Development of zona-free hamster ova to blastocysts in vitro

The zona pellucida (ZP) enclosing the mammalian ovum is important for its protection and for initial stages of fertilization, but the role of the ZP during embryo development is less clear. This study was designed to investigate if the hamster ZP is needed for embryo development from 1-cell to blastocyst in vitro, and to compare methods for removing the ZP. A total of 395 hamster pronucleate ova were collected 10 h post activation from superovulated, mated female hamsters. The ZP was removed from some ova using either 0.05% pronase, 0.05% trypsin or acid Tyrode's solution. To prevent ZP-free ova from sticking together, they were cultured singly in 30-50 microL drops of HECM-6 culture medium together with ZP-intact ova as controls. There was no significant difference among treatment groups in embryo development to blastocyst: 36/87 (42%) in the ZP intact group; 35/75 (47%) in the pronase-treated ZP-free group; 37/74 (50%) in the trypsin-treated ZP-free group; and 37/71 (52%) in the acid-treated ZP-free group. These results indicate that 1) the ZP is unnecessary for hamster embryo development in vitro from the pronucleate ovum stage to blastocyst; 2) none of the three ZP-removal methods was detrimental to embryo development; 3) embryos do not need to be cultured in groups during in vitro development from 1-cell to blastocyst.     

Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts

In vitro development of eight-cell hamster embryos to hatching blastocysts requires the presence of amino acids and a group of water-soluble vitamins in the culture medium. The present studies investigated the effect of type of macromolecule on blastocyst hatching and on the requirement for vitamins. Embryos were cultured for 3 days in the presence of the synthetic macromolecule polyvinylalcohol (PVA) and of different types of bovine serum albumin (BSA), both with and without vitamins. The results showed th at eight-cell embryos develop to hatching blastocysts in the presence of vitamins and amino acids with PVA as the only macromolecule in the medium. The presence of certain types of BSA reduced but did not eliminate the need for vitamins. Glutamine alone was as efficient as a complete amino acid supplement in supporting blastocyst hatching. These results demonstrate for the first time that eight-cell hamster embryos can be cultured to hatching blastocysts in a chemically defined medium.     

Reactivating tammar wallaby blastocysts oxidize fatty acids and amino acids.

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.     

Vitamin requirements for development of eight-cell hamster embryos to hatching blastocysts in vitro

We have shown in previous studies that development of 8-cell hamster embryos to hatching and hatched blastocysts in vitro is stimulated by the addition to the culture medium of a group of 11 water-soluble vitamins and growth factors from Ham's F10 medium. In the present study, the requirement for each of these vitamins for blastocyst hatching was examined by using a chemically defined protein-free medium. Eight-cell hamster embryos were cultured for 3 days either in medium with all 11 vitamins or in media with a single vitamin omitted at a time or in medium without any vitamins. The only vitamins whose omission caused a significant decrease in blastocyst hatching at any stage were inositol, pantothenate, and choline, with the omission of inositol having the most severe effect. This finding was confirmed in a subsequent experiment in which the addition of these 3 vitamins stimulated the same degree of hatching as all 11 vitamins.     

Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of pig blastocysts in vitro is altered by serum, bovine serum albumin and amino acids and vitamins

We tested the effects of the amino acids and vitamins in minimum essential medium (MEM) and Eagle's medium (BME) on pig blastocyst development and nuclei number. Embryos were recovered either 5 or 6 d after first detected estrus and were cultured for 96 h in U-bottomed wells (0.2 ml). In Experiment 1, addition of MEM amino acids and vitamins to modified Krebs-Ringer bicarbonate (MKRB) medium containing either bovine serum albumin (BSA, 4 mg/ml) or lamb serum (10%, v/v) resulted in fewer (P<0.001) nuclei and smaller (P<0.05) embryo volumes at the end of culture as compared to embryos cultured in MKRB without MEM-supplements. Addition of MEM-amino acids without glutamine (Experiment II) depressed blastocyst volume and rate of hatching, but glutamine (2 mM) had no effect on embryo development. Dialysis (molecular weight > 12,000 retained) of fetal bovine serum (Experiment III) did not affect blastocyst expansion but reduced (P<0.05) the number of nuclei/blastocyst at the end of the culture. Embryos cultured in MKRB with dialyzed serum and the amino acids and vitamins in BME were smaller (P<0.05) and had fewer (P<0.05) nuclei than embryos cultured in MKRB with dialyzed serum but without the BME-supplements. We conclude that, under our culture conditions, MEM and BME amino acids and vitamins are detrimental to the development of early pig blastocysts and that this effect is not due to glutamine. Also, dialysis of fetal bovine serum removes some component(s) that are important for cell division by pig embryos, but it does not affect blastocyst expansion.     

Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm

The mammalian blastocyst must hatch from its extracellular coat, or zona pellucida, to implant in the uterus and continue development normally. Results of experiments described here strongly suggest that a proteinase (74K Mr), called "strypsin," is directly involved in hatching of isolated mouse blastocysts in vitro. Strypsin is a trypsin-like proteinase, based on its substrate specificity and sensitivity to inhibitors, that is present in mouse blastocysts and exhibits certain properties characteristic of membrane-associated enzymes. Histochemical and autoradiographic evidence suggests that, prior to hatching of blastocysts, strypsin is found with cells of mural trophectoderm; not with polar trophectoderm or inner cell mass. Following hatching, strypsin is also found associated with empty zonae pellucidae, specifically at the opening through which the embryo emerged. These and other observations suggest that hatching of mouse blastocysts in vitro is initiated by limited proteolysis of the region of zona pellucida overlying mural trophectoderm.     

Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration

Addition of chymotrypsin or trypsin decreased the viscosity of highly viscous semen of subfertile patients. Besides that spermatozoal motility (in 50-80% of the case) and in vitro fertilizing ability was measured with zona-free hamster ova (in 35% of the cases) were increased of both normal and highly viscous semen.     

Insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonatal pigs

Infusion of physiological levels of insulin and/or amino acids reproduces the feeding-induced stimulation of muscle protein synthesis in neonates. To determine whether insulin and amino acids independently stimulate skeletal muscle protein synthesis in neonates, insulin secretion was blocked with somatostatin in fasted 7-day-old pigs (n = 8-12/group) while glucose and glucagon were maintained at fasting levels and insulin was infused to simulate either less than fasting, fasting, intermediate, or fed insulin levels. At each dose of insulin, amino acids were clamped at either the fasting or fed level; at the highest insulin dose, amino acids were also reduced to less than fasting levels. Skeletal muscle protein synthesis was measured using a flooding dose of l-[4-(3)H]phenylalanine. Hyperinsulinemia increased protein synthesis in skeletal muscle during hypoaminoacidemia and euaminoacidemia. Hyperaminoacidemia increased muscle protein synthesis during hypoinsulinemia and euinsulinemia. There was a dose-response effect of both insulin and amino acids on muscle protein synthesis. At each insulin dose, hyperaminoacidemia increased muscle protein synthesis. The effects of insulin and amino acids on muscle protein synthesis were largely additive until maximal rates of protein synthesis were achieved. Amino acids enhanced basal protein synthesis rates but did not enhance the sensitivity or responsiveness of muscle protein synthesis to insulin. The results suggest that insulin and amino acids independently stimulate protein synthesis in skeletal muscle of the neonate.     

Nonessential amino acids are not necessary to stimulate net muscle protein synthesis in healthy volunteers

The present study was performed to test the hypothesis that orally administered essential amino acids, in combination with carbohydrate, will stimulate net muscle protein synthesis in resting human muscle in vivo. Four volunteers ingested 500 mL of a solution containing 13.4 g of essential amino acids and 35 g sucrose (EAA). Blood samples were taken from femoral arterial and venous catheters over a 2-hour period following the ingestion of EAA to measure arteriovenous concentrations of amino acids across the muscle. Two muscle biopsies were taken during the study, one before administration of the drink and one approximately 2 hours after consumption of EAA. Serum insulin increased from normal physiologic levels at baseline (9.2 +/- 0.8 microU/mL) and peaked (48 +/- 7.1 microU/mL) 30 minutes after EAA ingestion. Arterial essential amino acid concentrations increased approximately 100 to 400% above basal levels between 10 and 30 minutes following drink ingestion. Net nitrogen (N) balance changed from negative (-495 +/- 128 nmol/mL) prior to consumption of EAA to a peak positive value (416 +/- 140 nmol/mL) within 10 minutes of ingestion of the drink. EAA resulted in an estimated positive net N uptake of 307.3 mg N above basal levels over the 2-hour period. Muscle amino acid concentrations were similar prior to and 2 hours following ingestion of EAA. We conclude that ingestion of a solution composed of carbohydrates to stimulate insulin release and a small amount of essential amino acids to increase amino acid availability for protein synthesis is an effective stimulator of muscle protein anabolism.     

Na(+)-dependent transport of anionic amino acids by preimplantation mouse blastocysts.

Negatively charged amino acids, such as aspartate and glutamate, were selected as substrates by low- and high-Km components of mediated Na(+)-dependent transport in preimplantation mouse blastocysts. These and other relatively small anionic amino acids with two carbon atoms between the negatively charged groups (or up to three carbon atoms when the groups were both carboxyl groups) interacted strongly with the low-Km component of transport, whereas larger anionic amino acids interacted weakly or not at all. The low-Km system was also stereoselective except in the case of aspartate. Moreover, transport was Cl(-)-dependent and slower at pH values outside the range 5.6-7.4. L-Aspartate, D-aspartate and L-glutamate each interacted strongly with the low-Km component of transport with Km values for transport nearly equal to their Ki values for inhibition of transport of one of the other amino acids. By these criteria, the low-Km component of transport of anionic amino acids in blastocysts appears to be the same as the familiar system X-AG that is present in other types of mammalian cells. In contrast, the high-Km component of transport in blastocysts preferred L-aspartate to L-glutamate, whereas the reverse is true for fibroblasts. Therefore, transport of anionic amino acids in blastocysts may occur via at least one process that has not been described in other types of cells. Roughly half of mediated glutamate and aspartate transport in blastocysts may occur via the high-Km component of transport at the concentrations of these amino acids that may be present in uterine secretions.     

