Association Between History of Psychiatric Disorder and Degree of Physical Injury Among Suicide Attempters: Secondary Data Analysis in a Japanese Rural Area.

INTRODUCTION: While there may be differences in the choice of suicide methods between attempters with and those without a history of psychiatric disorders, it is not clear whether these differences predict the actual degree of physical injury. The present study aimed to investigate the association between the history of psychiatric disorder and the degree of physical injury among suicide attempters in a Japanese rural area. METHODS: We conducted a cross-sectional study analyzing secondary data of 806 suicide attempters from April 2012 to March 2022 obtained from a Japanese rural city. The exposure variable was a history of psychiatric disorders. The primary outcome was the degree of physical injury of suicide attempters: moderate and severe. We conducted a multivariate Poisson regression analysis to estimate the prevalence ratios (PRs) and 95% confidence intervals (CIs). RESULTS: Among 806 suicide attempters, a significant negative association between the history of psychiatric disorder and the degree of physical injury was observed (PR = 0.40; 95% CI, 0.28-0.59). Those with and without psychiatric disorders were more likely to choose low- and severe-lethality suicide methods such as drug or psychotropic overdoses and hanging or deep wrist injuries, respectively (P < .001). CONCLUSIONS: The present study highlights the importance of considering suicide attempters, both with and without psychiatric disorders, while formulating targeted suicide prevention strategies.

Diverse forage improves lipid metabolism and antioxidant capacity in goats, as revealed by metabolomics.

It is well established that promoting the balance of nutrients and plant secondary metabolites (PSM) by feeding diverse forage physiologically improves ruminant production. However, the underlying mechanism remains unclear. To investigate the physiological mechanism related to the improvement of physiological stress tolerance, ruminants were fed diverse forage. Oxidative stress markers were quantified, and serum metabolomics was performed. Six crossbred Shiba wethers (32.8 ± 9.2 kg BW) were arranged in a replicated 3 × 3 Latin square design. The treatments were feeding only Sudan grass hay (100% SDN); feeding a mixture of Sudan grass and alfalfa hay (70:30, SDN-ALF); and feeding a mixture of Sudan grass, timothy grass, and alfalfa hay (35:35:30; SDN-TMT-ALF). Each diet group was fed its specific diet for 21 days with a 14-day adaptation period. Feed intake and digestibility, blood biochemistry, total antioxidant capacity (TAC), and superoxide dismutase (SOD) were analysed. In addition, blood serum metabolites were assessed by liquid chromatography-tandem mass spectrometry. The DM intake and DM, organic matter, and CP digestibility were higher (P < 0.05) in the SDN-TMT-ALF group than in the SDN group. The TAC was higher (P < 0.01) in the SDN-TMT-ALF and SDN-ALF groups (809.51 and 813.7 µM, respectively) than the SDN group (720.69 µM), while the SOD level was unchanged (P = 0.06) among the treatments. Total serum cholesterol and NH3 levels were higher (P < 0.05) in the SDN-TMT-ALF group (89.17 mg/dL and 242.42 µg/dL, respectively) than in the SDN group (71.00 mg/dL and 89.17 µg/dL). Additionally, the levels of nine metabolites in serum differed among the treatments (P < 0.05). Linoleic acid (LA) and cortisone, which are related to LA metabolism and the steroid biosynthesis pathway, were upregulated by the SDN-ALF and SDN-TMT-ALF diets compared to the SDN diet, suggesting the contribution of ALF to altering the metabolites. The levels of hippuric acid, which is a metabolite of phenolic compounds, were higher (P < 0.001) in the animals fed SDN, which contained higher phenolic and luteolin concentrations than the other diets. Pathway analysis suggested that the higher cortisone levels were derived from cholesterol due to upregulated glycolysis metabolism, which was positively related to increased ingestion, digestibility, and serum LA levels in animals given mixed forage. In conclusion, physiological stress tolerance in the animals was regulated by upregulation of LA and steroid hormone metabolism, which was associated with an increase in TAC rather than the ability of the animal to regulate its PSM intake.

Gluconeogenesis in the Yolk Sac Membrane: Enzyme Activity, Gene Expression, and Metabolites During Layer Chicken Development.

