Asparagine-linked oligosaccharide-independent secretion of egg envelope glycoprotein ZPC of the Japanese quail (Coturnix japonica)

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in the granulosa cells is cleaved at the consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the role of asparagine (N)-linked oligosaccharides in the proteolytic processing of proZPC and the subsequent secretion of ZPC by using site-directed mutagenesis of the consensus sequence for N-glycosylation, and tunicamycin, an inhibitor for N-glycosylation of glycoprotein. Western blot analysis demonstrated that tunicamycin did not block either proteolytic cleavage of proZPC or the subsequent ZPC secretion. Moreover, a site-directed mutant that possesses a mutated sequence for N-glycosylation was efficiently secreted from the cells. These results indicate that proteolytic cleavage of proZPC, and the subsequent ZPC secretion occur in the absence of N-linked oligosaccharides. Therefore, the addition of N-glycans to ZPC polypeptide is not required for quail ZPC secretion.     

Variant of perivitelline membrane glycoprotein ZPC of Japanese quail (Coturnix japonica) lacking its cytoplasmic tail exhibits the retention in the endoplasmic reticulum of Chinese hamster ovary (CHO-K1) cells

Avian perivitelline membrane, an investment homologous to the mammalian zona pellucida, is composed of at least two glycoproteins. Our previous studies demonstrated that one of its components, ZPC, which is synthesized in the ovarian granulosa cells, is secreted after carboxy-terminal proteolytic processing, and this event is a prerequisite event for ZPC secretion in quail. In the present study, we examined the role of the cytoplasmic tail, which is successfully removed after proteolytic processing, in membrane transport, proteolytic processing, and the secretion of quail ZPC. In pursuit of this, we produced a truncated ZPC mutant lacking the cytoplasmic tail located in its C-terminus and examined its expression in the mammalian cell line. Western blot analyses demonstrated that the cytoplasmic tail-deficient ZPC was neither secreted nor underwent proteolytic processing in the cells. Immunofluorescence analysis and the acquisition of resistance to endoglycosidase H digestion of the cytoplasmic tail-deficient ZPC demonstrated that the deletion of the cytoplasmic tail interferes with the intracellular trafficking of the protein from the endoplasmic reticulum to the Golgi apparatus. These results indicate that the cytoplasmic tail of quail ZPC might possess the determinant responsible for the efficient transport of the newly synthesized ZPC from the endoplasmic reticulum to the Golgi apparatus.     

Sensitivity of expression of perivitelline membrane glycoprotein ZP1 mRNA in the liver of Japanese quail (Coturnix japonica) to estrogenic compounds

Avian perivitelline membrane protein, ZP1, is synthesized and secreted by the liver with the stimulation of estrogens. In the present study, we measured the expression of ZP1 gene in the liver of immature male quail treated with various estrogenic compounds and in the liver of male quail embryos that were developed in the fertilized eggs laid by mother quail injected with various estrogenic compounds during vitellogenesis. Total RNA extracted from the liver was reverse-transcribed and cDNA was subjected to real-time PCR. Both diethylstilbestrol and ethinyl estradiol caused significant effect on the increase in mRNA in immature male quail. In contrast, diethylstilbestrol administered via the route of maternal injection was not effective for induction of embryonic mRNA, although the effect of ethinyl estradiol administered via the same route was prominent. These results showed that direct administration of estrogenic compounds, diethylstilbestrol and ethinyl estradiol, stimulates the induction of ZP1 gene, but the rate of accumulation of these compounds in the yolk is different during vitellogenesis. The present studies suggest that although ZP1 gene is a sensitive biomarker to evaluate the effects of endocrine disruptors, the route of administration is an important factor to compare the effectiveness.     

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica)

In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.     

Transplantation of ovaries in Japanese quail (Coturnix japonica)

This study aimed to extend techniques of ovarian transplantation from chickens to Japanese quail. Ovarian tissue was surgically transplanted from chicks obtained from a line of white-breasted (wb/wb) to those of wild-type (+/+) quail at 1-day of age or at 1 week, with or without subsequent administration of the immunosuppressant, mycophenolate mofetil. Only one out of seven quail (14.3%) transplanted at 1-day of age survived the surgery but 14 of 18 quail (77.8%) transplanted at a week of age survived and grew to sexual maturity. In the 8-week progeny test, among those quail that produced offspring, one of four in the non-immunosuppressed group and all five in the immuosuppressed group produced donor-derived offspring. The proportion of donor-derived to host-derived offspring from five quail in the immnuosuppressed group (173/57) was significantly higher than that from one quail that was not immunosuppressed (3/47). These results suggest that quail ovaries can be transplanted at the age of 1 week and that the immnunosuppressant, mycophenolate mofetil, used for mammalian organ transplantation can suppress immunological rejection in birds. These transplantation techniques could provide an efficient means of recovering a line of quail after cryopreservation.     

