The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.
Vertebrate eggs are surrounded by an extracellular matrix with similar functions and conserved individual components: the zona pellucida (ZP) glycoproteins. In mammals, chickens, frogs, and some fish species, we established an updated list of the ZP genes, studied the relationships within the ZP gene family using phylogenetic analysis, and identified ZP pseudogenes. Our study confirmed the classification of ZP genes in six subfamilies: ZPA/ZP2 , ZPB/ZP4 , ZPC/ZP3, ZP1 , ZPAX , and ZPD . The identification of a Zpb pseudogene in the mouse genome, Zp1 pseudogenes in the dog and bovine genomes, and Zpax pseudogenes in the human, chimpanzee, macaque, and bovine genomes showed that the evolution of ZP genes mainly occurs by death of genes. Our study revealed that the extracellular matrix surrounding vertebrate eggs contains three to at least six ZP glycoproteins. Mammals can be classified in three categories. In the mouse, the ZP is composed of three ZP proteins (ZPA/ZP2, ZPC/ZP3, and ZP1). In dog, cattle and, putatively, pig, cat, and rabbit, the zona is composed of three ZP proteins (ZPA/ZP2, ZPB/ZP4, and ZPC/ZP3). In human, chimpanzee, macaque, and rat, the ZP is composed of four ZP proteins (ZPA/ZP2, ZPB/ZP4, ZPC/ZP3, and ZP1). Our review provides new directions to investigate the molecular basis of sperm-egg recognition, a mechanism which is not yet elucidated.
The zebrafish nuclear progestin receptor (nPR; official symbol PGR) was identified and characterized to better understand its role in regulating reproduction in this well-established teleost model. A full-length cDNA was identified that encoded a 617-amino acid residue protein with high homology to PGRs in other vertebrates, and contained five domains characteristic of nuclear steroid receptors. In contrast to the multiplicity of steroid receptors often found in euteleosts and attributed to probable genome duplication, only a single locus encoding the full-length zebrafish pgr was identified. Cytosolic proteins from pgr -transfected cells showed a high affinity ( K d = 2 nM), saturable, single-binding site specific for a native progestin in euteleosts, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). Both 17,20beta-DHP and progesterone were potent inducers of transcriptional activity in cells transiently transfected with pgr in a dual luciferase reporter assay, whereas androgens and estrogens had little potency. The pgr transcript and protein were abundant in the ovaries, testis, and brain and were scarce or undetectable in the intestine, muscle, and gills. Further analyses indicate that Pgr was expressed robustly in the preoptic region of the hypothalamus in the brain; proliferating spermatogonia and early spermatocytes in the testis; and in follicular cells and early-stage oocytes (stages I and II), with very low levels within maturationally competent late-stage oocytes (IV) in the ovary. The localization of Pgr suggests that it mediates progestin regulation of reproductive signaling in the brain, early germ cell proliferation in testis, and ovarian follicular functions, but not final oocyte or sperm maturation.
Interleukin 10 (IL10) is a potent immune-regulating cytokine and inhibitor of inflammatory cytokine synthesis. To evaluate the anti-inflammatory role of IL10 in pregnancy, the response of genetically IL10-deficient mice to low-dose lipopolysaccharide (LPS)-induced abortion was examined. When IL10-null mutant C57Bl/6 ( Il10 â/â ) and control ( Il10 +/+ ) mice were administered low-dose LPS on Day 9.5 of gestation, IL10 deficiency predisposed to fetal loss accompanied by growth restriction in remaining viable fetuses, with an approximately 10-fold reduction in the threshold dose for 100% abortion. After LPS administration, inflammatory cytokines tumor necrosis factor-alpha (TNFA) and IL6 were markedly increased in serum, uterine, and conceptus tissues in Il10 â/â mice compared with Il10 +/+ mice, with elevated local synthesis of Tnfa and Il6 mRNAs in the gestational tissues. IL1A and IL12p40 were similarly elevated in serum and gestational tissues, whereas interferon gamma (IFNG) and soluble TNFRII content were unchanged in the absence of IL10. Recombinant IL10 rescued the increased susceptibility to LPS-induced fetal loss in Il10 â/â mice but did not improve outcomes in Il10 +/+ mice. IL10 genotype also influenced the responsiveness of mice to a TNFA antagonist, etanercept. Fetal loss in Il10 â/â mice was partly alleviated by moderate or high doses of etanercept, whereas Il10 +/+ mice were refractory to high-dose etanercept, consistent with attenuation by IL10 status of TNFA bioavailability after etanercept treatment. These data show that IL10 modulates resistance to inflammatory stimuli by downregulating expression of proinflammatory cytokines TNFA, IL6, IL1A, and IL12, acting to protect against inflammation-induced pathology in the implantation site.
All four CATSPER channel pore-forming subunits (CATSPER1â4) are localized in the sperm principal piece. They form an alkalization-activated Ca 2+ -permeable channel and are required for sperm-hyperactivated motility, egg coat penetration, and male fertility. Unlike many other ion channels, the composition of the CATSPER protein complex is poorly defined. Herein, we describe the novel protein CATSPERG associated with the CATSPER complex. CATSPERG is predicted to be a single transmembrane-spanning protein with a large extracellular domain and a short intracellular tail. Like all the CATSPERs and the previously identified CATSPER-associated protein CATSPERB, CATSPERG is only expressed in testis and is localized in the sperm principal piece. In CATSPER1-deficient sperm, the CATSPERG protein (but not the K + channel protein KCNU1) is also lost. Together with previous findings, our data suggest that the CATSPER protein complex contains pore-forming proteins and two additional proteins (CATSPERB and CATSPERG) and that the trafficking and/or assembly of these proteins depends on CATSPER1.
