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K J Betteridge

Publications and source records attributed to K J Betteridge.

At least 19 recordsLinked to original sources
PubMed2003

Production of capsular material by equine trophoblast transplanted into immunodeficient mice.

A novel xenogeneic transplantation approach was used to determine whether it is embryonic or maternal tissue that produces the material that gives rise to the mucin-like glycoprotein of the equine embryonic capsule. Endometrial biopsy samples and conceptuses from six mares at days 13-15 after ovulation were prepared as 1 mm(3) grafts of endometrium, trophoblast and capsule for transplantation, alone or in combination, into various sites in 88 immunodeficient (severe combined immunodeficient or RAG2/gamma(c) double mutant) mice. The overall recovery rate of grafts was over 50%, reaching 100% with experience and use of the renal subcapsular space exclusively. Periodic acid-Schiff (PAS) staining demonstrated capsule-like extracellular glycoprotein secretions at the graft site in 11 of 22 sites examined. Strong PAS-positive reactions (5-7 microm thick) were found in four of six sites containing trophoblast alone, five of six endometrium plus trophoblast sites, and zero of eight grafts of endometrium alone. Two recovered grafts of capsule were also PAS-positive. The secreted glycoprotein was identified as equine embryonic capsule material by using a monoclonal antibody (mAb) specific to equine capsule (mAb OC-1) in two experiments. In the first, in cryosections, this antibody bound to 19 of 19 recovered trophoblast graft secretions (including those in 12 from mice that had not received endometrium at any site), ten of ten recovered endometrium plus trophoblast grafts, and zero of 12 recovered endometrial grafts from mice in which trophoblast had been grafted to the same site or another site in the same mouse. In the second experiment, in paraformaldehyde-fixed sections of grafts from 11 mice, specific staining, identical to that shown by grafted capsule, was obtained with grafts of trophoblast (both alone and in combination with endometrium) but not with grafts of endometrium. These results support the contention that trophoblast is the principal source of equine embryonic capsule. In addition, they demonstrate that xenogeneic grafting is a useful means of culturing endometrium and conceptus tissues outside the mare when in vitro techniques do not suffice.

Animals↗
PubMed2003

Dose-response of RAG2-/-/gammac-/- mice to busulfan in preparation for spermatogonial transplantation.

Practical applications of spermatogonial transplantation require good rates of colonization by the donor cells. Recipient testes are usually depleted of competing endogenous spermatogonia by administration of 32-44 mg busulfan kg(-1) body weight before transplantation. However, it is not clear that this is the optimum dose, especially for immunodeficient mice. In the present study, the response of adult RAG2(-/-)/gamma(c)(-/-) (RAG2) male mice to treatment with 10-50 mg busulfan kg(-1) body weight was determined in terms of mortality rates, testicular masses and histology, and colonization of seminiferous tubules by transplanted spermatogonia. Mortality increased from 0 to 50% at doses between 20 mg busulfan kg(-1) and 40 mg busulfan kg(-1), whereas the maximum effects on testicular mass and histology were observed at 20 mg busulfan kg(-1). Colonization of testes by genetically marked spermatogonia after treatment of mice with 20 mg busulfan kg(-1) was equivalent to rates previously reported in recipients treated with 32-44 mg busulfan kg(-1). Thus, 20 mg busulfan kg(-1) appears to be the optimum dose for preparing RAG2 mice for spermatogonial transplantation. However, because the steepness of the dose-response curves indicates that direct administration of busulfan is not ideal for this purpose, 15 mg busulfan kg(-1) was administered to pregnant females at various times between day 10.5 and day 16.5 of gestation to determine whether this would deplete the number of germ cells in male offspring. Although there were large variations in testicular mass and histology, no mortality was observed and administration of busulfan at day 10.5 or 12.5 after mating delayed initiation of spermatogenesis, indicating that prenatal administration of busulfan combined with neonatal transplantation might be an effective method for further increasing rates of colonization by donor spermatogonia.

Alkylating Agents↗
PubMed2001

Comparisons of oocyte maturation times and of three methods of sperm preparation for their effects on the production of goat embryos in vitro.

