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J D Biggers

Publications and source records attributed to J D Biggers.

At least 19 recordsLinked to original sources
PubMed1992

Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media.

A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCl, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage. Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium. Even in the presence of 5 mM glucose, 70% of the embryos develop to at least four cells, and 60% reach the blastocyst stage. Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development. Moreover, the response to glutamine depends on the concentration of NaCl in the medium. When the NaCl concentration is low, glutamine inhibits development. In contrast, when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt. We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte.

Animals↗
PubMed1992

Expression of mouse PDGF-A and PDGF alpha-receptor genes during pre- and post-implantation development: evidence for a developmental shift from an autocrine to a paracrine mode of action.

We examined the expression of platelet-derived growth factor (PDGF)-A and the PDGF alpha-receptor in pre-implantation and early post-implantation mouse embryos. At two-cell and blastocyst stages, all cells express mRNA and protein for both ligand and receptor. In contrast, early post-implantation embryos express PDGF-A chain mRNA in both embryonic ectoderm and in the ectoderm lining the ectoplacental cavity, while mRNA for PDGF alpha-receptor is localized to the mesoderm layers of both embryonic and extra-embryonic membranes. At days 3.5 and 7.5, receptors are demonstrably functional in response to exogenous PDGF-AA. We propose that chronic autostimulation of PDGF alpha-receptors occurs in pre-implantation embryos, whereas, following implantation, early mesoderm development is dependent on stimulation by ectodermally produced PDGF-A.

Animals↗
PubMed1991

Relief from alkaline load in two-cell stage mouse embryos by bicarbonate/chloride exchange.

Mouse embryos at the two-cell stage are able to recover from an alkaline load. We found that this recovery is mediated by sodium-independent bicarbonate/chloride exchange: intracellular pH (pHi) recovery from alkaline load is inhibited by the anion exchange inhibitor 4,4'-diisothiocyanostilbene disulfonic acid, lack of bicarbonate, or lack of chloride. The dependence of the pHi recovery on extracellular chloride concentration exhibits Michaelis-Menten kinetics. Furthermore, uptake of chloride is inhibited in a dose-dependent manner by extracellular bicarbonate. The Km for external chloride was found to be about 3 mM, with a Ki for external bicarbonate of about 2 mM. The exchanger is active above approximately pH 7.15. These results demonstrate that mouse embryos at the two-cell stage possess a sodium-independent bicarbonate/chloride exchange mechanism that is similar to that found in other mammalian cells. This bicarbonate/chloride exchanger appears to be the sole pHi-regulatory mechanism in the two-cell stage mouse embryo, since our previous results have shown that there are apparently no specific mechanisms active in these cells for relieving acid loads.

Animals↗
PubMed1991

Two-cell stage mouse embryos appear to lack mechanisms for alleviating intracellular acid loads.

Mouse embryos at the two-cell stage, like other cells, can recover from an intracellular acid-load. Our previous work has shown, surprisingly, that there is no contribution to this recovery by Na+/H+ antiport activity. Here we show that the recovery similarly is not affected by inhibition of other known intracellular pH (pHi) regulatory mechanisms. Specifically, the recovery is unaffected by lack of external Na+, inhibition of anion exchange, or lack of bicarbonate, which eliminates the Na(+)-dependent HCO3-/Cl- exchanger as a possible mechanisms. These conditions also eliminate any possible Na+,HCO3- cotransporter operating to relieve acid-loading. Recovery is unaffected similarly by nonspecific inhibitors of H(+)-ATPase activity. These observations lead to the conclusion that recovery from acid-load is a passive process in the two-cell mouse embryo. Similarly, the mean base-line pHi (6.84) is not dependent on known pHi regulatory mechanisms. The embryos exhibit a marked intracellular alkalinization when exposed to Cl(-)-free medium in the presence of bicarbonate. This response is eliminated by an inhibitor of anion exchange and by lack of bicarbonate, but is independent of Na+. These results indicate that there is probably a Na(+)-independent HCO3-/Cl- exchanger active in these cells, presumably functioning to alleviate alkaline loads.

Animals↗
PubMed1991

Oxygen transport to embryos in microdrop cultures.

