Journal article: Application of modern molecular techniques to evaluate sperm sex

Sean P. Flaherty1 and Colin D. Matthews – [Fulltext]


Sperm and egg
Sperm and egg

Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital Woodville, SA 5011, Australia
The aim of this paper is to review modern approaches which have been used to evaluate sex preselection procedures. Two approaches can be used, polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH). FISH is currently the method of choice for evaluating sex selection procedures because: (i) FISH accurately identifies the sex chromosome of individual spermatozoa using specific probes for the X and Y chromosomes and a two-colour detection system; and (ii) large numbers of spermatozoa can be screened in a short period of time. Of the published sex pre-selection methods tested using FISH, only flow cytometry has been shown to produce a clinically significant enrichment of X- and/or Y-bearing human spermatozoa. Studies have shown that 12-step Percoll gradients produce a slight but clinically insignificant enrichment of X-bearing spermatozoa, swim-up techniques do not appgar to enrich either X- or Y-bearing spermatozoa, and discontinuous albumin gradients do not enrich Y-bearing spermatozoa. Despite this evidence, some of these methods continue to be used clinically, so it is vital that sex selection methods are properly evaluated using reliable methods such as double-label FISH before they are introduced for clinical use.


From the looks of it there is still no good method in sperm selection.

Invited Review: Effects of heat and cold stress on mammalian gene expression

Larry A. Sonna1, Jun Fujita2, Stephen L. Gaffin1, and Craig M. Lilly3 (J Appl Physiol 92: 1725-1742, 2002)


1 Thermal and Mountain Medicine Division, United States Army Research Institute of Environmental Medicine, Natick 01760; 3 Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston, Massachusetts 02115; and 2 Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 6068507, Japan

This review examines the effects of thermal stress on gene expression, with special emphasis on changes in the expression of genes other than heat shock proteins (HSPs). There are ~50 genes not traditionally considered to be HSPs that have been shown, by conventional techniques, to change expression as a result of heat stress, and there are <20 genes (including HSPs) that have been shown to be affected by cold. These numbers will likely become much larger as gene chip array and proteomic technologies are applied to the study of the cell stress response. Several mechanisms have been identified by which gene expression may be altered by heat and cold stress. The similarities and differences between the cellular responses to heat and cold may yield key insights into how cells, and by extension tissues and organisms, survive and adapt to stress.


This paper is a good review of past research on proten expression associated with freezing and cryopreservation. A good point to start for young reserachers who wish to start in this area .. Yan .. I think this a sutable paper for you!, I will write further on the paper when I have finished digesting through the whole 14 pages (minus the references)

Journal article: The quantification of lipid and protein oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and correlations with DNA strand breaks, classical seminal parameters and stallion fertility

Horse sperm Anim Reprod Sci. 2008 Jun;106(1-2):36-47 {Fulltext}

Authors: Morte MI, Rodrigues AM, Soares D, Rodrigues AS, Gamboa S, Ramalho-Santos J

The goal of this work was to correlate oxidative stress caused by reactive oxygen species (ROS) and DNA damage with classic semen parameters in spermatozoa and seminal plasma of fertile and subfertile stallions. Oxidation was measured in both lipids and proteins, using the thiobarbituric acid reactive species (TBARS) assay and the DNPH carbonyl groups assay, respectively. Sperm DNA damage was monitored using the TUNEL assay. These parameters were monitored in samples obtained during the breeding and the non-breeding seasons. In general, fertile stallions showed better classical semen parameters, and those parameters improved from the non-breeding to the breeding season, although an increase in sperm production was accompanied by a decrease in the semen quality from subfertile stallions in the breeding season. In terms of oxidation levels we found that there were clear differences whether lipids or proteins were considered. In the breeding season there seemed to be a tendency towards normalizing lipid oxidation in spermatozoa and seminal plasma, and protein oxidation in the seminal plasma, of both fertile and subfertile animals. Thus, differences monitored in the non-breeding season were no longer visible. Interestingly, a higher level of protein oxidation was found in the sperm of fertile animals in the breeding season. Considering that there were positive correlations between sperm protein oxidation and sperm motility and vitality, these results suggests that the oxidation of semen proteins may be important for sperm function. On the other hand, lipid oxidation in the seminal plasma seemed to be a general indicator for sperm damage. In the non-breeding season positive correlations between lipid and protein oxidation levels in both sperm and seminal plasma and several defects in sperm function were found, but only for subfertile animals, thus suggesting that lipid and protein oxidation may aid in the identification of subfertile stallions during the non-breeding season. Levels of ROS production never seemed to result in compromised sperm DNA integrity, indicating that measurements were within physiological levels and/or that there is an efficient antioxidant activity in stallion sperm cells.

PMID: 17451892 [PubMed – indexed for MEDLINE]


This paper is interesting as it discusses on oxidation of major components in a sperm – the infamous lipid and the less studied protein. It seemed that oxidation of protein would indicate viability (sperm quality) while an increase in lipid peroxidation would be a general indicator for sperm damage. Despite a comprehensive study, this study only looks lipid and protein peroxidation at the level of semen and not individual sperm. Hopefully his next study will look at individual sperm thus allow a better interpretation of sperm viability and quality.

Journal article: Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction

DNA damaged spermShamsi MB, Kumar R, Dada R. (Indian J Med Res. 2008 Feb;127(2):115-23.) [Full text]

Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India.

Diagnosis of sperm DNA integrity of semen sample is important for consistently high reproductive efficiency. The conventional parameters of semen analysis take into account morphology, motility, and concentration of spermatozoa in the sample, which are insufficient for evaluation of reproductive potential. Current studies have implicated abnormal organization of genomic material in sperms as a probable cause in 20 per cent cases of male infertility. This is especially important in the era of assisted reproduction technique (ART) when a majority of infertile couples opt for assisted reproduction and in where cases DNA integrity is a better diagnostic and prognostic marker as compared to routine semen analysis. This article reviews and discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. These different techniques include single cell gel electrophoresis (COMET assay), Terminal tranferase dUTP Nick End Labelling (TUNEL), sperm chromatin structure assay (SCSA), In situ nick translation (ISNT) and acridine orange test. These techniques are independent measure of sperm quality and assist in semen quality assessment by detecting defects in DNA integrity or chromatin structure. The discussed techniques vary in their level of accuracy, cost input, sophistication of analysis and their application depends upon the sensitivity required for analysis. The article also briefly outlines the DNA packaging and the causes of DNA damage in spermatozoa. During chromatin packing 85 per cent of the histones are replaced by protamine while the residual histones act as marker of genes which are expressed in early embryonic development. Among the different aetiological factors observed to be responsible for DNA damage in human spermatozoa increased reactive oxygen species (ROS), oxidative stress is highly correlated with greater DNA fragmentation index (DFI). Oxidative stress leads to single and double strand breaks in sperm DNA. Apoptosis and abnormal chromatin packing also contribute to DNA damage. The significance of chromatin structure studies is more stressed owing to the greater awareness to transmission of genetic diseases because of higher incidence of gene imprinting defects, increased cancer frequency and other congenital and non-congenital defects in children conceived through assisted reproduction techniques.

PMID: 18403788 [PubMed – indexed for MEDLINE]


ICSI or IVF is a technique which assume the manually selected sperm will have very minimal damage. Since selection of this procedure are usually a last alternative solution to male infertility, likelihood of selecting sperm with high DNA damage are very high. This paper discuss various techniques in assessing the severity of DNA damage a particular sperm is having. Causes of DNA damage to the DNA of the sperm are also discussed in detail. A good paper for those who are interested in causes of DNA damage. Hint: Incidentally that is my area if interest!