Cutaneous candidiasis in Japanese quail (Coturnix coturnix japonica)

Cutaneous candidiasis involving the foot pads of 5–30 weeks old Japanese quails was recorded in a large quail breeding unit. Histopathological lesions comprising granulomatous reaction in the dermis and hyperkeratosis resembled with those of candida granuloma of human beings. The causative fungus, isolated from the foot pad lesions on Sabouraud agar, was identified as Candida albicans on morphological and biochemical characteristics. Predisposing factors such as overcrowding and un-sanitary conditions were found to be apparently responsible for candidiasis in quails. These factors when taken care of resulted in a marked decrease in the disease incidence. This appears to be the first report of cutaneous candidiasis in avian species.     

A new chondrodystrophy mutation in Japanese quail (Coturnix japonica)

A second form of hereditary chondrodystrophy (ch-2) has been discovered in a selected line of Japanese quail, Coturnix japonica. This form of chondrodystrophy is autosomal and recessive, characterized by an overall shortening and bending of the long bones of the wings and legs, slight dwarfing of the trunk, bulging of the eyes, flattening of the head, and a parrot beak. The shortened long bones vary in regard to the amount of bending from nearly straight to bends of up to 90 degrees in the midshaft region. In severe cases, the bend is evident as a protuberance of the skin. Affected embryos usually survive the 18-day incubation period. Several have hatched, but most survived no longer than 4 days after hatching. Only one female has survived long enough to lay eggs. Testcrosses indicated that this mutation is not allelic to micromelia.     

Evidence for introgressive hybridization of wild common quail (Coturnix coturnix) by domesticated Japanese quail (Coturnix japonica) in France

Many cases of introgressive hybridization have been reported among birds, particularly following introduction to the natural environment of individuals belonging to non-native similar taxa. This appears to be the case for common quail (Coturnix coturnix) in France where wild populations artificially come into contact with domesticated Japanese quail (Coturnix japonica) raised for meat and egg production but sometimes released for hunting purposes. In order to highlight the possible existence of gene flows between both taxa, a comparison of nuclear (25 microsatellite loci) and mitochondrial (sequencing and RFLP) DNA polymorphisms was performed on 375 common quails (from France, Spain and Morocco) and 140 Japanese quails (from France and Japan). Genetic diversity was assessed, and analyses (Factorial Correspondence Analysis, Bayesian admixture) of molecular polymorphisms revealed clear differentiation between the two taxa, making it possible to detect for hybrids among quails sampled in the wild. Eight birds expected to be common quail were found to be two pure Japanese quail, one probable backcross to C. japonica, three F1/F2 hybrids, and two probable backcrosses to Coturnix coturnix. These results show that Japanese quails were released and suggest that the two taxa hybridize in the wild. They confirm the urgent need for preventing the release of pure Japanese or hybrid quails to preserve the genetic integrity of C. coturnix. The tools developed for this study should be useful for accurate monitoring of wild quail populations within the framework of avifauna management programs.     

Japanese quail (Coturnix japonica) as a laboratory animal model

For the past 50 years, the Japanese quail (Coturnix japonica) has been a popular animal model in numerous fields of research. The quail's 16-d developmental period and its easily accessible embryo make C. japonica a convenient model for studies of developmental biology. Because its lifespan is relatively short and its physiology is comparable to that of humans, the adult quail is useful for studies of aging and disease. The authors describe the Japanese quail as an animal model and, drawing on their experience raising a quail colony at the California Institute of Technology, present detailed guidelines for the husbandry of the species.     

The acute phase response in Japanese quail (Coturnix coturnix japonica)

Experiments were conducted to determine the effect of injection of lipopolysaccharide (LPS, from S. typhimurium) or muramyl dipeptide (MDP, N-acetylmuramyl-L-ala-isoglutamine) in Japanese quail. Doses of MDP between 0.3 and 10 mg/kg body wt. had no effect on body temperature. In contrast, doses of 1.0-22.5 mg LPS/kg body wt. caused significant increases in body temperature. None of the doses of LPS or MDP resulted in mortality. The febrile response to LPS was diminished following a second injection 48 h after the first, and was absent following a third injection. Plasma zinc, an indicator of the acute phase response, was significantly reduced by either LPS or MDP after the first injection (P<0.001), but not after the second or third injection. Splenic interleukin 1-beta (IL-1beta) mRNA expression was increased after the first and last injection of LPS (P<0.001), but only after the first injection of MDP (P<0.005). Hepatic IL-1beta mRNA expression was increased after the first, but not the third injection of LPS (P<0.001), while MDP had no effect. These data indicate that Japanese quail are less sensitive to MDP than LPS, and that quail demonstrate tolerance to LPS following repeated injections.     

A new mutant of Japanese quail (Coturnix coturnix japonica) characterized by generalized glycogenosis

A flock of Japanese quail with generalized glycogenosis has been established. Affected quail showed difficulty in raising their wings. Excessive accumulation of glycogen was seen in the liver, heart, skeletal muscle and brain, apparently due to decreased acid maltase activity. The condition appeared between 2 and 12 weeks of age and tissue deposition of glycogen increased with age. The growth of affected quail was normal and there were no deaths from the condition. Although genetic analysis has not yet been completed, an autosomal recessive inheritance is suspected.     

