Amino acids in oviduct and uterine fluid and blood plasma during the estrous cycle in the bovine

Up to 40% of cattle embryos die within 3 weeks of fertilization while they are nutritionally dependent on the maternal environment provided by the oviduct and uterine fluids for their development and survival. Despite this dependence there is limited information on the composition of these fluids in cattle. Amino acids are essential for the normal growth and development of the early embryo, acting as precursors of proteins and nucleic acids and as energy sources, osmolytes and signaling molecules. The objective of this study was to measure and compare the amino acid concentrations of oviduct and uterine fluid and blood plasma on different days of the estrous cycle. Oviduct fluid was collected in situ from anaesthetised heifers on Days 0, 2, 3, 4 and 6 and uterine fluid on Days 6, 8 and 14 of the estrous cycle and the concentrations of 19 amino acids determined. Glycine was the most abundant amino acid in both oviduct and uterine fluid. However, the concentrations of many amino acids differed between oviduct and uterus and many were present at higher concentrations in oviduct and uterine fluid than in blood plasma. Oviduct fluid concentrations of amino acids were not affected by day of cycle in contrast to uterine fluid for which there was a day of cycle effect on most of the amino acids. These results provide novel information on the amino acid concentrations in the maternal environment of the early cattle embryo and could form the basis for devising improved media for the production of embryos in vitro.     

Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy

Fulminant hepatic failure was induced in rabbits by intravenous administration of galactosamine hydrochloride. The animals were sacrified after 45 h and the hippocampus analyzed for free amino acids. In addition, free amino acids were measured in plasma and in the extracellular fluid of the hippocampus 20, 30 and 45 h after galactosamine injection. The extracellular fluid compartment was analyzed by slow perfusion of a thin dialysis tube which was implanted in the hippocampus one day prior to galactosamine administration. The amino acid concentration in the extracellular fluid agreed fairly well with that of the cerebrospinal fluid in the control situation. During development of hepatic failure, the plasma concentrations of all amino acids increased. The changes in extracellular amino acids were smaller, except for phosphoethanolamine and glutamate. The concentration ratio intra/extracellular amino acids decreased in the hippocampus for amino acids with a normally high concentration gradient.     

Energy substrates in bovine oviduct and uterine fluid and blood plasma during the oestrous cycle

Up to 40 percent of cattle embryos die within 3 weeks of fertilization but there is little or no published information on the composition of the oviduct and uterine fluids essential for their survival during this time. We have measured the concentrations of the energy substrates, glucose, lactate, and pyruvate in cattle oviduct fluid on Days 0, 2, 4, and 6 and uterine fluid on Days 6, 8, and 14 of the oestrous cycle and corresponding blood samples. Oviduct and uterine fluids were collected in situ. Glucose concentrations in oviduct and uterine fluids were similar on all days and lower than in plasma (P < 0.05). Oviduct lactate concentration was up to eightfold higher than uterine or plasma concentration (P < 0.01). Oviduct pyruvate concentrations were similar on all days and lower than plasma concentrations on Days 0 and 2 (P < 0.005). Pyruvate concentrations were similar in the uterus and in plasma except on Day 14 when the concentration in plasma was higher (P < 0.05). There were no associations between systemic progesterone or oestradiol and glucose, lactate or pyruvate. There was a linear positive relationship (P < 0.001) between oviduct fluid secretion rate and oviduct glucose concentration and a linear negative relationship (P < 0.001) between oviduct fluid secretion rate and oviduct lactate, but no association between uterine fluid secretion rate and energy substrates. The different concentrations and associations between the energy substrates in oviduct and uterine fluids and blood plasma indicate a differential regulation of the secretion of these energy substrates by the oviduct and uterine epithelium.     

THE CONCENTRATIONS OF FREE AMINO ACIDS AND OTHER ELECTROLYTES IN CEREBROSPINAL FLUID,IN VIVO DIALYSATE OF BRAIN,AND BLOOD PLASMA OF THE DOG*

The concentrations of free amino acids in plasma, CSF and in vivo dialysates of peripheral blood (neck sac fluid) and central nervous tissue (brain sac fluid) from each of five dogs (neck sac fluid from four of five dogs) were determined by ion-exchange chromatography. Dialysates were obtained by implanting small dialysis sacs filled with a dextran-saline solution into the subcutaneous tissue of the neck or the parenchyma of the brain at least 10 weeks before sample collection. The mean plasma concentration of most amino acids was within the range of values reported in the literature for human or dog plasma. The concentrations of most amino acids were higher in the neck sac fluid than in plasma; this discrepancy, however, was, for the most part, small and could most likely be accounted for by falling plasma free amino acid levels prior to sample taking. Previous conclusions that the CSF concentrations of most amino acids are lower than plasma concentrations are confirmed, although the present work indicates that there may be considerable individual variation in the CSF/plasma distribution ratio with respect to most amino acids. In the brain sac fluid the concentration of nearly every amino acid was consistently higher than that in CSF and lower than that in the neck sac fluid. The potassium concentration in the brain sac fluid was significantly higher than, and the total osmolality significantly lower than, those in the neck sac fluid. On the assumption that the brain sac fluid represents a dialysate of the brain extracellular fluid, these results contradict recent findings (Bito and Davson , 1965; 1966) indicating that the potassium concentration of the cortex extracellular fluid is lower than that of ventricular or cisterna magna CSF and certainly lower than that of plasma. Because of this and on the basis of consideration of the reaction of the brain to a foreign body, the possibility that the implanted brain sac lay on the‘blood side’of the bloodbrain barrier was suggested. Some implications of this possibility are discussed.     