Yolk sac membranes of layer eggs were collected daily (n = 7-9) from day three of incubation to day three post-hatch, and mRNA expression and activities were quantified for key gluconeogenesis enzymes (glucose-6-phosphatase, fructose-1,6-bisphosphatase, cytosolic and mitochondrial phosphoenolpyruvate carboxykinases, and pyruvate carboxylase). Lactate, triglycerides, non-esterified fatty acids, glycogen, and glucose in the yolk sac membrane, and blood glucose levels were also measured. The mRNA expression and activity were detected for all enzymes. Differences in expression levels and enzyme activities seemed to reflect the embryo's developmental environment and physiological demands at different developmental stages. During the first week to the mid-second week of incubation, the expression and activity of gluconeogenic enzymes and lactate concentrations were high, suggesting an active period of gluconeogenesis from lactate, reflecting possible hypoxia in the embryo before completed formation of the chorioallantoic capillaries. From the mid-second week to mid-third week, when embryos were in an aerobic state, the triglyceride and non-esterified fatty acid contents increased in the yolk sac. Triglycerides from yolk lipids are typically hydrolyzed to produce non-esterified fatty acids as an energy source, whereas the glycerol skeleton is used for gluconeogenesis. In the late third week, when embryos were considered to re-enter an anaerobic state, the mRNA expression and enzyme activity of only glucose-6-phosphatase were high and the amount of glycogen in the yolk sac was reduced. Therefore, it is suggested that gluconeogenesis activity is low during this period, and the carbohydrates stored in the yolk sac membrane are secreted into the blood as energy for hatching. This study confirmed the role of the yolk sac membrane as a vital gluconeogenic organ during chicken egg incubation.

Relaxin exerts a protective effect during ischemia-reperfusion in the rat model.

BACKGROUND: Testicular torsion, which causes ischemia-reperfusion (IR) injury, is a serious urological emergency that can lead to testicular dysfunction, including infertility, primarily among newborn and pubertal males; thus, effective drugs should be administered during or after ischemia. OBJECTIVES: Using a rat model of testicular IR injury, the present study investigated the protective effects of relaxin (RLN) against oxidative stress, testicular dysfunction, inflammation, histological damage, arrested spermatogenesis, and germ cell apoptosis as well as explored the usefulness of RLN as a potential protective drug for IR injury combined with surgical treatment. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to left testicular ischemia for 2 h, followed by 24 h of reperfusion. They were subsequently divided into three groups: sham, IR, and IR + RLN groups. Porcine RLN (500 ng/h) or saline was infused using an implanted osmotic mini-pump 90 min after inducing ischemia. The RLN dose used herein was that which resulted in serum RLN levels comparable to those in mid-pregnant rats based on previous studies. RESULTS: Testicular IR increased germ cell apoptosis and histological damage as well as promoted disorganized and arrested spermatogenesis, accompanied by a significant increase in oxidative stress and inflammation. However, RLN administration ameliorated the adverse consequences associated with IR injury by attenuating oxidative stress and mitigating apoptosis and inflammation. DISCUSSION AND CONCLUSION: The study findings clearly demonstrated that RLN exerts a protective effect against IR-induced testicular injury by attenuating oxidative stress, apoptosis, and inflammation, suggesting that RLN together with surgical treatment is a potentially efficacious approach toward ameliorating testicular dysfunction following testicular torsion.

Expression of thyroid hormone regulator genes in the yolk sac membrane of the developing chicken embryo.