Various times of in vitro maturation of oocytes, and three methods of separating spermatozoa from frozen-thawed semen (Percoll density-gradient centrifugation, swim-up, and glass-wool filtration), were compared for their effects on goat embryo production in vitro. Cumulus-oocyte-complexes (COCs) from abattoir ovaries were matured in M199 supplemented with 10% fetal calf serum and hormones. In Experiment 1, COCs were fixed at 4 h intervals from 0 to 27 h of culture to assess oocyte nuclear maturation. A higher proportion cultured for 27 h than for 24 h were in Metaphase II (27/37, 73% vs. 18/33, 55%, P < 0.05). In Experiment 2, the effects of separation methods on total numbers and numbers of membrane-intact spermatozoa, and the acrosome reaction were compared. Total numbers after Percoll density-gradient centrifugation were approximately 4 times higher than after swim-up and approximately 2 times higher than after glass-wool filtration (P < 0.001). Progression of the acrosome reaction was not affected differentially. In Experiments 3 and 4, after 27 h of culture the COCs were inseminated with sperm isolated by the three methods. In Experiment 3, presumptive zygotes were examined for pronucleus (PN) formation at 6, 12, 18 and 24 h post-insemination. At 12 h, male PN formation rate from Percoll-treated spermatozoa was higher than from sperm subjected to swim-up and glass-wool treatments (20/37, 54% vs. 6/37, 16% and 6/38, 16%, respectively; P < 0.001). In Experiment 4, embryos were compared for cleavage at 48 h and development into blastocysts, hatching rates and cell number at 192 h. The rates of cleavage and blastocyst formation in the Percoll-treated group were higher (P < 0.05) than in the swim-up and glass-wool groups (62% and 18% vs. 50% and 11%, and 45% and 8%, respectively). Similarly, the mean cell number in the Percoll group was higher (P < 0.05) than in the swim-up and glass-wool groups (167 +/- 5 vs. 149 +/- 4 and 126 +/- 4, respectively). We conclude that Percoll density-gradient centrifugation is superior to the other two methods for separating goat spermatozoa from frozen-thawed semen in preparation for IVF.

Acrosome↗
PubMed2001

Effect of blastomere sex and fluorescent labelling on the development of bovine chimeric embryos reconstituted at the four-cell stage.

The development rate of bovine chimeric embryos reconstituted at the 4-cell stage is relatively low. If chimerism is to be used as an approach in producing transgenic livestock, it is important to investigate whether this rate is affected by the sex of the blastomeres being combined and if all blastomeres survive equally well. In Experiment 1, blastomeres from 4-cell stage embryos were inserted into surrogate zonae pellucidae either in pairs to reconstitute 4-cell chimeras, or as the original sets of four to make handled controls. The development of chimeras with one pair of blastomeres labelled with PKH26-GL was also investigated. The rate of development into blastocysts was similar in chimeras with unlabelled blastomeres (23%) and in those in which one pair of blastomeres was labelled (26%) and was lower (P < 0.001) than in the handled and IVF control groups (43 and 58%, respectively). Labelled cells were distributed approximately evenly between ICM and trophoblast. In Experiment 2, the effect of sex differences between pairs of blastomeres in chimeras was investigated; chimeras were reconstituted from pairs of blastomeres taken from 4-cell embryos in which the remaining pair was sexed by PCR. No significant differences according to the sex of constituent blastomeres were detectable (mixed sex, 27%; males, 24%; females, 21%; P > 0.05). These results suggest that, in addition to the negative effects of micromanipulation, factors other than the sex of the blastomeres are involved in the reduced rate of development of chimeric bovine embryos. They also confirm the usefulness of PKH26-GL labelling for tracking the progeny of cleaving bovine blastomeres at least to the blastocyst stage.

Animals↗
PubMed2001

Comparative aspects of conceptus growth: a historical perspective.