Electrophoretic characterization of proteins in oviduct fluid of cows during the estrous cycle

This study was undertaken to examine the in vivo protein composition of the bovine oviduct during the estrous cycle. Oviduct fluid was collected daily from four oviduct-cannulated dairy cows for a total of four complete estrous cycles. Fluid secretion followed a definite cyclic pattern, with maximum secretion occurring at estrus in all cycles. Protein concentration fluctuated during the cycle and varied among animals. In general, protein concentration was lower at the time of estrus. Total protein in oviduct fluid, however, was higher around estrus, indicating increased transudation or secretion by the oviduct. One-dimensional SDS-PAGE separation revealed the protein pattern of oviduct fluid to be generally similar to that of blood serum and follicular fluid. Two proteins appeared to be oviduct-specific. The first, a protein of approximately 47 kDa, was evident in oviduct fluid throughout the estrous cycle. The second protein, evident as a broad diffuse staining band above albumin, appeared for only 3-4 days at or near ovulation. This protein had a molecular weight of 80-95 kDa and stained positive for carbohydrate with periodic acid-Schiff reagent. These studies indicate that the in vivo protein composition of oviduct fluid varies with the estrous cycle, and that around estrus, an oviduct-specific glycoprotein is present.     

Luminal secretion of myo-inositol by the rat epididymis perfused in vitro

A technique for perfusing the lumen of rat epididymal tubules maintained in vitro showed that [3H]inulin was largely excluded from the lumen of unravelled tubules from the cauda and tubules from the corpus if the connective tissue capsule was removed. The preparation transported [3H]inositol from the bath fluid for 3 h against a concentration gradient in both regions with activity rising (16-29% of bath fluid values) in the cauda and reaching a plateau (18%) in the corpus epididymidis. HPLC showed that radioactivity was solely associated with inositol and its movement to the lumen was reduced by raising inositol in the bath fluid from 50 microM (plasma levels) to 10 mM, but not affected by reducing the glucose concentration in the bath fluid or introducing physiological concentrations of inositol (30 mM) into the lumen. Secretion into the caudal lumen of unlabelled inositol measured by g.l.c. was maintained for 3 h at concentrations (300 microM) greater than those in the bath fluid and was not reduced when glucose or inositol were removed from the bath. In contrast, glucose was only detectable in the lumen when it was present in the bathing medium, reaching 1% of this concentration. Radioactivity appeared in the epididymal lumen reaching a plateau (19% of bath fluid values) in the corpus and cauda when [3H]glucose was added to the bath fluid, but no radiolabelled inositol was found in the lumen. We conclude that epididymal tissue is a major source of secreted inositol.     

Co-culture,oviduct secretion and the function of oviduct-specific glycoproteins.

Co-cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts. Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development. Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media. Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media. These glycoproteins are induced by oestrogen in vivo but not in vitro. It is our contention that co-cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells.     

Unilateral hydrosalpinx and absence of the infundibulum in a mare

Both ovaries and associated oviducts were removed from a mare via colpotomy. The right oviduct was greatly enlarged and filled with fluid. No infundibulum could be identified; rather, the blind distended end of the oviduct adhered to the ovary at the ovulation fossa. Two cysts were attached to the exterior of the oviduct a few centimeters from the fossa. Histologic examination of the oviduct and cysts revealed a dilated lumen, a decreased infolding of the wall into the lumen, and a lack of a defined muscle layer. Congenital absence of the infundibulum resulting in a blind ampullar terminus is proposed as the cause of the hydrosalpinx.     

Serum antigens detected in pars recta luminal fluid and coelomic envelope surrounding Bufo arenarum eggs

In anurans, protease activity from the pars recta portion of the oviduct (under regulation by 17β-estradiol), is known to cause ultrastructural alterations on the oocyte surface rendering fertilizability. In mammals, the presence of serum proteins in oviductal fluid via transudation is also well known. In the present study we determined the plasma proteins of the anuran Bufo arenarum that are present in pars recta fluid and oocyte extracellular matrix and characterized the 17β-estradiol-induced proteins synthesized de novo and secreted into the pars recta lumen. Rabbit polyclonal antibodies against the soluble proteins in pars recta fluid cross-reacted with anuran plasma proteins and with the extracellular matrix of coelomic eggs based on immunoelectrophoresis and immunohistochemistry, respectively. Using radiolabeled leucine in the absence and presence of 17β-estradiol, we show that a polypeptide of 66 kDa molecular mass is the principal protein synthesized and secreted into the pars recta lumen.     

