Parvathi K. Basrur

Genetic diseases of sheep and goats.

Congenital malformations and inherited disorders constitute a substantial proportion of the afflictions seen in sheep and goats. Of these, malformations tend to be similar in both species, whereas the genetic diseases encountered to date, with the exception of a few, are different. Of the 28 genetic diseases of sheep and goats described in this review, 60% and 62.5%, respectively, are monogenic disorders. For a majority of the monogenic recessive disorders encountered in these species, the carrier state is not detectable at present, whereas in others, in which a biochemical lesion is known (dermatosparaxis, erythrocyte glutathione deficiency, globoid cell leukodystrophy and glycogen storage disease), the carrier state is detectable with the aid of enzyme and surface protein markers. The latter group and the dominant disorders (anury, cataract, glomerulonephritis, and lethal grey in sheep; gynecomastia and anotia-microtia complex in goats) are easy to eliminate through selective breeding. The polygenic disorders (entropion, epidermolysis bullosa, hereditary chondrodysplasia, and muscular dystrophy of sheep, and udder problems in goats) are more difficult to eradicate, because the mutant genes responsible for these traits generally do not declare themselves until inbreeding brings together a critical concentration to create a health crisis in some, whereas others, which are only short of a few of these mutant genes, might go totally unaffected and therefore undetected. Chromosome defects of the structural nature (translocations) seen in sheep and goats generally create meiotic disturbances, which in a majority of cases lead to subfertility, whereas sex chromosome aneuploids are generally sterile.(ABSTRACT TRUNCATED AT 250 WORDS)

X-autosome translocation and low fertility in a family of crossbred cattle.

An investigation was carried out on a family of Limousin-Jersey crossbreds exhibiting low fertility in the females, to determine the impact of a previously identified X-autosome translocation (X-AT) on the reproductive performance of the carrier cows. Three of the identified translocation carriers, including a cow and two of her daughters, were maintained at our University Research Station and artificially inseminated periodically with semen from different bulls of known fertility. Attempts to breed the X-AT carriers resulted in high rates of return to estrus between days 28 and 60, abortions between days 121 and 235 after insemination, and a total of 13 live births including 4 translocation carrier calves. Results of superovulation and embryo retrieval trials on X-AT carriers revealed significantly higher proportions of unfertilized and uncleaved ova and abnormal embryos compared to those from normal cows, and no pregnancy in the recipients transferred with morphologically normal blastocysts from X-AT carriers. While the higher rates of failed fertilization and cleavage, abnormal embryos and return to estrus in X-AT carriers could be attributed to chromosome imbalance expected in their gametes, the relatively high prevalence of abortion (late in gestation) was unexpected. Our observations on the fetuses expelled by X-AT carriers after 5 months of gestation indicated that a majority (three out of four) of these fetuses were products of abnormal (3:1) segregation in meiosis I and that these chromosomally unbalanced (hyperdiploid) conceptuses were able to survive early embryogenesis and fetal life up to the end of the second trimester. We hypothesize that their relatively long in utero life and the absence of any overt birth defects may be attributable to the type of chromosomes over-represented in these fetuses and that their eventual expulsion may have been the result of selection against the clonal population of cells in which the altered X carrying a segment of chromosome 23 (Xp(+)), remained inactive.

Spatial distribution of histone isoforms on the bovine active and inactive X chromosomes.

The inactive X chromosome (Xi) in female mammals serves as an important model for studying the role of histone isoforms in directing specific nuclear processes leading to inherited differences in transcription. In the present study, we investigated the distribution of some histone isoforms known to be involved in the process of human X inactivation on their bovine counterparts. To ascertain the identity of active and inactive X chromosome, their distribution was investigated on the X chromosomes in a cell line derived from a bovine female carrying an X;autosome translocation rcp(Xp+;23q–) which allowed the recognition of the maternal (translocated) and paternal (normal) X chromosome. The distribution patterns of histone H3 trimethylated at lysine 9 (H3K9me3) and trimethylated at lysine 27 (H3K27me3), and histone macroH2A1 and macroH2A2 (isoforms specific to heterochromatin) were determined by immunocytochemistry and compared to the temporal pattern of replication using BrdU pulse labeling prior to staining. Immunostaining revealed that H3K9me3, H3K27me3, and macroH2A1 are preferentially concentrated on the Xi, whereas the histone variant macroH2A2 is not a marker for this chromosome. H3K9me3, H3K27me3, and macroH2A1 were consistently located in bands along the Xi, while H3K9me3, macroH2A1 and macroH2A2 localized in the pericentromeric regions of the autosomes. H3K27me3 identified two intense bands on the Xi at Xp22 and Xq31, representing the early replication regions of the chromosome. H3K27me3 and macroH2A1 overlapped in the Xq31 region. It was concluded that different heterochromatin regions on the bovine inactive X chromosome can be identified by their histone isoform composition.

