Macrocephaly in Bull Spermatozoa Is Associated with Nuclear Vacuoles, Diploidy and Alteration of Chromatin Condensation

Abstract

Spermatozoa from 2 dairy AI (artificial insemination) bulls (A and B), identified by their abnormal spermiogram with cells depicting frequent macrocephaly, double tails and nuclear vacuoles, were case-investigated and compared to normal spermatozoa from a control AI sire (C). Head sizes were measured and morphological abnormalities scored using brightfield and differential interference contrast microscopy. The degree of sperm maturation and of resistance to acid-induced DNA denaturation in situ were determined after uploading of acridine orange using flow cytometry of 5,000 cells/sample. Nuclear fragmentation, i.e. the ratio of red to total (red + green) fluorescence, reached 7.1% and 31% in bulls A and B, compared to 2% in bull C. The proportion of immature spermatozoa, i.e. those with incomplete histone-protamine exchange and depicting higher green fluorescence compared to the main population of the control bull, reached 9.54% in A and 7.75% in B, compared to only 0.47% in the control. In the second part of this study the previously unknown chromosomal constitution of large-headed spermatozoa of bull A was investigated by fluorescence in situ hybridization using an X-Y painting probe set. The 7.5% XY-bearing cells and the presence of diploid spermatozoa detected by flow cytometry indicate a meiotic arrest in the first division in bull A, becoming the first proven case of association of macrocephaly and M1 diploidy. The diverse approaches used for the investigation of spermatozoal DNA provide insights into the etiology of macrocephaly.

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…170 Besides evaluating sex chromosome content of individual spermatozoa, FC offers a precise tool to do quick cytogenetic analyses on sperm: aneuploidy can be detected based on DNA content of individual sperm cells. 171,172 THE 'DAMAGE UNDERESTIMATION' PROBLEM As it was indicated throughout the present review, one of the biggest challenges in applying FC in spermatology is the correct recognition of spermatozoa and the clear separation from signals of other particles present in the actual samples. With assays like the SYBR14/PI dye combination for viability measurements, it is relatively easy as every sperm cell is labeled with a DNA-specific fluorochrome, while other sperm-sized particles (like lipid droplets) are not.…”

Genetic Dissection of a Key Reproductive Barrier Between Nascent Species of House Mice

“…In addition to reduced testis weight and sperm density, F 1 hybrid males from M. m. musculus PWD mothers presented abnormal sperm types similar to those previously connected with sterility. Abnormal sperm head shapes often arise as byproducts of aneuploidy (Prisant et al 2007;Perrin et al 2008;Revay et al 2009). In rodents, alterations of the sperm hook are correlated with decreases in fertilization success (Immler et al 2007;Firman and Simmons 2009), and severely amorphous sperm heads are inefficient at penetrating the ova (Krzanowska and Lorenc 1983;Oka et al 2007;Styrna 2008).…”

X–Y Interactions Underlie Sperm Head Abnormality in Hybrid Male House Mice

“…During this final stage of spermatogenesis, chromatin is progressively remodeled and condensed, and nuclear morphology undergoes a dramatic transformation, culminating in the highly differentiated structure of mature spermatozoa (reviewed in Oliva and Castillo 2011). Incomplete chromatin compaction is a common cause of abnormal sperm head morphology in mammals (Balhorn 2007;Revay et al 2009). While autosomal genes play the major roles in chromatin repackaging and condensation (e.g., transition nuclear proteins and protamines; reviewed in Sassone-Corsi 2002), a small subset of X-and Y-linked genes are highly transcribed in postmeiotic spermatids (Namekawa et al 2006;Mueller et al 2008), and several are required for normal sperm differentiation (e.g., Cocquet et al 2009Cocquet et al , 2012Vernet et al 2012).…”