Immunomagnetic purification of viable primordial germ cells of Japanese quail (Coturnix japonica).

Immunomagnetic cell sorting (MACS) with the monoclonal antibody (mAb) QCR1 was compared with the Ficoll density-gradient centrifugation system (FICS) in terms of the efficiency of enrichment of quail (Coturnix japonica) primordial germ cells (PGCs) from blood. The purified PGCs were tested for their ability to settle in the chick (Gallus domesticus) embryonic gonad. Blood containing 60-100 PGCs microliter-1 was taken from the dorsal aorta of quail embryos at Hamburger and Hamilton's stages 14-16. The amount and concentration of PGCs in the PGC-rich fraction purified by MACS were greater than in the fraction purified by FICS. Purified quail PGCs were transfused into chick embryos at stages 14-16 and immunohistochemically stained with mAb QCRI on day 8 of chick development. Transfused PGCs purified by either MACS or FICS were positively stained in the chick embryonic gonads.     

Enhanced yolk testosterone influences behavioral phenotype independent of sex in Japanese quail chicks Coturnix japonica

Studies have demonstrated an effect of yolk testosterone levels on the physiology and behavior of nestling birds. In order to investigate this phenomenon experimentally in a precocial bird, we enhanced yolk testosterone, but within the physiological range, by injecting 50 ng testosterone in ethanol into Japanese quail Coturnix japonica eggs prior to incubation. The chicks hatching from these as well as from control eggs that had received the carrier-only or were left unmanipulated were subject to a number of behavioral tests from hatching to the age of 3 weeks. In addition, fecal samples were taken during a 90-min isolation period to determine a physiological response to a stressor. Experimental chicks performed a detour task faster and approached novel objects sooner than did the controls. Chicks from treated eggs took a longer time to start distress vocalizing and also produced less distress calls during open-field trials, took on average a larger number of trials for them to show tonic immobility and also excreted lower levels of corticosterone metabolites (BM) than did the controls. In response to a stressor, excreted BM was initially higher in the control chicks, as compared to the experimental birds. Induced behavioral effects were independent of sex with no sex treatment interactions found. In sum, experimentally enhanced testosterone levels in the eggs shifted individual behavioral phenotype towards "bold" or "proactive", irrespective of sex. We conclude that testosterone in the yolk influences the coping style of hatchlings and may be a potential means of maternal influence on offspring phenotype.     

Effects of 2 G hypergravity exposure on Bobwhite (Colinus virginianus) and Japanese quail (Coturnix coturnix japonica)

We compared reproductive fitness and early postnatal growth of Bobwhite (Colinus virginianus) and Japanese (Coturnix coturnix japonica) quail incubated and hatched during 2 G centrifugation. Fertilized Bobwhite and Japanese quail eggs were placed in portable incubators on the 8-ft International Space Station Test Bed (ISSTB) Centrifuge at NASA Ames Research Center. The quail eggs were incubated throughout hatching and reared until Postnatal day (P)4 at either 1.0, 1.2 or 2.0 G. Two days before hatching, candling revealed significantly greater numbers of viable Bobwhite than Japanese quail eggs at all g-loads. Bobwhite quail exhibited significantly better hatching success at all g-loads than did Japanese quail. Bobwhite hatchlings were sensitive to gravitational loading as evidenced by reduced postnatal body mass and length of 2 G hatchlings relative to 1 G control hatchlings. In contrast, mass and length of Japanese quail hatchlings were unaffected by 1.2 or 2 G exposure. Together, our findings provide evidence for superior viability and hatching success in Bobwhite quail relative to Japanese quail, coupled with greater sensitivity of postnatal body growth and development to 2 G loading. Bobwhite quail may be better suited than Japanese quail for scientific studies on space biology platforms.     

AFLP linkage map of the Japanese quail Coturnix japonica

The quail is a valuable farm and laboratory animal. Yet molecular information about this species remains scarce. We present here the first genetic linkage map of the Japanese quail. This comprehensive map is based solely on amplified fragment length polymorphism (AFLP) markers. These markers were developed and genotyped in an F2 progeny from a cross between two lines of quail differing in stress reactivity. A total of 432 polymorphic AFLP markers were detected with 24 TaqI/EcoRI primer combinations. On average, 18 markers were produced per primer combination. Two hundred and fifty eight of the polymorphic markers were assigned to 39 autosomal linkage groups plus the ZW sex chromosome linkage groups. The linkage groups range from 2 to 28 markers and from 0.0 to 195.5 cM. The AFLP map covers a total length of 1516 cM, with an average genetic distance between two consecutive markers of 7.6 cM. This AFLP map can be enriched with other marker types, especially mapped chicken genes that will enable to link the maps of both species and make use of the powerful comparative mapping approach. This AFLP map of the Japanese quail already provides an efficient tool for quantitative trait loci (QTL) mapping.