Anatomical and Cytological Sex of a Saanen Goat

A Saanen goat registered as female was found to have a pair of abdominal testes, a well developed uterus and a prominent clitoris. The chromosome features of peripheral leukocytes and the nuclear sex

Meiosis and apoptosis in germ cells of X-autosome translocation carrier boars.

Meiotic features and fate of germ cells were studied using electron microscopy on surface spread spermatocytes and in situ tests for apoptosis on testicular tissues of normal boars and X‐autosome translocation (X‐AT) carrier boars. Histological sections of the translocation t(Xp+; 14q−) carrier boars showed accumulation of degenerating germ cells including binucleate and multinucleate cells, as well as pyknosis and nuclear fragmentation characteristic of apoptosis. Synaptonemal complex analysis of X‐AT carrier boars revealed 19 bivalents including a large complex made up of the altered X (Xp+) and normal chromosome 14, and a smaller element representing the Y chromosome in synapsis with the derived chromosome 14 (14q−) in most (89.3%) of the germ cells. In situ tests for apoptotic DNA fragmentation revealed positive signals exclusively among early spermatocytes and degenerating germ cells. These findings and the absence of stages beyond pachytene suggest that the meiocytes are arrested at pachytene and eliminated through apoptotic process in spite of the complete synapsis displayed by the chromosomes involved in this translocation. Failure of meiotic progress in our X‐AT carriers would appear to be the result of the disruption of gene sequence (or function) caused by the involvement of the X chromosome in this rearrangement, rather than the deleterious consequences of abnormal segregation anticipated in reciprocal translocation carriers. We hypothesize that this disruption could have affected the induction of stage‐specific gene products in meiosis such as heat shock proteins and caused the excessive release of endonucleases normally produced by early prophase meiocytes, leading to their apoptosis in our X‐autosome translocation carrier boars. Mol. Reprod. Dev. 56:448–457, 2000.

Veterinary cytogenetics: past and perspective

Cytogenetics was conceived in the late 1800s and nurtured through the early 1900s by discoveries pointing to the chromosomal basis of inheritance. The relevance of chromosomes to human health and disease was realized more than half a century later when improvements in techniques facilitated unequivocal chromosome delineation. Veterinary cytogenetics has benefited from the information generated in human cytogenetics which, in turn, owes its theoretical and technical advancement to data gathered from plants, insects and laboratory mammals. The scope of this science has moved from the structure and number of chromosomes to molecular cytogenetics for use in research or for diagnostic and prognostic purposes including comparative genomic hybridization arrays, single nucleotide polymorphism array-based karyotyping and automated systems for counting the results of standard FISH preparations. Even though the counterparts to a variety of human diseases and disorders are seen in domestic animals, clinical applications of veterinary cytogenetics will be less well exploited mainly because of the cost-driven nature of demand on diagnosis and treatment which often out-weigh emotional and sentimental attachments. An area where the potential of veterinary cytogenetics will be fully exploited is reproduction since an inherited aberration that impacts on reproductive efficiency can compromise the success achieved over the years in animal breeding. It is gratifying to note that such aberrations can now be tracked and tackled using sophisticated cytogenetic tools already commercially available for RNA expression analysis, chromatin immunoprecipitation, or comparative genomic hybridization using custom-made microarray platforms that allow the construction of microarrays that match veterinary cytogenetic needs, be it for research or for clinical applications. Judging from the technical refinements already accomplished in veterinary cytogenetics since the 1960s, it is clear that the importance of the achievements to date are bound to be matched or out-weighed by what awaits to be accomplished in the not-too-far future.