The Booroola fecundity gene ( FecB ) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23â31, which is syntenic to human chromosome 4q21â25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-Î² (TGF-Î²) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-Î² pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.
ABSTRACT The ovalbumin gene family in Gallus gallus is composed of three homologous genes located within a 46 kb locus on chromosome 2: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX) genes. The expression of these genes in hen oviduct is under estrogen control, but their relative hormonal responsiveness and subsequent protein concentration in egg, is distinctive. Interestingly, all three proteins lack the classical signal peptide for secretion. Ovalbumin, OVAX, and OVAY belong to the serine protease inhibitor (serpin) family whose members share a common tertiary structure. Ovalbumin and OVAX are one of the few members of this family that do not express any protease inhibition activity whereas OVAY has been predicted to be inhibitory, by comparison with the consensus sequence for inhibitory serpins. In contrast to ovalbumin and OVAY, OVAX interacts with heparin, a negatively charged glycosaminoglycan, via a positively charged domain exposed at the surface of the molec...
There are two estrogen receptor (ER) subtypes in fish, ERÎ± and ERÎ², and increasing evidence that the ERÎ² subtype has more than one form. However, there is little information on the characteristics and functional significance of these ERs in adults and during development. Here, we report the cloning and characterization of three functional ER forms, zfERÎ±, zfERÎ²1, and zfERÎ²2, in the zebrafish. The percentages of identity between these receptors suggest the existence of three distinct genes. Each cDNA encoded a protein that specifically bound estradiol with a dissociation constant ranging from 0.4 nM (zfERÎ²2) to 0.75 nM (zfERÎ± and zfERÎ²1). In transiently transfected cells, all three forms were able to induce, in a dose-dependent manner, the expression of a reporter gene driven by a consensus estrogen responsive element; zfERÎ²2 was slightly more sensitive than zfERÎ± and zfERÎ²1. Tissue distribution pattern, analyzed by reverse transcription polymerase chain reaction, showed that the three zfER mRNAs largely overlap and are predominantly expressed in brain, pituitary, liver, and gonads. In situ hybridization was performed to study in more detail the distribution of the three zfER mRNAs in the brain of adult females. The zfER mRNAs exhibit distinct but partially overlapping patterns of expression in two neuroendocrine regions, the preoptic area and the mediobasal hypothalamus. The characterization of these zfERs provides a new perspective for understanding the mechanisms underlying estradiol actions in a vertebrate species commonly used for developmental studies.
To study the rapid action of estrogen on the male reproductive system in teleost, a full-length cDNA homologous to the seven-transmembrane receptor GPER of humans and rodents was cloned from the testis of zebrafish. Biological characterization of this cloned zebrafish gper was performed based on its functional expression in cultured eukaryotic cells. Saturation analysis and Scatchard plotting of [ 3 H]-estradiol binding to plasma membranes of gper -transfected COS-7 cells and cAMP response element transactivation assay demonstrated the biological function of the cloned gper as an estrogen receptor. In addition, treatment of gper -transfected COS-7 cells with 17beta-estradiol increased the phosphorylation of MAPK3/MAPK1. However, the inactivity of Gper in the FOS promoter transactivation study indicated some functional difference between the zebrafish and human receptors. We found gper to be highly expressed in the brain and testis by RT-PCR analysis. Results of in situ hybridization demonstrated the localization of gper in specific brain regions and in early germ cells of the testis, including the spermatogonia, spermatocytes, and somatic cells such as Sertoli cells in adult male zebrafish. Subsequent RT-PCR analysis in cells derived from laser capture microdissection microscopy further confirmed the high expression of gper in early germ cells of the testis. The present study demonstrates the existence of a functionally active Gper in zebrafish and suggests a putative role in mediating the rapid action of estrogen in male reproduction.
Androgens play an important role in male sexual differentiation and development. They exert their function by binding to and activating the androgen receptor (Ar), a member of the steroid hormone receptor superfamily. Here, we report on the isolation and characterization of zebrafish Ar. The complete transcript of zebrafish ar is 5.3 kb long encoding a putative polypeptide of 868 amino acids. Our experimental and bioinformatic analysis has found a single ar locus in zebrafish. Phylogenetic analysis using the ligand-binding domain showed that the zebrafish Ar clustered with its cyprinid orthologs to form a separate group, which was closer to the beta clade than to the alpha clade. Tissue-specific expression analysis revealed that the ar mRNA was expressed ubiquitously in all adult tissues tested, with sexually dimorphic expression in the gonad and muscle. While the ar transcript was maternally deposited into the embryo, signs of zygotic expression could be detected as early as 24 h after fertilization, and the expression level increased substantially afterwards. When analyzed during gonad development, the expression level of ar mRNA at 4 wk after fertilization was similar in both developing gonads but later became higher in the transforming testis, suggesting a potential role during male gonad differentiation. We also combined theoretical modeling with in vitro experiments to show that the zebrafish Ar is preferentially activated by 11-ketotestosterone.