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Biomedical subjects

L M Wiley

Publications and source records attributed to L M Wiley.

At least 55 records · Page 3Linked to original sources
PubMed1987

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay.

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

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PubMed1986

Immunofluorescent localization of immunoglobulins on the cell surface of mouse oocytes and preimplantation embryos.

Living mouse oocytes and preimplantation embryos were assayed by indirect immunofluorescence for their ability to adsorb heterologous serum proteins from culture media to their cell surfaces. Bovine and human immunoglobulins of the IgG class were adsorbed by the oocytes and all stages of preimplantation embryos, while IgG of mouse or goat origin was not. In contrast, none of the serum albumins was adsorbed to the cell surfaces of mouse oocytes and preimplantation embryos. From the differential binding of IgG of some, but not all, of the species that were tested, we concluded that these cell surface IgG "receptors" on mouse oocytes and preimplantation embryos are likely to be heterophilic in nature. Similar observations were made irrespective of the strain of mice used to provide the oocytes and embryos. These observations raise the question of whether human oocytes and preimplantation embryos should also be assayed for their ability to adsorb animal serum proteins that are possible candidates as a substitute protein supplement for human serum in culture medium used in human in vitro fertilization/embryo transfer programs.

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PubMed1986

Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro.

Mouse preimplantation embryos were used to test the effects of three culture parameters on development of 2-cell stage embryos to the late morula stage (40 to 43 hours of culture, 65 to 80 hours after fertilization): concentration of potassium (K)--0.6 mM, 1.4 mM, and 6.0 mM; type of protein supplement, bovine serum albumin (BSA), and bovine serum immunoglobulins (BSIG); and number of embryos (2 or 20) cultured per 10- to 12-microliter drop of T6 culture medium under paraffin oil. Cell number per embryo of cultured late morulae was compared with that of same-age embryos developing in vivo. The developmental rate to late morula stage with 20 embryos per drop of medium was media containing 0.6- and 1.4-mM K, 0.15% BSA, 0.15% BSIG greater than medium containing 1.4-mM K, 0.3% BSA greater than all media containing 6.0-mM K (P less than 0.01 comparing media containing 0.6- or 1.4-mM K with media containing 6.0-mM K). For a given medium, developmental rate was significantly slower for 2 embryos per drop than for 20 embryos per drop of medium with those media containing either 0.3% BSA (for all concentrations of K tested) or 6.0-mM K (for 0.3% BSA and for 0.15% BSA + 0.15% BSIG). Finally, cell number per embryo was: in vivo-developed embryos greater than medium containing 1.4-mM K, 0.15% BSA, 0.15% BSIG much greater than 6.0- or 1.4-mM K, 0.3% BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1985

Polarity of isolated blastomeres from mouse morulae: detection of transcellular ion currents.

Eight- to sixteen-cell stage mouse morulae were dissociated with Ca2+-free medium into blastomeres that were labeled with fluoresceinated-succinylated Con A (FS-Con A) to mark their apical-basal axes. The vibrating probe was then used to map their extracellular current patterns. The average current density around normal blastomeres approached the resolution of the probe system (0.2 microA/cm2) and was undetectable in the majority of blastomeres. Since the current density at the measuring point outside the cell is known to increase with cell size in other systems, enlarged blastomeres were created by fusing together blastomeres of 4-cell stage embryos in 45% polyethylene glycol. Enlarged blastomeres were then aggregated with normal blastomeres using phytohemagglutinin and cultured to the 8- to 16-cell stage to allow them to become polarized. Such aggregates were then dissociated with Ca2+-free medium to recover polarized, enlarged blastomeres. The enlarged blastomeres were 30-65 microns in diameter and 70% of them generated a detectable current; currents were detected around 83% of those blastomeres larger than 40 micron in diameter. The current pattern in these most reliable cases was predominantly inward apical (11/16 or 69%) and outward basal (15/16 or 94%), with lateral currents about three-fold smaller in amplitude than these apical-basal currents. Lateral currents were undetectable in 53% of the cases. Preliminary data suggest that the inward current is carried in part by Na+ influx and is independent of the Na+,K+-ATPase over the short term. Transcellular ion currents were detectable as long as 4 hr after dissociation, and the apical-basal current pattern was usually stable during that time. In contrast, the fluorescent cap of FS-Con A faded within 7-30 min at 35 degrees C but remained stable in 0.1% azide or 1.5 micrograms/ml cytochalasin D. The electrical polarity therefore persisted after the apical cap of Con A fluorescence was no longer visible. We propose that these transcellular ion currents may be involved in the establishment of blastomere polarity and describe a mechanism of action in an "ion current polarization" hypothesis.

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PubMed1984

Cavitation in the mouse preimplantation embryo: Na/K-ATPase and the origin of nascent blastocoele fluid.

This study tested the proposition that Na/K-ATPase activity could be involved in the morphogenetic aspects of mouse blastocyst formation by facilitating the localization of certain organelles to apposed borders, the production of nascent blastocoele fluid, and cavitation. It was assumed that such Na/K-ATPase activity would be sensitive to varying concentrations of external K (Ko)--which would alter plasma membrane potentials--and to ouabain--which would directly alter Na/K-ATPase function. Morulae were cultured for 40 hr in varying concentrations of Ko and/or ouabain and were observed for their ability to form blastocoeles (cavitate) and to localize mitochondria to apposed cell borders. Cavitation was accelerated when Ko was decreased from the control value of 6.0 to 0.6 mM and was delayed when Ko was increased to 25 mM. With Ko at 6.0 mM, 10(-5) M ouabain accelerated cavitation while 10(-4) M ouabain delayed cavitation and reduced the total number of embryos that cavitated by the end of the 40-hr culture period. With Ko at 0.6 mM, 10(-5) M ouabain now delayed cavitation while 10(-4) M ouabain almost completely inhibited it. When Ko was increased to 25 mM, 10(-5) M ouabain again accelerated cavitation while 10(-4) M ouabain delayed-rather than inhibited--cavitation. Morphometric analyses at the electron microscopic level showed changes in the distances of mitochondria from apposed cell borders with conditions that accelerated or delayed cavitation and these changes differed for inside and outside cells of the morula. These observations are consistent with the proposition that Na/K-ATPase activity could be involved in the localization of organelles to apposed cell borders, the production of nascent blastocoele fluid, and in cavitation during mouse blastocyst development.

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PubMed1978

Apparent trophoblast giant cell production in vitro by core cells isolated from cultured mouse inner cell masses.

Mouse ICM's that were isolated from blastocysts by immunosurgery and subsequently cultured for four to five days were tested for their ability to form trophoblast giant cells (TGC's) in vitro. The cores were isolated from such ICM's by a second immunosurgery and cultured for 7 to 14 days. After two days of culture a variable percentage (30%--90%) of the ICM cores produced outgrowths containing cells that resembled those of cultured ectoplacental cone. Then the outgrowths became amitotic and produced vascuolated cells that enlarged to dimensions of 200 X 375 micrometer with nuclei as large as 53 micrometer in diameter. The time course of their development from the ICM cores, their morphology and their nuclear diameters suggest that these large cells were TGC's.

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PubMedDate not supplied

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accelerates differentiation of murine preimplantation embryos in vitro.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent teratogen in several animal species, especially during the period of organogenesis. The purpose of this study was to test the hypothesis that TCDD has direct effects on the earliest stages of murine embryonic development, namely the preimplantation stages. Three endpoints were measured: 1) embryo cell number, a measure of embryo viability, 2) competitive embryonic cell proliferation utilizing chimeric embryos, another measure of embryo viability, and 3) cavitation rate, a functional measure of trophectoderm differentiation. Neither embryo cell numbers nor competitive embryonic cell proliferation (chimera assay) were affected by TCDD, either when the mother was dosed in vivo (prior to fertilization), or when 2-cell embryos were dosed in vitro. However, cavitation rates of in vitro-dosed embryos, in the presence of 10% fetal bovine serum, were significantly higher for TCDD than controls, suggesting that TCDD accelerated differentiation of murine preimplantation embryos. Taken together, these results demonstrate that: 1) TCDD can act directly on the murine preimplantation embryo, and 2) TCDD's actions are primarily on accelerated differentiation and not on embryo viability. To our knowledge, this is the earliest stage of mammalian development during which TCDD has been shown to exert an effect.

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