Thyroid hormones (THs) are essential for the correct development of nearly every structure in the body from the very early stages of development, yet the embryonic thyroid gland is not functional at these stages. To clarify the roles of the egg yolk as a source of THs, the TH content in the yolk and the expression of TH regulator genes in the yolk sac membrane were evaluated throughout the 21-day incubation period of chicken embryos. The yolk TH content (22.3 ng triiodothyronine and 654.7 ng thyroxine per total yolk on day 4 of incubation) decreased almost linearly along with development. Real-time PCR revealed gene expression of transthyretin, a principal TH distributor in the chicken, and of a TH-inactivating iodothyronine deiodinase (DIO3), until the second week of incubation when the embryonic pituitary-thyroid axis is generally thought to start functioning. The TH-activating deiodinase (DIO2) and transmembrane transporter of thyroxine (SLCO1C1) genes were expressed in the last week of incubation, which coincided with a marked increase of circulating thyroxine and a reduction in the yolk sac weight. DIO1, which can remove iodine from inactive THs, was expressed throughout the incubation period. It is assumed that the chicken yolk sac inactivates THs contained abundantly in the yolk and supplies the hormones to the developing embryo in appropriate concentrations until the second week of incubation, while THs may be activated in the yolk sac membrane in the last week of incubation. Additionally, the yolk sac could serve as a source of iodine for the embryo.

Temporal changes in liver tissue metabolome of lambs fed low-quality roughage.

Early experience with low-quality roughage may induce adaptations in ruminants' metabolism. This study was conducted to explore the variation in the hepatic metabolomes of lambs fed low-quality roughage beginning early in life. Five lambs were fed Sudan grass hay (crude protein (CP): 4.6% of the dry matter (DM), neutral detergent fiber, 68.5% of DM) for 6 months during time periods P1, P2 and P3, which consisted of 2 months each. The metabolizable energy (ME) and CP intake satisfied lambs' maintenance requirements in P1 and P2, but the ME intake was 78.5% of the maintenance ME requirement in P3. Liver metabolomics was carried out in P2 and P3 by the capillary electrophoresis and time-of-flight mass spectrometry system. Eight amino acids and six amino acid metabolism-related metabolites were altered between P2 and P3. Several intermediates of glycolysis/gluconeogenesis decreased, while nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate increased in P3. Taurocholic acid and taurine increased, while glycocholic acid decreased in P3. The results suggested that amino acid utilization and the efficiency of glycolysis/gluconeogenesis might be adjusted to accommodate the low-quality roughage fed to the lambs during early stages of life. The composition of bile acids might also be optimized to promote the efficiency of lipid absorption.

Ligand-binding characteristics of feline insulin-binding immunoglobulin G.

Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e(-4) M for fraction B (low affinity IgGs) and 2e(-5) M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin.

Natural anti-insulin autoantibodies in cats: enzyme-linked immunosorbent assay for the determination of plasma anti-insulin IgG and its concentrations in domestic cats.

Anti-insulin immunoglobulin G (IgG) has been found in the sera of healthy cats. To determine the concentrations of these antibodies, an enzyme-linked immunosorbent assay (ELISA) for anti-insulin IgG was developed. ELISA maintained the linearity of a standard concentration line between 67.5 and 2160 ng/ml. The coefficients of variances (CVs) of intra-assays in two different plasma samples were 4.0% and 3.7%, respectively. The inter-assay CVs in two different plasma samples were 5.1% and 6.9%, respectively. The dilution curves of two samples were rectilinear. Anti-insulin IgG was detected in all 84 of the healthy cats that were tested. Plasma anti-insulin IgG concentrations ranged from 80 to 1578 µg/ml, with a median concentration of 221 µg/ml, and this value correlated positively with total plasma IgG concentrations (r=0.383, p<0.01). In an intravenous glucose tolerance test, plasma anti-insulin IgG concentrations did not alter, even with changes in plasma glucose and insulin concentrations. The ELISA that was developed was able to determine plasma anti-insulin IgG in domestic cats, and confirmed that all healthy cats had plasma anti-insulin IgG. Determining the plasma concentrations of anti-insulin IgG in cats with various pathological conditions might clarify the role of anti-insulin IgG.

Hatching mechanism of the Chinese soft-shelled turtle Pelodiscus sinensis.