Amino acid transport in the intestine of the caiman

Seventeen amino acids were fed singly to small caimans and the rates of their disappearance from the gut lumen, and of their appearance in intestinal mucosa, whole intestine, whole stomach, and plasma were determined. The results were compared with those in which massive amounts of protein were fed. When single amino acids were fed, only traces of arginine, ornithine, lysine, aspartate and asparagine were absorbed intact. Glycine, alanine and serine were absorbed rapidly reaching mucosal concentrations as high as 40 mM. The others were not concentrated as highly and most were absorbed by the mucosa more slowly than the glycine group. Protein feeding did not result in high amino acid concentrations in the mucosa. Whether amino acids were ingested as protein or in the free state, glycine, alanine and glutamine increased in the mucosa, suggesting these three incorporate nitrogen released from the others. It appeared that several transport systems operate if amino acids are given singly, and that a different more efficient transport system operates during protein digestion.     

Regulation of Substances in Allantoic and Amniotic Fluid of the Chicken Embryo

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.     

Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx

Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to an endoparasitoid, C. plutellae. Despite rich information on CpBV genome, there has been little known on its viral replication mode from proviral to episomal form. This study illustrates fine structures of the epithelial cells producing CpBV with a reference to non-producing ovarian epithelial cells. The ovarian epithelial cells of teneral females (within 12 h after emergence) were characterized by large nucleus and rich rough endoplasmic reticulum. CpBV particles were present only at the calyx region, in which follicle epithelial cells exhibited virogenic structures. Though a matured CpBV particle found in the calyx lumen was encapsidated in a single envelop containing multiple nucleocapsids, numerous free nucleocapsids were observed in the calyx epithelial cells and appeared to undergo assembly step to a final multiple capsid form. The multiple capsid forms appeared to be released into the oviduct lumen. The epithelial cells bordering the oviduct lumen showed phagocytosis presumably due to clearing cellular debris. At the calyx area close to the common oviduct, the epithelial cells appeared to maintain protein synthetic activity due to highly developed rough endoplasmic reticulum, but showed a marked decrease in the viral production.     

Interference between leucine, isoleucine and valine during intestinal absorption

1. The reciprocal interference between l-leucine, l-isoleucine and l-valine during absorption was studied in rats both in vivo and with an everted-sac preparation in vitro. 2. After feeding with the amino acids alone there was a considerable increase in their concentration in the intestinal lumen followed by a rapid disappearance, indicating efficient absorption. Absorption was reflected by a high concentration of the respective amino acids in the portal plasma. Isoleucine and valine inhibited the absorption of leucine, and leucine inhibited the absorption of isoleucine and valine. Inhibition of absorption by the interfering amino acid was generally partly overcome after 30–60min., probably through the absorption of the interfering amino acid. At that time the rise in the concentration of the amino acid in portal plasma began. 3. These results were confirmed by experiments in vitro: isoleucine and valine inhibited the absorption rate of leucine, and leucine that of isoleucine and valine. 4. Active absorption of amino acids was rapid at low concentrations and depressed at higher concentrations.     

AMINO ACID TRANSPORT INTO THE CEREBROSPINAL FLUID OF THE RAT1

The concentrations of glycine and 6 other amino acids have been assayed in the CSF and plasma of the rat, and regional heterogeneity of CSF amino acid concentration has been found. Steady state flux rates into the cranial and spinal fluid compartments were determined during perfusion with amino acid free medium. The transfer of glycine from blood into both the cranial and spinal subarachnoid fluids was saturable with only several-fold elevations of plasma glycine. The results are discussed with regard to the putative neurotransmitter function of glycine in the spinal cord.     

