Nanoscale Differences in the Shape and Size of X and Y Chromosome-Bearing Bovine Sperm Heads Assessed by Atomic Force Microscopy

José O Carvalho,Roberto Sartori,Luciano P Silva,Margot A N Dode,Laurent Kreplak

doi:10.1371/journal.pone.0059387

José O Carvalho, Roberto Sartori + Show 3 more

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https://doi.org/10.1371/journal.pone.0059387

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Abstract

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing.

Highlights

Since the discovery by Painter [1] that sperm cells carry either an X or Y chromosome, there has been continuing interest in technologies that enable sperm sorting into X and Y chromosomebearing fractions
Sorting by flow cytometry is based on differences in the DNA content of X and Y chromosome-bearing sperm cells; the differences in X and Y sperm is approximately 4% in bovine [4]
The reduced pregnancy rates or embryo production in artificial insemination (AI) and embryo transfer (ET) programs using sexed sperm by flow cytometry [5,6,7,8,9,10,11] has motivated the search for alternative methods

Summary

Introduction

Since the discovery by Painter [1] that sperm cells carry either an X or Y chromosome, there has been continuing interest in technologies that enable sperm sorting into X and Y chromosomebearing fractions. Several methods have been developed for sperm sex determination, the only method effective for routine use is uorescence-activated cell sorting using ow cytometry [3]. Sorting by flow cytometry is based on differences in the DNA content of X and Y chromosome-bearing sperm cells; the differences in X and Y sperm is approximately 4% in bovine [4]. The reduced pregnancy rates or embryo production in artificial insemination (AI) and embryo transfer (ET) programs using sexed sperm by flow cytometry [5,6,7,8,9,10,11] has motivated the search for alternative methods. The development of new sorting methods could eliminate the potential hazards that result from the flow cytometry technique, which requires DNA staining and UV excitation

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