Hormonal correlates of ovarian alterations in bovine freemartin fetuses

Abstract Gonadal morphology and steroidogenic patterns of female bovine fetuses in twin pregnancies were compared with those of single male and female fetuses to determine the morphogenetic and functional correlates of ovarian alteration in freemartins. In male fetuses, seminiferous cords were detected by 40 days and Leydig cells by 45 days. The ovaries of 50-day fetuses showed clusters of steroidogenic cells forming medullary cords, which appeared to be the precursors of the follicular cells detected in the ovarian cortex of fetuses over 90 days old. Among the steroids produced by the testis of single male fetuses, testosterone was the most prominent, androstenedione was intermediate and estrone and estradiol were in trace amounts, whereas ovaries secreted low levels of testosterone and moderate amounts of androstenedione, estradiol and estrone. Ovarian estradiol secretion in the fetus peaked during 50 to 70 days of development while estrone was produced at elevated levels during 75 to 95 days. During the peak period for estradiol, steroidogenesis in the ovary appeared to follow the androstenedione-to-estrone-to-estradiol pathway. From the relative concentrations of steroids detected, it would appear that steroid synthesis declines in fetal ovaries, probably due to the inhibition of 17β-hydroxysteroid dehydrogenase, prior to that of aromatase. In contrast, in female fetuses of heterosexual twins (freemartins) an interruption of ovarian differentiation was evident as early as 55 days whereas testicular cords and cells resembling Leydig cells were apparent only in fetuses over 70 days old. The morphologic evidence of ovarian alteration was preceded by functional alterations in that the levels of estrogen were significantly reduced and androstenedione was elevated in freemartins as early as 40 days of fetal development. These initial changes were followed by elevated levels of testosterone and androstenedione after 75 days in virilized freemartin gonads coincident with the induction of Leydig cells in the ovary under the influence of the male co-twin. The higher levels of androstenedione secreted by freemartin gonads in early stages seem to further confirm that the androstenedione-to-testosterone pathway may be inherently inactive in normal bovine ovaries.

Chromosome studies in bovine quintuplets

Chromosome analysis has been carried out on the four surviving calves of a bovine quintuplet set. Blood samples from all calves exhibited lymphocyte chimerism with greater percentages of male cells. Chromatid breaks and the presence of a hypodiploid cell carrying a metacentric marker were noted in varying proportions in all four calves. The possible relationship between chromosome abnormalities and immunological diversity between the calves have been discussed.

Fetal fluid steroids and their relationship to gonadal steroid secretion in single and twin bovine fetuses.

Steroid concentrations in the fetal fluids of 153 single and 69 twin bovine pregnancies, ranging in age from 35 to 125 d of gestation, were studied to compare gonadal steroid secretions in vitro with the concentrations found in amniotic and allantoic fluids during the early stages of sex differentiation. Among the steroids measured in fetal fluids, only the testosterone level showed a correlation with the amount secreted by the gonads. Significantly higher concentrations of testosterone were associated with male fetuses than with female fetuses. The concentrations of androstenedione, estradiol and estrone in both fetal fluid compartments were generally correlated with age, reflecting the extra-gonadal source of steroids in these fluids. Androstenedione levels in fetal fluids were significantly higher in twins than in singletons, suggesting that this parameter may be useful for the diagnosis of fetal sex and/or type of pregnancy.

Tritiated thymidine uptake by ehrlich ascites cells exposed to ethyl heptyloxyacetate

An investigation on the pattern of DNA synthesis was conducted on treated and untreated Ehrlich ascites tumor cells in order to determine the nature of mitotic block induced by ethyl heptyloxyacetate. Ascites cells, aspirated from treated and control mice at 2, 4, 6, 24 and 48 hours after the administration of ethyl heptyloxyacetate, were incubated with tritiated thymidine for 20 min at 37°C and autoradiographic analyses were carried out. The percentage of labeled cells and the average grain count did not vary appreciably in the controls during the experimental period; however, the percentage of labeled cells was the lowest at 4 hours and the grain count approximately 2.5 times that of control at 48 hours in ethyl heptyloxyacetate treated samples. The marked drop in S phase cells in the treated samples at 4 hours is considered to be due to the destruction of cells in mitosis at the time of treatment. The absence of mitotic stages in samples taken before 48 hours and the increased grain count at 48 hours in treated ascites cells seem to be attributable to a delay of G2 phase by ethyl heptyloxyacetate.