Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection

Kazuo Yamagata1, Rinako Suetsugu, and Teruhiko Wakayama

Human Reproduction, Vol.24, No.10 pp. 2490 – 2499, 2009

Intracytoplasmic sperm injection (ICSI) is a technique in which sperm are injected directly into unfertilized oocytes, whereby offspring can be obtained even with dysfunctional sperm. Despite its advantages in human and animal reproductive technology, the low rate of resultant live offspring is perturbing. One major cause is thought to be embryonic chromosomal abnormalities. However, there is no direct evidence of how these occur or how they affect pregnancy outcomes.

Chromosomal dynamics during the first mitotic division of mouse embryos were analyzed using a new live-cell imaging technology. After imaging, the embryos’ developmental capacities were determined.

When ICSI-generated embryos were monitored for their chromosome integrity, some embryos with apparent normal morphology seen by conventional light microscopy had abnormal chromosome segregation (ACS) at the first mitotic division. Chromosomal fragments were misaligned during the first metaphase and formed micronuclear-like structures at the interphase of the 2-cell stage. Similar ACS was also found in mouse embryos produced by microinjecting round spermatids, with even higher frequency. Giemsa staining and immunostaining revealed that these fragments were derived from double-strand DNA breaks in the paternal genome. About half of the embryos with ACS developed into normal-looking morulae or blastocysts and implanted, but almost all of them aborted spontaneously before embryonic day 7.5.

ACS during first mitosis appears to be a major cause of early pregnancy losses in ICSI-generated mouse embryos. Moreover, this novel imaging technology could be applicable as a method for the assessment of embryo quality.




Isodicentric Y Chromosomes and Sex Disorders as Byproducts of Homologous

Recombination that Maintains Palindromes

Julian Lange,1 Helen Skaletsky,1 Saskia K.M. van Daalen,2 Stephanie L. Embry,3 Cindy M. Korver,2 Laura G. Brown,1 Robert D. Oates,4 Sherman Silber,5 Sjoerd Repping,2 and David C. Page1

Cell 138, 855–869, September 4, 2009 ª2009 Elsevier Inc. DOI 0.1016/j.cell.2009.07.042


Massive palindromes in the human Y chromosome harbor mirror-image gene pairs essential for spermatogenesis. During evolution, these gene pairs have been maintained by intrapalindrome, arm-to-arm recombination. The mechanism of intrapalindrome recombination and risk of harmful effects are unknown. We report 51 patients with isodicentric Y (idicY) chromosomes formed by homologous crossing over between opposing arms of palindromes on sister chromatids. These ectopic recombination events occur at nearly all Y-linked palindromes. Based on our findings, we propose that intrapalindrome sequence identity is maintained via noncrossover pathways of homologous recombination. DNA double-strand breaks that initiate these pathways can be alternatively resolved by crossing over between sister chromatids to form idicY chromosomes, with clinical consequences ranging from spermatogenic failure to sex reversal and Turner syndrome. Our observations imply that crossover and noncrossover pathways are active in nearly all Y-linked palindromes, exposing an Achilles’ heel in

the mechanism that preserves palindrome-borne genes.



Effect of Cryopreservation on Nitric Oxide Production by Stallion Spermatozoa

C. Ortega Ferrusola, L. González Fernández, B. Macías García, C. Salazar-Sandoval, A. Morillo Rodríuez, H. Rodríguez Martinez, J. A. Tapia and F. J. Peña

Published online July 15, 2009, doi: 10.1095/ biolreprod.109.078220


Ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe 4,5-diaminofluorescenin diacetate (DAF-2 DA). The presence of nitric oxide synthase (NOS) was investigated by western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS, antibodies. While NO was detected both in fresh and FT-sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 antibody (Ab) intensively labelled a single band with an apparent molecular weight (MW) of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. Nitric oxide production was positively correlated with sperm motility and velocity post thaw, suggesting NO role for the functionality of cryopreserved stallion spermatozoa, but visibly compromised in egg yolk-containing extenders.


Epigenetics: Ready for the marks

Robert Feil

Nature 461, 359-360 (17 September 2009) | doi:10.1038/461359a; online 16 Sept 2009


Genomic imprinting, in which genes are expressed from either the maternal or paternal genome, involves the addition of methyl marks to DNA. Paradoxically, demethylation of histone proteins is an essential first step.

ìFor its development in the womb, a baby needs the genetic information inherited from both mother and father. This is because some of our genes are expressed from only one of the two inherited parental genomes. This remarkable phenomenon — genomic imprinting — is controlled by marks placed on the genome in germ cells (oocytes and sperm), in particular by the methylation of DNA. In this issue, Ciccone and colleagues explore how other methyl marks — not on the genome itself, but on the histone proteins around which the DNA fibre is wrapped — contribute to genomic imprinting. The authors find that methyl marks on histones need to be removed for the genome to 'get ready' for the establishment of imprints.