preovulatory follicles as a result of the luteinizing hormone (LH) surge. Recently, signaling by C-type natriuretic peptide (CNP) through its receptor, natriuretic peptide receptor 2 (NPR2), was found to be essential for meiotic arrest at the late antral stage. Whether or not CNP/NPR2 signaling maintains oocyte meiotic arrest in earlier follicular stages and how it is associated with meiotic resumption induced by the LH surge is unclear. In this study, we examined the expression of Nppc and Npr2, respectively encoding CNP and NPR2, in the ovaries of immature mice. Nppc and Npr2 mRNA were specifically expressed in the outer and inner granulosa cell layers, respectively, in early antral follicles. Histological analysis of mice with a mutation in Npr2 revealed precocious resumption of oocyte meiosis in early antral follicles. Ovaries of mice treated with excess human chorionic gonadotropin (hCG) exhibited markedly decreased Nppc mRNA levels in granulosa cells of preovulatory follicles. Moreover, we found that amphiregulin, a mediator of LH/hCG activity through epidermal growth factor receptor (EGFR), suppressed Nppc mRNA levels in cultured granulosa cells. These results suggest that CNP/NPR2 signaling is essential for oocyte meiotic arrest in early antral follicles and that activated LH/amphiregulin/EGFR signaling pathway suppresses this signal by downregulating Nppc expression.

Skeletal analysis of the long bone abnormality (lbab/lbab) mouse, a novel chondrodysplastic C-type natriuretic peptide mutant.

Long bone abnormality (lbab/lbab) is a strain of dwarf mice. Recent studies revealed that the phenotype is caused by a spontaneous mutation in the Nppc gene, which encodes mouse C-type natriuretic peptide (CNP). In this study, we analyzed the chondrodysplastic skeletal phenotype of lbab/lbab mice. At birth, lbab/lbab mice are only slightly shorter than their wild-type littermates. Nevertheless, lbab/lbab mice do not undergo a growth spurt, and their final body and bone lengths are only ~60% of those of wild-type mice. Histological analysis revealed that the growth plate in lbab/lbab mice, especially the hypertrophic chondrocyte layer, was significantly thinner than in wild-type mice. Overexpression of CNP in the cartilage of lbab/lbab mice restored their thinned growth plate, followed by the complete rescue of their impaired endochondral bone growth. Furthermore, the bone volume in lbab/lbab mouse was severely decreased and was recovered by CNP overexpression. On the other hand, the thickness of the growth plate of lbab/+ mice was not different from that of wild-type mice; accordingly, impaired endochondral bone growth was not observed in lbab/+ mice. In organ culture experiments, tibial explants from fetal lbab/lbab mice were significantly shorter than those from lbab/+ mice and elongated by addition of 10(-7) M CNP to the same extent as lbab/+ tibiae treated with the same dose of CNP. These results demonstrate that lbab/lbab is a novel mouse model of chondrodysplasia caused by insufficient CNP action on endochondral ossification.