The physiological role of an orphan G protein-coupled receptor, LGR5, was investigated by targeted deletion of this seven-transmembrane protein containing a large N-terminal extracellular domain with leucine-rich repeats.LGR5 null mice exhibited 100% neonatal lethality characterized by gastrointestinal tract dilation with air and an absence of milk in the stomach. Gross and histological examination revealed fusion of the tongue to the floor of oral cavity in the mutant newborns and immunostaining of LGR5 expression in the epithelium of the tongue and in the mandible of the wild-type embryos. The observed ankyloglossia phenotype provides a model for understanding the genetic basis of this craniofacial defect in humans and an opportunity to elucidate the physiological role of the LGR5 signaling system during embryonic development.The leucine-rich repeat-containing, G protein-coupled receptors (LGRs) designated LGR4 through LGR8 are structurally similar to receptors for gonadotropins and thyrotropin (11,12). These receptors are characterized by a large N-terminal extracellular domain containing leucine-rich repeats followed by a seven-transmembrane region. Phylogenetic analyses showed three LGR subgroups: the glycoprotein hormone receptors; the subgroup of LGR4, LGR5, and LGR6; and a third subgroup represented by LGR7 and LGR8 recently found to be receptors for the relaxin family ligands (13,15,23). Evolutionary analyses suggested that these three subgroups of LGRs existed before the divergence of vertebrates and invertebrates (10). LGR5, also known as GPR49, HG38, or FEX, is a large protein consisting of 18 extracellular leucine-rich repeats together with a seven-transmembrane region (8,12,17). UnlikeLGRs in the other two subgroups, the ligands and physiological functions for the LGR4, LGR5, and LGR6 genes are unclear.Ankyloglossia is a rare human craniofacial defect associated with difficulties in an infant's ability to breast-feed and defective speech articulation (6,16,18). In patients with this disorder, the lingual frenulum, which attaches the tongue to the floor of the oral cavity, extends to the tip of the tongue, thereby preventing optimal tongue movement. Limitation of tongue movement may vary from very mild to complete fusion of the tongue to the floor of the mouth. Ankyloglossia in breastfeeding infants can cause ineffective latch, inadequate milk transfer, and maternal nipple pain, resulting in slower weight gain and untimely weaning. Some cases of ankyloglossia are associated with cleft palate (CPX; MIM 303400; OMIM database, Johns Hopkins University, Baltimore, Md.) and are inherited as an X-linked disorder caused by mutations in TBX22, a T-box transcription factor gene located in Xq21 (3).In an attempt to elucidate the physiological roles of the subgroup of orphan LGRs consisting of LGR4, LGR5, and LGR6, we performed gene deletion experiments with LGR5 using a mouse model. Here, we report that the LGR5 null mice exhibit neonatal lethality associated with ankyloglossia characterized by fusion of the ...
Glycoprotein hormones play important roles in thyroid and gonadal function in vertebrates. The glycoprotein hormone alpha-subunit forms heterodimers with different beta-subunits to activate TSH or gonadotropin (LH and FSH) receptors. Recent genomic analyses allowed the identification of another alpha-subunit, GPA2, and another beta-subunit, GPB5, in human, capable of forming heterodimers to activate TSH receptors. Based on comparative genomic searches, we isolated the fly orthologs for human GPA2 and GPB5, each consisting of 10 cysteine residues likely involved in cystine-knot formation. RT-PCR analyses in Drosophila melanogaster demonstrated the expression of GPA2 and GPB5 at different developmental stages. Immunoblot analyses further showed that fly GPA2 and GPB5 subunit proteins are of approximately 16 kDa, and coexpression of these subunits yielded heterodimers. Purified recombinant fly GPA2/GPB5 heterodimers were found to be glycoproteins with N-linked glycosylated alpha-subunits and nonglycosylated beta-subunits, capable of stimulating cAMP production mediated by fly orphan receptor DLGR1 but not DLGR2. Although the fly GPA2/GPB5 heterodimers did not activate human TSH or gonadotropin receptors, chimeric fly GPA2/human GPB5 heterodimers stimulated human TSH receptors. These findings indicated that fly GPA2/GPB5 is a ligand for DLGR1, thus showing the ancient origin of this glycoprotein hormone-seven transmembrane receptor-G protein signaling system. The fly GPA2 also could form heterodimers with human GPB5 to activate human TSH receptors, indicating the evolutionary conservation of these genes and suggesting that the GPA2 subunit may serve as a scaffold for the beta-subunit to activate downstream G protein-mediated signaling.