This review provides a historical background for a series of articles arising from a symposium on Early Regulation of Mammalian Development. It begins by tracing connections between the work of J. Cossar Ewart and the first cloning of mammals by somatic cell nuclear transfer. Historical scientific landmarks over the last century that have affected our attitudes to 'fetal autonomy' and conceptus development are reviewed briefly, as are salient studies that have established the importance of nutrition to pre- and postnatal life. Finally, attention is drawn to some differences in conceptus development among various mammals, and the argument made that understanding these differences might be of benefit to the development of reproductive technology.

Animals↗
PubMed2000

Comparative aspects of equine embryonic development.

The developmental changes in the equine conceptus, its maternal environment and their interaction during the first 4 weeks following fertilization are reviewed. Attention is drawn to species-specific events to show why the horse is such a valuable model in which to study early pregnancy.

Animals↗
PubMed2000

Reflections on the golden anniversary of the first embryo transfer to produce a calf.

In the belief that the vitality of a discipline benefits from a knowledge of its roots, events surrounding the first embryo transfer to produce a live calf (by Willett and his colleagues in Wisconsin fifty years ago) have been reviewed. That first success is discussed in the context of those times (including a brief review of the first international conference on embryo transfer, held in Texas in 1949) and in relation to subsequent events that led to the founding of the International Embryo Transfer Society in 1974.

Animals↗
PubMed1998

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro.

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.

Animals↗
PubMed1998

Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection.

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6-dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3-h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co-cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI.

Adenine↗
PubMed1998

Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes.

This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.

Animals↗
PubMed1997

Biochemical changes in the equine capsule following prostaglandin-induced pregnancy failure.

The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of "fixation," or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These "defective" conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with 3H on sialyl-oligosaccharides and 125I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule.

Abortion, Induced↗
PubMed1997

Ultrastructural and immunocytochemical analysis of diploid germ cells isolated from fetal rabbit gonads.

Germ cells were isolated from rabbit fetal gonads between 18 and 22 days post coitum and examined morphologically, ultrastructurally and for immunocytochemical and cytochemical characteristics. Observations were compared with the information available from the corresponding cells of other mammalian species. The general morphology and ultrastructure of healthy isolated rabbit fetal germ cells were found to be very similar to those of the rabbit and mouse diploid germ cells in situ. Moreover, rabbit fetal germ cells shared common immunocytochemical characteristics with mouse undifferentiated embryonic stem cells or embryonic carcinoma cells, such as the presence of TEC-1 (SSEA-1) antigens, a peripheral network of F-actin, the absence of cytokeratins 8/18 and lamins A/C and an alkaline phosphatase activity. No difference between the sexes was observed. Morphological and physiological similarities with the migrating and cultured primordial germ cells of the mouse also suggest that diploid rabbit germ cells would be good candidates for deriving pluripotential embryonic germ cells (EG cells) if favourable culture conditions could be found. In conclusion, the rabbit may be a suitable model for investigations on EG cells in domestic mammals with delayed meiosis.

Alkaline Phosphatase↗
PubMed1997

Birth of live mice resulting from oocytes fertilized in vitro with cryopreserved spermatozoa.

The objective of this study was to develop a reliable method to cryopreserve mouse spermatozoa, using in vitro fertilization (IVF) of oocytes, development of resultant zygotes into blastocysts, and the birth of live young after embryo transfer as criteria of sperm survival. Epididymal sperm were frozen in a raffinose-glycerol solution containing either egg yolk (EY) or BSA. Sperm frozen in the presence of EY yielded higher survivals than those frozen in the presence of BSA; when used for IVF, the former resulted in more blastocysts (26% vs. 5%). Comparison between one-step and stepwise dilution of the cryoprotectant revealed no differences in sperm viability as assayed by a vital stain. Nevertheless, IVF using frozen sperm recovered by one-step or stepwise dilution resulted in 33% and 17% blastocyst formation, respectively. When embryos produced with cryopreserved sperm were transferred into recipients, pregnancies (12 of 15 recipients) and the birth of live young (57 pups from 167 embryos transferred) resulted. These results were comparable to those obtained from transferral of embryos produced by IVF with fresh sperm (17 pregnancies from 19 recipients, 87 live young from 197 embryos). Thus, this study has resulted in the development of a simple and efficient cryopreservation procedure for mouse sperm.