The standard method for culturing small preimplantation mammalian embryos is a system in which they are placed into microdrops of culture medium under oil. Thus the source of oxygen for the embryos lies beyond two liquid phases--medium and oil. If transport of oxygen is not sufficiently rapid to replace that consumed by the embryos, the medium could become depleted of oxygen. Over small distances, the dominant means by which oxygen is transported through liquid is by diffusion. Our calculations show that diffusion alone is sufficient to supply oxygen to mouse embryos; there is virtually no perturbation of the oxygen concentration when there are up to 10 embryos in the drop, and 50 embryos produce a drop in oxygen tension that is not large enough to have a deleterious effect. Furthermore, diffusion is probably not the dominant mechanism by which oxygen is transported to the embryos; on the scale of these microdrops, convection is faster and would serve to mix the drop so that anoxic regions cannot develop. Therefore, we conclude that even a relatively large number of embryos in a culture drop do not significantly deplete oxygen.

Blastocyst↗
PubMed1991

Growth and DNA replication in rabbit blastocysts.

DNA content and DNA polymerase activity were measured on rabbit blastocysts removed from the uterus at 24-hr intervals over the period of days 4-7 postcoitum (pc). Median DNA content increased 53 times over the 72-hr period, from 25.3 ng on day 4 to 1,360 ng on day 7. Median DNA polymerase activity (fmole of radiolabeled nucleotide incorporated in 30 min at 37 degrees C) increased 393-fold from day 4 to day 7: 32.8 to 12,900. These embryos also increased in surface area and volume by 334-fold and 6,078-fold, respectively. Litters containing individuals with high DNA content also tended to have similar individuals with high DNA polymerase activity. Therefore, DNA polymerase activity may be a useful measure of the potential for the next cell division. A large amount of variation existed between blastocysts in all parameters measured. An analysis of variance, conducted to partition variation between litters and within litters, determined that within-litter variation was actually greater than that between litters, resulting in intraclass correlation coefficients less than 0.5. There was also a positive regression of DNA content and DNA polymerase activity on surface area in 6- and 7-day-old blastocysts after eliminating variation attributable to litters. The developmental pattern of DNA polymerase activity in the rabbit may be quantitatively different from that described in the mouse. The pattern in mammals is very different from that described in several nonmammalian species.

Animals↗
PubMed1991

Postnatal compensatory growth of manipulated mouse embryos.

As an extension of the biometrical analysis of data described in an earlier paper (Papaioannou et al., 1989, Development, 106, 817-827), we have examined the extent of postnatal compensatory growth in body weight and tail length of genetically identical F1 mice from the following groups: controls--the natural offspring of timed matings; transferred controls--offspring from embryos flushed at the 2-cell stage and transferred to recipient foster mothers; and transferred half embryos--offspring developing from one blastomere from the 2-cell stage transferred to recipient foster mothers. There was little difference between the postnatal growth curves of transferred controls and mice produced from half embryos, indicating regulation during prenatal development is complete and that halving an embryo at the beginning of gestation has no lasting effects on its size or postnatal growth rate. In contrast, embryo transfer itself had a significant effect, resulting in smaller litters and larger, more variable animals after birth. There was then a period of postnatal compensatory growth in body weight and tail length after 4 weeks of age. We have provided direct evidence that animals that become relatively large during early growth curtail their growth rate compared to small animals as they approach maximum size and the reverse occurs with small animals. A similar phenomenon was observed for tail length. Although the overall variance of embryo transfer mice is high during postnatal growth, the intraclass correlation coefficient is also high, making them useful for comparative experiments.

Animals↗
PubMed1991

Quality of oocytes from superovulated rhesus monkeys.

The parameters, serum oestradiol (E2) response, follicle size and cumulus morphology, which are commonly used to determine in-vivo oocyte maturation in human in-vitro fertilization and embryo transfer (IVF-ET) programmes, were shown to be unreliable predictors of maturation of rhesus oocytes. In two groups of rhesus, one stimulated with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) and the other with human menopausal gonadotrophin (HMG) and HCG, the three parameters varied widely within and between protocols. Triple fluorochrome staining (TFS) for chromatin, microtubules and filamentous actin (f-actin) in oocytes at the time of collection and following 24 h in culture showed major differences in their maturation both in vivo and in vitro following priming with PMSG and HMG. In evaluating IVF protocols, TFS provides a valuable assay for the meiotic status of fixed oocytes of non-human primates.