Nutrient concentrations in murine follicular fluid and the female reproductive tract

The culture of murine oocytes and preimplantation embryos in vitro has been used successfully for many years. However, this practice can result in cellular stress and reduced viability. Since this phenomenon is partly attributable to differences in nutrient composition between culture media and maternal tract fluids, we determined the concentrations of glucose, pyruvate, lactate and 19 amino acids in murine preovulatory follicles and oestrous oviductal and uterine fluids. Follicular fluids were aspirated from hyperstimulated ovaries, whereas oviductal fluids (with/without oocyte-cumulus complexes) and uterine fluids were collected from naturally cycling animals. Glucose, pyruvate and lactate concentrations were analysed using ultramicrofluorometric methods, whilst amino acid profiles were determined by reverse-phase high performance liquid chromatography. Mean glucose concentrations in follicular, oviduct (with/without cumulus cells) and uterine fluids were 0.46, 1.09/1.65 and 0.61 mmol l(-1), respectively. Pyruvate concentrations were 0.38, 0.37/0.17 and 0.25 mmol l(-1), respectively, and lactate concentrations were 17.34, 10.92/11.68 and 9.41 mmol l(-1), respectively. Oviductal pyruvate concentration was significantly higher, and glucose significantly lower, in the presence of cumulus cells. Taurine, glycine, alanine, glutamine and glutamate were the major amino acids detected. Concentrations of amino acids differed among fluids, with highest levels being found in the oviduct. The follicular fluid and tract nutrient profiles differed from those of murine maturation, fertilisation and embryo culture media. These data extend our understanding of cellular metabolism and of nutritional environments of the oocyte and early embryo as they progress along the reproductive tract in vivo. These results may also contribute to the formulation of nutritionally more physiological media for mouse oocyte maturation and embryo culture.     

Studies on the catabolism of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine in the rabbit.

1. The routes of elimination of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine were investigated in the rabbit. 2. Analyses showed low plasma concentrations of these amino acids (around 1 nmol/ml) and ratios similar to those found in tissue proteins. The concentrations of these amino acids in extracts of brain, kidney, liver and spleen were similar except that liver had a lower concentration of Ng-methylarginine and Ng, Ng-dimethylarginine. Cerebrospinal fluid contained traces of each amino acid.     

FREE AMINO ACID LEVELS IN THE CEREBROSPINAL FLUID OF NORMAL HUMANS AND THEIR VARIATION IN CASES OF EPILEPSY AND SPIELMEYER-VOGT-BATTEN DISEASE

Abstract— The concentrations of free amino acids in the plasma and cerebrospinal fluid from control subjects and from patients suffering from epilepsy and Spielmeyer-Vogt-Batten disease were determined using an automatic amino acid analyser. It was found that the plasma/cerebrospinal fluid ratio of amino acid concentrations showed the variation in the amounts of free amino acids in epilepsy more clearly than the cerebrospinal fluid levels alone.     

STUDIES OF THE INTER-RELATIONSHIP BETWEEN CEREBROSPINAL FLUID AND PLASMA AMINO ACID CONCENTRATIONS IN NORMAL INDIVIDUALS

Abstract— —The concentration of free amino acids has been determined in lumbar CSF in 37 fasting normal subjects. The values obtained have been compared with the concentration of the same amino acids measured in venous plasma collected simultaneously and with ventricular CSF amino acid concentrations. Twenty-three amino acids have been identified and quantitated in CSF and plasma. Trace quantities of eight other amino acids have been also detected.
The concentration of 13 amino acids in CSF has been shown to be directly related to the plasma concentration. No such relationship was noted for the other 7 amino acids. Significant variations in the concentration of individual amino acids relating to both age and sex have been noted. A large number of unidentified ninhydrin positive compounds have been found in CSF. Preliminary studies have identified one of these as ɛ-aminobutyric acid (GABA).     

Molecular cloning of the chicken avidin cDNA.

A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides. Four recombinant clones were characterized and each contained the sequence of the oligonucleotide probes used in screening. They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised against avidin. The longest clone, lambda cAV4, contained the entire coding sequence of avidin along with a signal peptide of 24 amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated using the cDNA probe.     

Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout (Zoarces viviparus)

Estrogenic chemicals such as alkylphenols (APs) have been shown to disrupt the reproductive system of male fish. In the present study, the effects of the estrogenic chemical octylphenol (100 microg g(-1)) and 17 beta-estradiol on the semen production and the biochemical composition of the seminal fluid of the viviparous eelpout (Zoarces viviparus) were investigated at the time of spawning. After 10 days of octylphenol or estrogen treatment, vitellogenin (Vtg) synthesis was induced as indicated by increased plasma vitellogenin concentration. In accordance with the increased vitellogenin concentration, hepatosomatic index (HSI), total protein concentration, and total calcium concentration were also increased, and free amino acids concentration was decreased in blood plasma. Octylphenol treatment caused a decrease in the gonadosomatic index (GSI) and the milt volume and an increase in the spermatocrit. The histological examination revealed that octylphenol affected the normal lobular structure, including the Sertoli cells. In the majority of the octylphenol-treated fish, trapped sperm cells were observed in parts of the seminiferous lobules and the sperm ducts. The biochemical composition of the seminal fluid was also affected by the octylphenol or estrogen. The seminal plasma concentrations of magnesium, calcium, and total protein were elevated, and the concentration of free amino acids was reduced in the treated fish. This study indicates that octylphenol inhibits the seminal fluid production and changes its biochemical composition in eelpouts.