Animals↗
PubMed1997

Development of bovine nuclear transfer embryos made with oogonia.

The pluripotency of embryonic germ cells in the mouse suggests that mitotic bovine fetal germ cells might also be a source of pluripotent cells. To investigate the pluripotency of bovine oogonia, the development in vitro of bovine embryos reconstructed by fusing oogonia with enucleated oocytes was compared with that of embryos made similarly with either blastomeres or granulosa cells. The donor cells (fresh oogonia, cryopreserved oogonia, 16- to 32-cell-stage blastomeres, or granulosa cells) were fused to the enucleated oocytes electrically. The proportions of reconstructed embryos that had cleaved at 40 h after fusion using these types of donor cells were not significantly different (37%, 33%, 56%, and 31%, respectively; p > 0.05). However, the proportions of cleaved reconstructed embryos that developed to the blastocyst stage were 9%, 13%, 36%, and 3%, respectively, significantly higher (p < or = 0.05) with blastomeres than with the other three types of donor cells. After transfer of 3 morulae and 4 blastocysts made with oogonia into three recipient heifers, embryonic and extra-embryonic tissues developed in one animal. On recovery after 43 days gestation, this conceptus was shown to be genetically identical, at 11 microsatellite loci, to the fetus that had provided the oogonia. Cytological analysis of the embryos made with oogonia at 40-44 h after fusion and at the morula and blastocyst stages revealed that aberrant cytokinesis and nucleokinesis had given rise to multinucleated, anucleate, and polyploid cells in the reconstructed embryos. It is concluded that limited pluripotency of bovine oogonia has been demonstrated, warranting further study in this area.

Animals↗
PubMed1997

Transcription and translation in bovine nuclear transfer embryos.

The development of bovine embryos reconstructed by nuclear transfer (NT) is poor compared to that of embryos produced by in vitro fertilization. One reason for this could be incomplete reprogramming of the transferred nucleus. Therefore, with a view to optimizing the conditions for NT, the reprogramming of blastomere nuclei from 16- to 32-cell-stage in vitro-fertilized (IVF) embryos was investigated following NT by fusion of individual blastomeres with cytoplasts prepared from oocytes at two different stages of maturation. Heterogeneous RNA (hnRNA) production, nucleolar ultrastructure, and protein profiles of the NT embryos up to the 8-cell stage were analyzed. In all NT embryos analyzed for their hnRNA production (n = 133), [3H]uridine incorporation was higher at the 1-, 2-, and 4-cell stages than in control IVF embryos (n = 50). Ultrastructural examination of 11 NT embryos revealed evidence of transcriptional activity; fibrillar and granular components were seen in the nucleolus at the 1-cell stage. At the 2-, 4-, and 8-cell stages, fibrillar components were still evident but granular components had become scarce. The hnRNA synthesis, however, was not reflected in the one-dimensional electrophoretic patterns of protein production in the NT embryos (n = 56); these were largely similar to those of IVF embryos (n = 34) of corresponding stages. Thus, NT embryos made in this way do not behave like equivalent IVF embryos, suggesting that reprogramming of the transferred nucleus is absent or incomplete.

Animals↗
PubMed1997

Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus.

The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and beta1 subunits are both present in Day 15 conceptuses. Trophoblast tissues from 19 conceptuses between 8 and 31 days after ovulation were stained immunohistochemically using primary antibodies against the alpha1 and beta1 subunit isoforms of the Na+,K+-ATPase. Both isoforms were detected in all sections. Trophoblastic vesicles, prepared from 6 conceptuses between 12 and 14 days after ovulation, were used to investigate the inhibition of blastocyst expansion with ouabain after collapse induced with cytochalasin D. In normal medium there was a mean 3-fold increase, and in ouabain (10(-6) M) a mean 3-fold decrease, in the volume of vesicles that had been partially collapsed with cytochalasin D. We therefore conclude that, despite the hypotonicity of the blastocyst fluid in the early horse conceptus, the Na+,K+-ATPase plays a role in its accumulation, as in other species.

Animals↗
PubMed1996

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells.

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18-22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (alpha-1 antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8- to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines.

Animals↗