Animals↗
PubMed1991

Overcoming the 2-cell block by modifying standard components in a mouse embryo culture medium.

The 2-cell block may be caused by inappropriate concentrations of commonly used constituents of embryo culture media. Almost all zygotes obtained by fertilizing CF1 ova with hybrid B6D2F1/CrlBR sperm did not develop beyond the 2-cell stage when cultured in Whittingham's medium M16. This 2-cell block was overcome by lowering the concentrations of NaCl, KCl, KH2PO4, glucose, and pyruvate, either individually or in combination. The effects of changing the concentration of either NaCl or KCl depend on the concentration of NaHCO3 in the medium. Although a high percentage of embryos grew to the 4-cell stage in several media with lowered concentrations of certain components, the media are not optimal for complete preimplantation embryo development since the yield of blastocysts is low.

Animals↗
PubMed1991

Optimization of mouse embryo culture media using simplex methods.

Culture media were developed for pronuclear-stage mouse embryos using simplex optimization, which has the benefit of being able to optimize several components simultaneously. Initially, several different media were generated. All media contained the same components, yet each medium was characterized by having a different component at a high concentration. The simplex procedure identified 4 components (NaCl, pyruvate, KH2PO4 and glucose) which at high concentrations were detrimental to embryo development, compared to the other components tested. For example, all embryos cultured in a medium with high NaCl blocked at the 2-cell stage. The optimization method then adjusted each medium by lowering the concentration of the component or removing it entirely, which resulted in a significant increase in development. In an experiment comparing 8 media generated from the simplex optimization, along with 7 other media, removal of KH2PO4 resulted in the largest increase in development; 88% of embryos were greater than or equal to 4 cells on Day 3 after hCG, and 53% developed into blastocysts by Day 5. Another experiment compared 4 of the best media generated from the simplex optimization. In 3 out of the 4 media, 90% or more of the embryos were greater than or equal to 4 cells on Day 3. In 3 of the media, approximately 60% or more of the embryos developed into blastocysts. The simplex optimization procedure is an efficient method for developing culture media and determining requirements for development in vitro.

Animals↗
PubMed1990

Polar trophoblast (Rauber's layer) of the rabbit blastocyst.

The germinal area of the rabbit blastocyst between 108 h postcoitum (pc) and 168 h pc has been examined by scanning electron and transmission electron microscopy. At 108 h and 120 h pc the polar trophoblast (Rauber's layer) is an intact epithelium overlying the epiblast of the inner cell mass. By 132 h pc the polar trophoblast cells begin to separate at multiple foci, exposing the underlying epiblast. Most of the polar trophoblast cells have become individually separated at 144 h pc. The villous, electron-dense polar trophoblast cells can be easily distinguished from the cells of the epiblast, which have smooth apical surfaces. By 162 h pc the polar trophoblast cells have disappeared from the germinal area. Before the polar trophoblast breaks up, the underlying epiblast cells are only loosely attached to one another. Concurrent with the disintegration of the trophoblast epithelium, the epiblast cells change in shape so that their lateral borders become closely apposed, and junctions develop to form a new epithelium. The epiblast becomes contiguous with the mural trophoblast, and thus the blastocyst does not lose its turgidity as the permeability seal is maintained. There are two classical theories on the fate of the polar trophoblast: the cells die, or they become incorporated into the epiblast as living cells. In newly exposed epiblast the presence of very large phagosomes, which are not found when the polar trophoblast is still intact, favors the first hypothesis and indicates that in the rabbit the epiblast is involved in the phagocytosis of the polar trophoblast.

Animals↗
PubMed1990

Apparent absence of Na+/H+ antiport activity in the two-cell mouse embryo.

We have used the pH-sensitive dye BCECF to investigate the regulation of intracellular pH (pHi) by two-cell stage mouse embryos in bicarbonate-free medium. There is no indication of a Na+/H+ antiport active in regulating pHi, as recovery from acid-loading was insensitive to amiloride, ethylisopropylamiloride, or the absence of extracellular Na+. Instead, protons appear to be in equilibrium across the plasma membrane, as indicated by the response of pHi to changes in external K+. The embryos have an intracellular buffering power in the normal range (25.3 mM/pH); their apparent permeability to protons is, however, very high (0.22 cm/sec).

Animals↗
PubMed1990

Arbitrary partitions of prenatal life.

The word 'embryo' and the closely related word 'fetus' have been used since the 14th century to partition prenatal life. Very recently two new compound words have been introduced to denote a further partition of early development. This article stresses that the definitions of these words denote arbitrary phases in a continuous process, and therefore have little precise scientific use. In rigorous ethical debate such arbitrary, terminology, particularly if it is used to assign moral values, should be avoided.

Embryo, Mammalian↗
PubMed1989

Development of a noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos.

A noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos is described. A linear relationship was found between fluorescence intensity of NADH produced and glutamine concentration (R2 = 0.985). Single embryos were placed in 20 nl drops of medium containing 0.5 mM glutamine, and the medium was sampled after 2 hr incubation at 37 degrees C. Changes in net glutamine uptake were determined in one-cell, two-cell, and eight-cell embryos and blastocysts incubated in medium containing no energy substrates (glucose, pyruvate, and lactate). The median glutamine uptake increased significantly from 0.480 and 0.270 pmoles/embryo/2 hr at the one-cell and two-cell stages, respectively, to 1.610 pmoles/embryo/2 hr at the blastocyst stage. Mean glutamine uptake was compared in the presence or absence of energy substrates at several developmental stages. A highly significant reduction of glutamine uptake in the presence of substrates was observed at the one-cell and two-cell stages of development. At the eight-cell stage, glutamine uptake was only marginally reduced in the presence of substrates, and no effect was found at the blastocyst stage. These data may partially explain the beneficial effect of glutamine on the culture of early mouse embryos through the two-cell block of development.

Animals↗
PubMed1989

The effect of heterologous gonadotropins on the fecundity of rhesus monkeys.

Ovarian stimulation with heterologous gonadotropins over a period of a single month did not depress the fecundity or fecundability of 50 rhesus monkeys as compared to 60 untreated monkeys in a timed breeding colony. The birth rates, seasonality of births, interbirth intervals, and waiting time to the next pregnancy were not significantly different before treatment, after treatment, or among the untreated. The stillbirth rate was not increased after treatment.

Animals↗
PubMed1989

Development and phenotypic variability of genetically identical half mouse embryos.

The growth and development of three groups of genetically identical F1 C57BL/6J female x SJL/J male mice were compared to examine whether embryo manipulation affects subsequent postnatal growth and development of mammalian embryos: (1) controls--the natural offspring of timed matings, (2) transferred controls--offspring from 2-cell embryos transferred to recipients 1 day asynchronous, and (3) transferred half embryos--offspring developing from one blastomere from the 2-cell stage transferred to recipients 1 day asynchronous. The recipients were C57BL/6J females. No differences were found in the age at eye opening and vaginal opening. At 5 days after birth the median body weights of the controls were lower than the weights of the transferred groups. This result could be explained by the larger litter size in the control group. The overall variances of the body weights did not differ between the groups. By the second week after birth a marked increase in overall variances of body weights of the transferred groups, compared with the control group, was observed. At 5 days after birth, the median tail lengths did not differ between groups, and overall variances were the same. By the second week, the overall variances of the tail lengths of the transferred groups were significantly greater than that of the control group. Possibly the increased overall variances of the body weight and the tail length of the transferred groups are related to the smaller litter size in these groups which affects competition for food and the ambient temperature in the nest. The overall results suggest newborn mice that have developed from half embryos have compensated for their initial deficiency. The intraclass correlation coefficients for body weight and tail length are approximately the same in all groups. Thus, producing artificial identical twins by embryo bisection may not affect their potential usefulness in the design of experiments.

Animals↗