In mammals, xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO) either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated that atoms in the highly charged active-site loop (Gln-423-Lys-433) around the FAD cofactor underwent large dislocations during the conversion, blocking the approach of the NAD ؉ substrate to FAD in the XO form as well as changing the electrostatic environment around FAD. Here we identify a unique cluster of amino acids that plays a dual role by forming the core of a relay system for the XDH͞XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and sitedirected mutagenesis analysis experiments showed that Phe-549, Arg-335, Trp-336, and Arg-427 sit at the center of a relay system that transmits modifications of the linker peptide by cysteine oxidation or proteolytic cleavage to the active-site loop (Gln-423-Lys-433). The tight interactions of these residues are crucial in the stabilization of the XDH conformation and for keeping the solvent channel closed. Both oxidative and proteolytic generation of XO effectively leads to the removal of Phe-549 from the cluster causing a reorientation of the bulky side chain of Trp-336, which then in turn forces a dislocation of Arg-427, an amino acid located in the active-site loop. The conformational change also opens the gate for the solvent channel, making it easier for oxygen to reach the reduced FAD in XO.X anthine oxidoreductase (XOR) is a homodimer of molecular weight 290,000, and each subunit of the enzyme contains one molybdo-pterin (Mo-pt) cofactor, two distinct [2Fe-2S] centers, and one flavin adenine dinucleotide (FAD) cofactor (1, 2). The mammalian XORs catalyze the hydroxylation of hypoxanthine or xanthine at the Mo center, and reducing equivalents thus introduced into the enzymes are transferred via two [2Fe-2S] centers to FAD, where the reduction of NAD ϩ or molecular oxygen occurs (3). These enzymes are synthesized as the dehydrogenase form [xanthine dehydrogenase (XDH)] but can be readily converted to the oxidase form [xanthine oxidase (XO)] reversibly by oxidation of sulfhydryl residues or irreversibly by proteolysis (1, 2, 4-6). XDH shows a preference for NAD ϩ reduction at the FAD reaction site (although it still displays considerable reactivity with oxygen), whereas XO fails to react with NAD ϩ and exclusively uses dioxygen as its substrate, leading to formation of superoxide anion and hydrogen peroxide (1, 7). Previous investigations have suggested that the XDH͞XO conversion is related to milk lipid secretion (8, 9) and is implicated in diseases characterized by oxygen radical-induced tissue damage such as postischemic reperfusion injury (10-13). Thus, the XDH͞XO transition has attracted much attention from both basic and clinical researchers, not only because of the mechanistic interest in the different reactivity of FAD toward NAD ϩ or oxygen subs...
Since the first baby in Japan conceived as a result of in vitro fertilization (IVF) was born in 1983, the number of assisted reproductive technology (ART) cycles has increased dramatically each year. According to the latest preliminary report from the International Committee Monitoring Assisted Reproductive Technologies for ART worldwide in 2016, Japan was the second largest user of ART worldwide in terms of the annual total number of treatment cycles performed. 1
PurposeThe Japan Society of Obstetrics and Gynecology (JSOG) has collected cycle‐based assisted reproductive technology (ART) data in an online registry since 2007. Herein, we present the characteristics and treatment outcomes of ART cycles registered during 2017.MethodsWe collected cycle‐specific information for all ART cycles implemented at participating facilities and performed descriptive analysis.ResultsIn total, 448,210 treatment cycles and 56,617 neonates (1 in 16.7 neonates born in Japan) were reported in 2017, increased from 2016; the number of initiated fresh cycles decreased for the first time ever. The mean patient age was 38.0 years (standard deviation 4.6). A total 110,641 of 245,205 egg retrieval cycles (45.1%) were freeze‐all cycles; fresh embryo transfer (ET) was performed in 55,720 cycles. A total 194,415 frozen‐thawed ET cycles were reported, resulting in 66,881 pregnancies and 47,807 neonates born. Single ET (SET) was performed in 81.8% of fresh transfers and 83.4% of frozen cycles, with singleton pregnancy/live birth rates of 97.5%/97.3% and 96.7%/96.6%, respectively.ConclusionsTotal ART cycles and subsequent live births increased continuously in 2017, whereas the number of initiated fresh cycles decreased. SET was performed in over 80% of cases, and ET shifted from using fresh embryos to frozen ones.
Purpose The Japan Society of Obstetrics and Gynecology records online annual cycle‐based information for assisted reproductive technology (ART). This report presents the characteristics and treatment outcomes of ART cycles registered during 2019. Methods The Japanese ART registry includes cycle‐specific information from 619 participating facilities, including treatment and pregnancy outcomes. Descriptive analyses were conducted for cycles registered during 2019. Results In 2019, 458 101 treatment cycles and 60 598 neonates were reported, both of which increased from 2018. The number of fresh cycles, including in vitro fertilization and intracytoplasmic sperm injection, decreased, while frozen‐thawed embryo transfer (ET) cycles increased. The mean maternal age was 37.9 years (standard deviation ± 4.7). Of 239 348 oocyte retrievals, 123 690 (51.7%) involved freeze‐all‐embryos cycles; fresh ET was performed in 41 831 cycles (a decreasing trend since 2015). In 2019, there were 211 597 frozen‐thawed ET cycles, resulting in 74 882 pregnancies and 54 168 neonates born. Single ET was performed in 82.6% of fresh transfers and 85.1% of frozen‐thawed cycles, with singleton live birth rates of 97.3% for both. Conclusions The number of fresh cycles decreased but frozen cycles increased in 2019. Single ET was performed in >80% of cases, and the proportion of babies born from frozen‐thawed ET increased.
Purpose Since 1986, the Japan Society of Obstetrics and Gynecology assisted reproductive technology (ART) registry system has collected data on national ART use and outcomes trends in Japan. Herein, we describe the characteristics and outcomes of ART cycles registered during 2020 and compare the results with those from 2019. Methods and Results In 2020, 621 ART facilities participated in the registration. The total number of registered cycles was 449 900, and there were 60 381 live births, which decreased from the previous year (1.79% and 0.36% decrease, respectively). The number of freeze‐all in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles increased in 2020, and the number of neonates born was 2282 for IVF‐embryo transfer (ET) cycles and 2596 for ICSI cycles, which had decreased from the previous year. Frozen–thawed ET (FET) cycles had slightly increased from 2019 (0.04%). In 2020, 215 285 FET cycles were conducted, resulting in 76 196 pregnancies and 55 503 neonates. Single ET was performed in 81.6% of fresh transfers and 85.1% of frozen–thawed cycles, respectively, resulting in over 97% singleton pregnancies/livebirths rates. Conclusion Despite the COVID‐19 pandemic during 2020, the overall number of ART cycles and neonates born demonstrated only a slight decrease in 2020 compared with 2019.
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