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Shooting Blanks

Mutant sperm can result in male infertility.

Dr Frank Y.T. Sin

Male infertility accounts for about 30% of all couples who come to infertility clinics for fertility assessment, yet the causes of male infertility remain largely unknown.

Infertility may be a short-term response to environmental influences, such as stress, drugs, alcohol or illnesses. Once such factors are eliminated, normal fertility returns.

Another type of infertility has a permanent effect on the men involved. These infertile men may have a genetic predisposition, where they carry a defective nuclear gene which affects fertility. This gene may be transmitted from one generation to the next, or the individual may carry a mutation in the mitochondrial genome of the sperm which will not be transmitted to the next generation, but which may affect normal sperm function.

Male fertility assessment is generally based on guidelines set by the World Health Organization. The parameters used routinely in semen analysis include semen volume, sperm motility, sperm counts per millilitre of semen, and sperm morphology. However, these criteria do not always provide an accurate assessment of an individual's fertility status. Men with severe defects in their semen may still contribute to normal pregnancy rates. So additional parameters are often used.

Seminal plasma analysis is one aspect that has been used for fertility assessment, checking levels of citric acid, zinc level and acid phosphatase. However, plasma protein has not been used for this purpose. Whether plasma protein patterns can be used as a marker to predict fertility status needs to be examined.

In a pilot study, we are using polyacrylamide gel electrophoresis to determine the variability of seminal plasma protein in semen from donors. This method separates proteins according to the net charges on the protein and the molecular size. Once the variability among different individuals with a normal fertility status has been established, we can attempt to identify plasma proteins that may be specific to individuals with a particular fertility status.

One of our group, Sue Richards, has analyzed semen samples from over 50 fertile males. At least 20 million sperm per millilitre is required for fertilization, and fertile men may have 50-100 million sperm per millilitre of semen.

Richards has found that seminal plasma proteins vary quite a bit between individuals, but that there are certain basic protein species that can be easily recognized. She has also analyzed semen samples from two infertile men (sperm counts of less than three million per millilitre of semen), and found two proteins which are not present in fertile males. At this stage, we do not know the function of these proteins. We intend to find out more about them and to see whether these are related to infertility.

At the other extreme, Richards has discovered three proteins in two men with high sperm counts (80 and 200 million per ml). These preliminary studies suggest that semen plasma proteins are worth further investigation.

Mutations Responsible

Our second approach in the study of male infertility is to look for mutations in the genetic blueprints of the mitochondria of sperm. Mitochondria are small organelles which are the powerhouse of the sperm. They produce the energy required by sperm to swim to and fertilize the egg. If there is a mutation in these energy-producing organelles, the swimming ability may be impaired, stopping the sperm from reaching the egg and fertilizing it.

The mitochondrial genome is a circular molecule consisting of over twenty genes. We are analysing two particular regions of the mitochondrial genome from patients with known infertility status, using a technique which can discriminate DNA sequences which have just a single base change.

Another member of our research group, Andrew Holyoake, has analyzed more than 30 subfertile males and obtained preliminary results suggesting that point mutations can be detected by this method. More importantly, he has found a mutation in the mitochondrial genome of a man who is severely oligozoospermic and asthenozoospermic -- that is, his sperm are smaller and weaker than usual. Whether these mutations are responsible for the fertility status of this man has yet to be established.

Can a single mutation in the mitochondria of sperm be the cause of infertility? Even a male with normal fertility status may have a large number of abnormal sperm. We believe the proportion of sperm in a semen sample carrying the mutation is an important factor. To test this, Holyoake has developed a method of isolating single sperm from semen for mutational analysis. This work is in progress.

No matter what the outcome of the findings, we are learning more about the genetic diversity of the male population in Christchurch and the possible roles of seminal plasma proteins and mitochondrial mutations in male infertility. We believe that, with increasing understanding of these relationships, we will be able to use protein markers and mitochondrial mutations, together with the conventional assessment criteria, to predict more accurately the fertility status of a man with a given semen profile.

Dr Frank Y.T. Sin is senior lecturer in molecular genetics at the University of Canterbury.