The mechanism by which the embryo hatches out of the egg envelope, the vitelline membrane and egg white, was studied in the Chinese soft-shelled turtle Pelodiscus sinensis. The cDNA of the turtle hatching enzyme (HE) was 1555bp-long and a mature enzyme of 321 amino acids. The mature HE was composed of an astacin protease domain of 200 amino acids and a CUB domain of 121 amino acids, and the estimated molecular size was 35,311. The protease domain contained two active site consensus sequences, HExxHxxGFxHExxRxDR and MHY. An immunoblotting test of an extract of allanto-chorions revealed a 40-kDa band by cross-reaction with the anti-Xenopus HE antiserum. The first change in the envelopes was the appearance of a hole, 1mm in diameter, at the location around the animal pole of day 8 incubation eggs. A cluster of tall cells, forming a circle in the avascular chorion of day 8 embryos and facing the edge of the hole, had various sizes of inclusion bodies and secretory granules that were labeled by immuno-electron microscopic staining with the antiserum. The egg envelopes were degraded gradually from the animal pole side towards the vegetal pole side in accordance with translocation of the avascular site of the chorion in the same direction. Labeled cells degenerated, presumably when the chorion was underlain by allantois in succeeding developmental stages. The vitelline membrane and egg white were totally digested, presumably by secreted HE, during the hatching period and were consumed for embryonic growth.

Cuticle formation in quail eggs.

The present study was conducted to determine both the site at which cuticle materials are produced and the critical period for their production in the oviductal uterus of the Japanese quail, Coturnix japonica. An antiserum was produced against the 32-kDa band in electrophoretic profiles of cuticle materials obtained from eggshells decalcified with EDTA. Immunofluorescence and immunoelectron microscopic observations revealed that the 32-kDa protein was synthesized in luminal ciliated epithelial cells of the uterus until 21 h after the previous oviposition (the first phase) and then secreted during the 4 h before the next oviposition (the second phase). Scanning electron microscopic observations revealed that 10-microm-wide posts appear on the surface of the luminal epithella during the first phase, and that they disappear during the second phase. During the second phase, air canals are formed in the eggshell by the retreat of the posts, and a cuticle layer forms on the eggshell. Our results indicate that the cuticle may function as a lubricant that facilitates egg rotation in the uterus.

Thyrotrophin in the pars tuberalis triggers photoperiodic response.

Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin (TSH) beta-subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV) administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor-cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long-day photoinduced seasonal breeding.

Mechanism of chalaza formation in quail eggs.

The present study was conducted to determine the mechanism of chalaza formation in eggs of the Japanese quail Coturnix japonica and to determine the production site of chalaza materials in the oviduct. Electrophoretic profiles of the chalaza materials showed six bands of 480, 320, 210, 180, 96, and 58 kDa following Coomassie Blue staining and one band of 600 kDa after immunoblotting. An antiserum was produced against the 180-kDa band. This antiserum and an antiserum generated against the 600-kDa protein were used as probes to detect chalaza materials. Immunofluorescent and immunoelectron-microscopic observations revealed that chalazae and chalaziferous layers overlaid to approximately 40 microm upon the vitelline membrane of the ovum were composed of the same materials as those produced by both types of secretory cells in the luminal and glandular epithelia at the infundibulum. We propose that the mechanism of chalaza formation is as follows: (1) chalazae first appear as fine filaments at the presumptive sharp and blunt ends of the ovum at the infundibulum; (2) these filaments are twisted into a lead fiber while the ovum is rotating and descending in the magnum; (3) at the posterior end of the magnum, the lead fiber is anchored to the thick egg white and lifted outward with the chalaziferous sublayers when the inner egg white is liquefied by absorbing water; (4) the lead fiber and chalaziferous sublayers are twisted further into the chalaza in the uterus.

The asymmetry of avian egg-shape: an adaptation for reproduction on dry land.

The present study describes the biological meaning of the asymmetrical shape in avian reproduction using quail. During the incubation of eggs, water was gradually lost and the air chamber which appeared in between the inner and outer shell membranes at the blunt end expanded, so that the angle made by the long egg-axis and the horizontal line increased, presumably because the centre of gravity of the egg contents moved toward the sharp end. The increase in angle occurred in both fertile and infertile eggs, suggesting that this phenomenon occurs irrespective of fertility and is due to the asymmetrical shape. The increase in the volume of the air chamber resulted in an increase in the area of the inner shell membrane at the chamber to satisfy the amount of gas exchange needed by the developing embryo for better hatching. We isolated a 300-kDa protein from the inner shell membrane. It was produced by cells in the luminal epithelium of the oviductal isthmus and was found in the cortex of the fibres of shell membranes and a lining surrounding the air chamber. The lining comprised a medial layer between the inner and outer shell membranes in uterine eggs. The asymmetrical ellipsoid produces the air chamber at the blunt end of the avian egg during its sojourn in the oviductal isthmus, to maintain the blunt end up after oviposition and to raise that end during incubation in a dry environment, leading to high hatchability.