Phorbol esters stimulate the transport of anionic amino acids in cultured human fibroblasts

The effect of phorbol esters on the transport of amino acids has been evaluated in cultured human fibroblasts. The activity of the Na(+)-dependent system XAG- for anionic amino acids is selectively and markedly stimulated by phorbol esters. The effect is maximal within 15 min; it is attributable to an increase in transport maximum (Vmax) and not prevented by inhibitors of protein synthesis. The half-maximal stimulation is observed at concentrations of phorbol 12,13-dibutyrate lower than 100 nM. Prolonged incubations in the presence of 1 microM phorbol 12,13-dibutyrate lower the binding of the ligand to its receptor with a loss of the stimulatory effect on transport. The results presented indicate that the stimulation of amino acid transport through system XAG- by phorbol esters requires the activation of protein kinase C.     

Transport of naturally occurring amino acids and alpha-amino isobutyric acid by normal and diapausing mouse blastocysts

Uptake of ¹⁴C-labelled α-amino isobutyric acid and a mixture of naturally occurring amino acids by normal mouse preimplantation blastocysts was found to be twice that of delayed implantation blastocysts. With both groups of embryos concentration of the amino acids against a gradient occurred. The results may explain, in part, the lower amino acid incorporation rates found with diapausing vs. normal blastocysts and open the question of “metabolic dormancy” of the diapausing embryo.     

Exogenous amino acids stimulate human muscle anabolism without interfering with the response to mixed meal ingestion

We sought to determine whether ingestion of a between-meal supplement containing 30 g of carbohydrate and 15 g of essential amino acids (CAA) altered the metabolic response to a nutritionally mixed meal in healthy, recreationally active male volunteers. A control group (CON; n = 6, 38 +/- 8 yr, 86 +/- 10 kg, 179 +/- 3 cm) received a liquid mixed meal [protein, 23.4 +/- 1.0 g (essential amino acids, 14.7 +/- 0.7 g); carbohydrate, 126.6 +/- 4.0 g; fat, 30.3 +/- 2.8 g] every 5 h (0830, 1330, 1830). The experimental group (SUP; n = 7, 36 +/- 10 yr, 87 +/- 12 kg, 180 +/- 3 cm) consumed the same meals but, in addition, were given CAA supplements (1100, 1600, 2100). Net phenylalanine balance (NB) and fractional synthetic rate (FSR) were calculated during a 16-h primed constant infusion of L-[ring-2H5]phenylalanine. Ingestion of a combination of CAA supplements and meals resulted in a greater mixed muscle FSR than ingestion of the meals alone (SUP, 0.099 +/- 0.008; CON, 0.076 +/- 0.005%/h; P < 0.05). Both groups experienced an improvement in NB after the morning (SUP, -2.2 +/- 3.3; CON, -1.5 +/- 3.5 nmol x min(-1) x 100 ml leg volume(-1)) and evening meals (SUP, -9.7 +/- 4.3; CON, -6.7 +/- 4.1 nmol x min(-1) x 100 ml leg volume(-1)). NB after CAA ingestion was significantly greater than after the meals, with values of 40.2 +/- 8.5 nmol x min(-1) x 100 ml leg volume(-1). These data indicate that CAA supplementation produces a greater anabolic effect than ingestion of intact protein but does not interfere with the normal metabolic response to a meal.     

Attachment of porcine blastocysts to fibroblast monolayers in vitro

The objective of this study was to compare the ability of porcine blastocysts to attach to various cellular and non-cellular substrates $\text{in vitro}$. One hundred twenty-two hatched blastocysts were collected from 17 handmated gilts and sows at slaughter. Blastocysts were randomly assigned to one of four treatments: Minimal Essential Medium (MEM) supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibroblasts in MEM + 10% HTFCS (Btes), and MEM + 10% HTFCS exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Embryos were cultured individually in 24 well Linbro culture plates at 37 C in a humidified gas atmosphere of 5% CO₂ in air. Embryos were observed at 24 hr intervals by phase contrast microscopy (100X) and measured with an ocular micrometer. Blastocyst attachment was greater (P < .01) in Buf ($\text{21}\text{35}$) compared to MEM ($\text{7}\text{32}$), cMEM ($\text{9}\text{28}$), and Btes ($\text{1}\text{27}$). Embryo diameter was greater (P < .05) 24 hr prior to attachment in Buf compared to the other treatments. In addition, trophoblast monolayers continued to proliferate for 20 days when cocultured with uterine fibroblasts. These observations suggest that uterine fibroblasts provide a superior substratum for blastocyst attachment and the maintenance of swine trophoblast cells $\text{in vitro}$.     

Transformation of some hydroxy amino acids to other amino acids

It has been observed that -hydroxy--amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehyde reactions, glycine also reacts with acetone to give -hydroxyvaline under prebiologically feasible conditions. It is suggested, therefore, that the formation of -hydroxy--amino acids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions.