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Biomedical subjects

K J Betteridge

Publications and source records attributed to K J Betteridge.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differential ability of male and female rabbit fetal germ cell nuclei to be reprogrammed by nuclear transfer.

The pluri- or totipotency of gonial cells, isolated from rabbit fetuses at 18-20 days of pregnancy, has been investigated by transferring their nuclei into enucleated oocytes and following the development of the resulting reconstituted embryos both in vitro (in a total of 726 embryos) and in vivo (in 135 embryos). The gonial cells exhibited pseudopodial activity like that of primordial germ cells and ultrastructural studies confirmed that neither male nor female cells had entered meiosis. When the gonial cells were used immediately after isolation, about 37% of the reconstituted embryos of both sexes cleaved, with no significant difference according to sex. However, after a further 4-day culture of the cleaved embryos, the blastocyst formation rate was four times higher in those made with male (16%) than with female (4%) gonial cells. No implantation sites were detected following transfer of reconstituted embryos into recipient females. These results show that the nuclei of male and female rabbit diploid germ cells differ in their capability to be "reprogrammed" and bring about development to the blastocyst stage following nuclear transfer. The origin of this difference, which is evidenced long before the onset of meiosis is discussed.

Animals↗
PubMed1996

Cryopreservation of germ cells from bovine fetal ovaries.

Bovine fetuses at stages required for studies of female germ cells (primordial germ cells and oogonia) become available from the abattoir at unpredictable times. To alleviate this logistical problem, a procedure to cryopreserve these ovarian germ cells has been devised. Fetal ovarian cells were dispersed and suspended in 1.5 M dimethyl sulfoxide (DMSO) prepared in modified TCM 199 medium. The suspensions were aspirated into plastic semen straws, cooled, seeded to induce ice formation at -7 degrees C, and then cooled at 1 degree C min-1 to -70 degrees C before being plunged into liquid nitrogen at -196 degrees C for storage. The straws were thawed at a moderate rate of approximately 250 degrees C min-1, the DMSO was diluted 28-fold with culture medium, and then the cells were cultured for > 2 h before their viability was tested or they were used for nuclear transfer. No statistically significant difference in viability before and after cryopreservation was detected by vital staining with fluorescein diacetate (P > 0.05). When frozen-thawed germ cells were fused to cytoplasts, the cleavage rate of the resultant reconstructed embryos 44 h after fusion was 31%, although none developed into blastocysts. It is concluded that cryopreservation of bovine fetal ovarian germ cells is feasible and can play a major role in facilitating future experimentation.

Animals↗
PubMed1996

Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy.

Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmolalities and Na+ concentrations increased gradually with age but the former never exceeded 255 mOsm kg-1. Fluids from Group B were obtained from eight conceptuses exposed to saline solutions of different osmolalities for various periods of time. An increased perivitelline space of 1-3 mm became evident at the lower pole of the floating conceptus after 45 min of exposure to solutions with osmolalities of > or = 300 mOsm kg-1, suggesting that Na+ and Cl- diffuse freely through the capsule but not through the trophoblast. The significance of the hypo-osmolality of equine blastocyst fluid is discussed but remains unclear.

Animals↗
PubMed1996

The use of prostaglandins and oxytocin for transcervical recovery of bovine fetuses at days 33-58 of gestation.

The study of bovine germ cells of known developmental stage calls for alternatives to the recovery of fetuses by surgery or slaughter. Fetuses were therefore obtained during the second month of pregnancy by aborting 49 animals using a progressively modified treatment regimen of cloprostenol, prostaglandin E2 (PGE2) and oxytocin. The viability of fetuses was monitored by ultrasonography throughout treatment. Intracervical treatment with PGE2 led to cervical dilation in all treated animals. However, retrieval of the fetuses by subsequent flushing of the uterus was successful in only two of six animals. When i.m. injections of cloprostenol were given 20-40 h before PGE2 treatment, fetuses < or = 40 days of gestation were expelled spontaneously, while the majority of fetuses > or = 50 days of gestation were retained. When i.m. injections of oxytocin were given in relation to clinical signs of impending fetal expulsion after cloprostenol and PGE2 treatment, 20 of 22 fetuses were expelled 42-53 h after the cloprostenol injection. Of these 20 fetuses, 19 were expelled 0-7 h after the cessation of fetal heartbeat. The subsequent fertility of animals was not affected. Thus, the final protocol allowed bovine fetuses to be retrieved at predictable times, within a few hours of death, with little maternal trauma and without affecting subsequent fertility.

Abortion, Induced↗
PubMed1995

Production of four identical calves by the separation of blastomeres from an in vitro derived four-cell embryo.

The blastomeres of two in vitro derived four-cell embryos were separated and transferred individually into surrogate zonae pellucidae, then co-cultured with bovine oviductal epithelial cells for five days until blastulation. Pairs of the quarter blastocysts were co-transferred with trophoblastic vesicles into each of four synchronised Holstein heifers, three of which were diagnosed pregnant at 28 days gestation, carrying twin fetuses. Four genetically identical bull calves were delivered by elective caesarean section at term pregnancy. One pregnancy was terminated at 56 days.

Animals↗
PubMed1995

Comparison of the effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos.

The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitro fertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 +/- 6.1 versus 56.7 +/- 3.3; P < or = 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 +/- 1.0 versus 1.5 +/- 0.4 pmol per embryo per 3 h, P < or = 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Isolation and identification of germ cells from fetal bovine ovaries.

Gonadal cell suspensions were made from bovine fetuses of 35-55-, 56-80-, and 80-130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35-55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56-80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80-130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation.

Animals↗
PubMed1993

Mucin-like glycoproteins in the equine embryonic capsule.

The equine embryonic capsule replaces the zona pellucida and envelopes the conceptus during the second and third weeks of pregnancy. Although this capsule was described more than 100 years ago, its molecular structure has not been characterized. Here we present evidence that the glycoprotein(s) of the equine capsule resembles those of the mucin glycoprotein family. The resistance of the capsule to chemical and enzymatic solubilization was confirmed, and, as in mucins, protein constituted only 35-40% of its total dry mass. Determination of the sugar composition of the capsule using colorimetric assays and high-performance anion-exchange chromatography also showed it to have mucin-like characteristics. Gal, GalNAc, sulfated sugars, and sialic acid make up a high proportion of the capsular carbohydrate, while GlcNAc, Glc, and Man are minor components. These findings were verified using lectin histochemical staining of frozen sections of conceptuses. The results of amino acid analysis were also consistent with the proposal that the capsular glycoproteins belong to the mucin family. Removal of the covalently bound carbohydrate by beta-elimination under reducing conditions demonstrated that the capsule is O-glycosylated mainly on threonine residues. Affinity chromatography on jacalin-agarose confirmed that, like mucins, the capsular glycoproteins are heavily O-glycosylated. SDS-PAGE analysis revealed a prominent 21-kDa band, specific to the capsule, in preparations solubilized by trypsin but not by other proteases. Characterization of its constituent glycoprotein(s) should be helpful in elucidating the role of the capsule (and analogous blastocyst coverings in other species) during early pregnancy.

Amino Acids↗
PubMed1993

Relationships between the completion of first cleavage and the chromosomal complement, sex, and developmental rates of bovine embryos generated in vitro.

One thousand eighty-four two-cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24, 30, 40, 48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight-cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the "early" cleaving (24 and 30 hpi) and "late" cleaving (40-62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight-cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X- and Y-bearing sperm within the zygote or to very early differential expression of genes derived from X- and Y-bearing sperm.

Animals↗
PubMed1993

Embryo transfer and related techniques in domestic animals, and their implications for human medicine.

This report was commissioned by the Canadian government on the relevance of new techniques in animal embryology to the social, ethical and clinical applications of assisted human reproduction. It briefly describes the history of animal breeding, and the regulation of the female reproductive tract, ovulation and fertilization in laboratory and veterinary species. Embryo transfer is described in detail, including the synchronization of reproductive cycles, superovulation and embryo growth in vitro. Methods of experimental embryology, including bisection, sexing of spermatozoa and embryos, cloning and gene therapy are described. The relevance of these studies to human IVF are considered briefly.

Animals↗
PubMed1993

Developmentally regulated changes in the glycoproteins of the equine embryonic capsule.

The embryonic capsule, which covers the equine blastocyst after it loses its zona pellucida, is composed of mucin-like glycoproteins. In the present study, we investigated both macroscopic and molecular changes in the capsule during development. The weight of the capsule increased from day 11-12 of pregnancy and reached a maximum at about day 18, coinciding with the time during which the conceptus migrates extensively throughout the uterus. The sialic acid content of the capsule declined markedly from about day 16, the time of conceptus 'fixation' in the uterus, which suggests a unique developmentally regulated mechanism for the control of embryo mobility. These results lead us to propose that the capsule may have an anti-adhesion function in the developing conceptus, and that this effect could be regulated by the sugar side chains of the capsular glycoproteins. The glycosylation characteristics of the blastocyst coverings also underwent changes at about day 9 of pregnancy, which may be related to loss of the zona pellucida. An anti-capsule monoclonal antibody was raised and shown to recognize a tissue-specific antigen present only on the capsule and trophoblast. This antigen was present on the trophoblastic cells soon after the blastocyst is formed, reached a maximum concentration at about day 18, and was absent after day 22, coinciding with the disappearance of the capsule. Immunohistochemical studies indicate that the mucin-like capsular glycoproteins are secreted, at least in major part, by the trophoblast.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

Application of in vitro fertilisation techniques to obtain calves from valuable cows after slaughter.

The ovaries of two infertile cows of high breeding value were recovered after slaughter, and a total of 222 oocytes were obtained. Of these, 156 were classified as of good or fair quality and were subjected to in vitro maturation, in vitro fertilisation (using frozen semen from three bulls of high breeding value) and in vitro culture procedures. After eight days, 27 embryos were obtained, of which 13 were transferred fresh, and 14 were frozen. Three recipients of fresh embryos became pregnant; two calved and one aborted at four months. One of eight recipients of frozen-thawed embryos became pregnant but aborted at three months.

Abattoirs↗
PubMed1992

Sex-related differences in developmental rates of bovine embryos produced and cultured in vitro.

The classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex-determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex-related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.

Animals↗
PubMed1992

Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro.

The metabolism of, and retention of radioactivity from, radiolabelled glucose, glutamine and pyruvate were measured in individual cattle embryos produced in vitro from the 2-cell to hatched blastocyst stage. Uptake was defined as the numeric sum of metabolism and retention of radiolabel. Glucose metabolism increased significantly between the 8- and 16-cell stages, but was accompanied by a much larger increase in glucose uptake. Consequently, the proportion of glucose uptake that was metabolized through the pentose-phosphate and Embden-Meyerhof pathways reached a minimum at those stages. From the compacted morula stage onward, the calculated uptake of [14C]glucose was only 25 to 33% of that calculated for [5-3H]glucose. This suggests that 66 to 75% of glucose carbon leaves the embryo, after metabolism to phosphoenolpyruvate, in some form other than CO2. Little or no glucose metabolism by the Krebs cycle could be detected at any stage. Both glutamine and pyruvate metabolism were relatively high at the 2- and 4-cell stages, declined to a minimum at the compacted morula stage and then increased with blastulation. Glutamine metabolism continued to increase with expansion and hatching of the blastocyst, but pyruvate metabolism did not. This suggest that, relative to the activity of the pathway from pyruvate to 2-oxoglutarate, the activity of the 2-oxoglutarate-to-oxaloacetate segment of the Krebs cycle is of increasing significance during expansion and hatching of the cattle blastocyst.

Analysis of Variance↗
PubMed1992

Development and viability of bovine embryos derived from oocytes matured and fertilized in vitro and co-cultured with bovine oviducal epithelial cells.

A co-culture system using a suspension of detached bovine oviducal epithelial cells (BOEC) has been developed as an effective culture method for supporting the development of bovine embryos derived from oocytes matured and fertilized in vitro. Four commercially available culture media (Waymouth's, Ham's F-10, TCM 199 and Ménézo's B2) supplemented with 10% oestrous cow serum, and a modified Tyrode's medium (TALP) supplemented with 0.6% bovine serum albumin were used. Ménézo's B2 resulted in the highest percentages of total uncleaved presumptive zygotes, and of the cleaved zygotes that reached at least the morula stage (31-46% and 66-74%, respectively). The embryos produced in vitro in B2 with BOEC resembled embryos produced in vivo with regard to numbers of cells (averaging 45.4 in morulae, 101.5 in blastocysts, 174.7 in hatching blastocysts and 195.9 in hatched blastocysts), rate of development (hatching on Day 8-9 of culture in vitro), rate of hatching (66% of cleaved zygotes) and pregnancy rates (63%) resulting from the transcervical transfer of selected embryos.

Animals↗
PubMed1992

Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro.

The metabolism of radiolabelled glucose and glutamine was measured in individual cattle embryos produced by in vitro maturation and fertilization of oocytes, and culture with bovine oviductal epithelial cells. Metabolism of glucose through the pentose-phosphate pathway increased almost 15 times and the total metabolism of glucose 30 times, during development from the two-cell to the expanded blastocyst stage. The first marked increase in glucose metabolism did not occur until between the eight- and 16-cell stages, the time of activation of the embryonic genome. Conversely, the metabolism of glutamine was high in two- and four-cell embryos and then decreased to reach a minimum at the compacted morula to blastocyst stage, possibly because of degradation of maternally derived enzymes. Blastocyst expansion was accompanied by significant increases in the metabolism of glucose and glutamine, presumably reflecting the increased energy demands of Na(+)-K+ ATPase necessary for formation and maintenance of the blastocoel.

Analysis of Variance↗