The magnum-isthmus junction of the fowl oviduct participates in the formation of the avian-type shell membrane.

Avian eggs possess a shell membrane in the shape of an asymmetrical ellipsoid and with a limiting membrane that is a smooth layer of homogeneous, dense materials. We describe the role of the magnum-isthmus junction (MIJ) of the oviduct in the formation of the avian-type shell membrane in the domestic fowl Gallus domesticus. The narrow width of the lumen at the MIJ indirectly participates in the determination of the asymmetrical ellipsoid shape of eggs that are encased by the egg-white layer and subsequently by the peri-albumen layer (PL) and the shell membrane. The PL reacts with Alcian blue and exists between the egg white and the limiting membrane. It is added to the ovulating egg at the MIJ and covers the outermost surface of the egg-white layer. The function of the PL is to provide a smooth surface by covering the irregular surface of the egg-white layer. The materials of the PL consist of an Alcian blue-positive polysaccharide (or glycoprotein) of 240 kDa and five proteins of 135, 116, 72, 49, and 46 kDa. The isolated materials have an affinity to bind with the egg-white mass. An antiserum against quail PL materials stains the domestic fowl PL and secretory cells of the luminal epithelium at the MIJ, and cross-reacts with the molecules of 240, 135, and 116 kDa.

Roles of His9 (P2 subsite) and His13 (P3' subsite) in angiotensinogen for catalytic reaction of renin.

Roles of His9 (P2) and His13 (P3') of angiotensinogen for the catalytic reaction of renin were investigated using purified recombinant ovine angiotensinogen and its mutants H9Q and H13Q. The pH depended reaction of human renin with angiotensinogens of wild-type, H9Q and H13Q showed peaks at pH 6.5 and 8.5, but the altitude of each peak was different. The Vmax of the reaction between H9Q and H13Q with human renin was decreased by about 50 and 70%, respectively, in comparison to wild-type angiotensinogen, at pH 6.5. At pH 8.5, the Vmax of H9Q and H13Q was 50 and 100% of that of wild-type, respectively. At pH 6.5, it was revealed that the catalytic efficiency of renin (Vmax/Km) reduced to 20 and 60% after mutation of angiotensinogen at His9 and His13 with Gln, respectively. At pH 8.5, the catalytic efficiency decreased to 10 and 70% after these mutations, respectively. These findings, therefore, indicate that histidine residues at both P2 and P3' positions probably associate with the renin catalytic reaction for angiotensin I generation.

Development of gonadotropes in the chicken embryonic pituitary gland.

Although a number of immunohistochemical studies have been carried out on the differentiation of chicken gonadotropes during embryogenesis, the temporal and spatial properties of appearance of gonadotropes are not clear. In this study, we studied the appearance and morphological characteristics of gonadotropes in the embryonic and adult chicken anterior pituitary glands using RT-PCR, in situ hybridization and immunohistochemistry. For this purpose, we raised specific antisera against chicken follicle-stimulating hormone beta-subunit (cFSHbeta) and chicken luteinizing hormone beta-subunit (cLHbeta) based on each putative amino acid sequence. RT-PCR analysis revealed that cFSHbeta mRNA was expressed from embryonic day 7 (E7). Chicken FSHbeta mRNA-expressing (-ex) and -immunopositive (-ip) cells started to appear in the ventral part of the caudal lobe in the anterior pituitary gland at E8. Chicken LHbeta-ip cells were also first observed there at E8, but cLH mRNA expression was confirmed from E4 by RT-PCR analysis. The distribution of these chicken gonadotropin-ex and -ip cells spread from the ventral part to dorsal part in the caudal lobe around E10 and subsequently expanded to the cephalic lobe from E12 to E20. These cells were morphologically classified into two types (round- and club-shaped cells). It was found that the density of gonadotropin-ip cells in the caudal lobe was always higher than that in the cephalic lobe throughout the period of development. To the best of our knowledge, this is the first report focusing on the differentiation of chicken gonadotropes by assessment of both protein and mRNA of chicken gonadotropin.

