ULTRASTRUCTURAL FEATURES OF BOVINE EMBRYOS DERIVED FROM CARRIERS OF AN X-AUTOSOME TRANSLOCATION

Basrur, P.K., Bouvet, A., Pinheiro, L.E.L., Berepubo, N.A. and Baird, J.D. Department of Biomedical Sciences and Department of Clinical Studies, University of Guelph, Guelph, Ontario, NlG 2Wl, Canada.

Introduction

Structural anomalies of chromosomes lead to reproductive problems in man and domestic animals. In cattle, structural alterations of the Robertsonian type have been known to cause varying degrees of subfertility in the male and female (1). Female carriers of these defects (translocations) which are generally more severely affected than males,tend to be repeat breeders (2). Translocations involving a sex chromosome and an autosome are relatively rare in cattle probably because of their fertility reducing impact on the females and the probable non-viability of the males.

In our cytogenetic survey of cattle in Southern Ontario, an X-autosome translocation was detected exclusively in the female line of a pedigree. The female carriers of this translocation were noted to be repeat breeders with a tendency to return to heat generally within 30 to 35 days after service.

Since this pattern of return to service is consistent with early embryo mortality, we undertook to examine the embryos collected from the translocation carriers in order to determine whether a consistent ultrastructural alteration foretelling the eventual destruction of these embryos is detectable at the time of embryo collection.

Materials and Methods

The herd investigated consisted of Limousine-Jersey crossbreds from which a 4 year old cow and her daughter were used as donors for embryo collection.

The heifer and the cow were karyotyped using the whole blood culture procedure prior to subjecting them to embryo collection. Three collections from the cow and 2 from the heifer, were made using the non-surgical collection procedure following superovulation with gonadotropins (3).

The embryos were rated on the basis of phase contrast morphology as "normal", "unfertilized" and "abnormal" and some of the normal embryos were transferred to synchronized recipients while the others were fixed in glutaraldehyde for light and electron microscopic observations .

Results

Altogether 20 embryos were recovered in 5 uterine flushings. Five of these were in blastocyst stage. Three blastocysts which appeared normal, and 4 normal embryos representing earlier stages, were transferred to recipients. All but one of the recipients returned to heat within 30 to 60 days. The exception concerns a cow receiving a late blastocyst which produced a live bull calf at term. The bull calf was normal in appearance and karyotype.

The abnormal blastocysts showed a tendency of the cells to be irregular in size, reduced in number and to aggregate tightly in the center or towards one enc of the zona pellucida which was often fragile (Fig. la).

Abnormal embryos of earlier stages exhibited blastomeres of varying size and number, and cytoplasmic blebs (Fig. lb). On light microscopic examination, 3 of the "embryos" previously classified as unfertilized, proved to contain two or more unequal sized cells (Fig. 2). Ultrastructural examination on the abnormalblastocysts showed very few microvilli on the cells exposed to the perivltelline space, irregularly distributed electron dense depositions on the zona pellucida, interruptions of the sub-zonal space and vacuolations of varying size and number (Fig. 3). One of the common features noted in blasstocysts was the presence of a juxtanuclear crescent (Fig. 4) probably resulting from the dilatation of the outer nuclear membrane (Fig. 4a).

Other anomalies noted included wide separation of the junctional complexes (Fig. 4b), aggregation of cellular constituents in lysosome-like bodies (Fig. 3) and enlargement and shattering of mitochondria (Fig. 4a).

A majority of the abnormal embryos and some of those identified as unfertilized, exhibited abundant lysosomes, lipids and vacuoles of various sizes.

These structures were more striking in the cytoplasmic blebs (Figs lb and 5a) which also carried distorted and hooded mitochondria (Figs. 5a and 5b).

Discussion

The ultrastructural deviations and the results of embryo transfer to recipient cows indicate that approximately 80% of the embryos from the translocation carriers are abnormal exhibiting either failure of syngamy or interruption of cleavage divisions. The fine structural abnormalities including the reduced distribution and abnormal morphology of the microvilli and the retention of oocyte type mitochondria (4) in blastocysts indicate interrupted cellular differentiation. The failure to detect indications of cytoplasmic differentiation which in normal embryos leads to the formation of a blastocoele, suggests that a majority of the embryos are defective and probably incapable of implantation. At present it is not clear what type of chromosomal imbalance is associated with the ultrastructural aberrations noted althouFh a Food proportion of the embryo from these translocation carriers are expected to be genetically unbalanced on the basis of the segregation patterns in the X-autosome translocation carriers (3).

It is worthy of note that some of the morphological changes have also been noted in embryos of karyotypically normal cows (5) although translocation carrier cows consistently exhibit retarded growth and mortality of embryos, and varying degrees of subfertility (3,5,6).

Acknowledgements

We are indebted to Mr. Ed. Reyes, Mrs. H. Randall, and Mrs. M. John for their technical assistance and to the Natural Science and Engineering Research Council and the Ontario Ministry of Agriculture and Food for financial support for this project.

References

1. Gustavsson, I. (1969) Hereditas 63: 68-16 0.

2. Refsdal, A.O. (1976) Acta vet scand. 17: 190-195.

3. KinF, N.A., and Linares, T. (1983) Can. Vet. Jour. 24: 112-115.

4. SenFer, P.L. and Saacke, R.G. (1970) J. Cell Biol. 46: 405-408.

5. Linares, T. (1991) Ph.D. Thesis. Uppsala, Sweden.

6. Swartz, H.A., and Vogt, D.W. (1983) J. Heredity 78: 320-324.

Figs 1-5: Bovine embryos from carriers of an X-autosome translocation.

la: A blastocyst showing fragile zona (arrow) and aggregation of "uncompacted" cells. lb: An abnormal embryo showing unequal sized blastomeres and a cytoplasmic bleb (cb). Phase contrast XY0. Fig. 2: Thin sections of embryos (categorized as unfertilized on phase microscopy), showing unequal blastomeres X120. Fig. 3: Electron micrographs of the blastocysts showing abnormality in number and morphology of microvilli (mv) projecting into the perivitelline space (pv), interruption of zona pellucida (arrows), sequestration of cytoplasmic material in lysosome-like bodies and vacuoles of various sizes. a: X1000j~b: X5000; C: X20,000. Fig. 4: Portions of cells from abnormal blastocysts showing juxta-nuclear crescents (cr). Note the wide separation and disruption of junctional complexes (jc) and indications of dilatation of the outer nuclear membrane (arrows) and mitochondria (m). a: X10,000: b: X1500. Fig 5: A cytoplasmic bleb from an embryo rated as unfertilized showing the abundance of vacuoles, lysosomes and distorted mitochondria (5a) and a higher magnification of mitochondria (Sb) with and without "hooded" appearance (hm and mt). a: X3500; b: X22,000.


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