Human prorenin has "gate and handle" regions for its non-proteolytic activation.

We investigated the mechanism for non-proteolytic activation of human prorenin using five kinds of antibodies. Each of the antigens, L1PPTDTTTFKRI11P, T7PFKRIFLKRMP17P, I11PFLKRMPSIRESLKER26P, M16PPSIRESLKER26P, and G27PVDMARLGPEWSQPM41P, was designed from the tertiary structure of predicted prorenin. These antibodies were labeled anti-01/06, anti-07/10, anti-11/26, anti-16/26, and anti-27/41, respectively, for their binding specificities. Inactive recombinant human prorenin (0.1 nM) bound to various concentrations of anti-01/06, anti-11/26, and anti-27/41 antibodies at 4 degrees C with equilibrium dissociation constants of 138, 41, and 22 nM, respectively. However, intact prorenin (0.1 nM) did not show significant binding to 200 nM anti-07/10 and anti-16/26 antibodies for 20 h. Ninety percent of prorenin (0.1 nM) was found to be non-proteolytically activated by incubation with anti-11/26 antibodies (200 nM) at 4 degrees C for 20 h. Prorenin was not active even under complex with either anti-01/06 or anti-27/41 antibodies. Prorenin was also reversibly activated at pH 3.3 and 4 degrees C for 25 h. The acid-activated prorenin bound to anti-07/10 and anti-16/26 antibodies as well as to anti-01/06, anti-11/15, and anti-27/41 antibodies at neutral pH and 4 degrees C in 2 h. Their dissociation constants were 13, 40, 8.6, 3.6, and 14 nM, respectively. The acid-activated prorenin was re-inactivated by incubation at pH 7.4 and 4 degrees C in 50 h. Anti-07/10 and anti-11/26 antibodies inhibited such re-inactivation at 25 degrees C by more than 90% and 50%, respectively, whereas other kinds of antibodies did not prevent the re-inactivation at 25 degrees C. These results indicate that prorenin has "gate" (T7PFKR10P) and "handle" (I11PFLKR15P) regions critical for its non-proteolytic activation.

Absorption, transportation and digestion of egg white in quail embryos.

The present study was done to reveal how egg white is taken up by embryonic tissues, the pathway through which egg white is transported, and the location where it is digested during the development of the quail Coturnix japonica. Antiserum against quail ovalbumin was raised in rabbit and used as a probe. By immunoelectron microscopy, the uptake of ovalbumin on a small scale by receptor-mediated endocytosis was observed in the ectodermal cells of the yolk sac on days four to seven of incubation. The uptake of egg white on a large scale by fluid-phase endocytosis took place in the cells generally referred to collectively as the 'albumen sac'. The ovalbumin was transported through the albumen sac into the extraembryonic cavity during days eight to 10, and then into the amniotic cavity through the amnion approximately on day 10. Ovalbumin was present in the intestinal lumen on days 11 and 14, but it was not digested in the intestinal epithelial cells. The ovalbumin was detected in the yolk of embryos after day 10. Immunoblot testing, as well as a fluoroimmunoassay, revealed that the location where the amount of ovalbumin was highest changed chronologically from the extraembryonic cavity on day 10 to the amniotic cavity on day 11, the intestinal lumen on day 12 and then to the yolk on day 13. Several low molecular proteins which cross-reacted with the antiserum were observed in the extracts of the yolk. The reaction producing these proteins depended on low pH (approximately 3.0) and was inhibited by pepstatin A. The ovotransferrin was similarly digested. These results indicate that egg white is, for the most part, transported through the albumen sac to the yolk via the extraembryonic cavity, the amniotic cavity, and the intestinal lumen, and is digested in the yolk by aspartic proteinases.