Male infertility may be due to failure of calcium signal

A key protein in sperm causing some men to be infertile, appears to be phospholipase C zeta or PLC-zeta. When PLC-zeta is ineffective due to poor volume or a mutation, sperm fail to initiate calcium signalling and egg development. The discovery could enable early diagnosis and treatment.

Investigators found through clinical studies, failed lab fertilization attempts could be restarted by injecting eggs with a higher volume of PLC-zeta protein than found in male patient contributions. And for some, mutated PLC-zeta was found and replaced with unmutated PLC-zeta found in those patients, also restoring previously failed fertilizations. All this suggests infertility may be reversed.

"Our ultimate goal is to use the tools that we are currently developing not only to treat, but also diagnose, the cases of male infertility associated with absent or dysfunctional PLC-zeta protein in human sperm."

Michail Nomikos MSc PhD, College of Medicine, Qatar University, Doha, Qatar College of Biomedical and Life Sciences, School of Biosciences, Cardiff University, Cardiff, UK and lead author of the paper.

The work appears in the March 7th issue of Biochemical Journal.

An essential part of human fertilization is the repetitive and dramatic increase in calcium levels in the egg — known as calcium oscillations — triggered by a signal from one sperm upon fusion with the egg. This calcium increase is vital for all early events in fertilization and continued early embryo development. Over the last decade, research carried out by Professor Lai, a senior author of the study, has shown that PLC-zeta is a key trigger beginning calcium oscillations. Then, in 2002 Professor Tony Lai (Cardiff University, UK) and colleagues discovered the sperm-specific PLC-zeta protein.

A number of genetic defects in encoding the PLC-zeta gene have been linked to male infertility in the past. A recent study by Jessica Escoffier and colleagues identified a new mutation (SNP, single nucleotide polymorphism - I489F) located in the C2 domain of the PLC-zeta gene in two infertile brothers from Tunisia.

Dr Nomikos and collaborators have now investigated the effects of this C2 domain mutation and found that injecting the abnormal PLC-zeta protein into mouse eggs at levels comparable with those found in sperm of infertile men — resulted in no calcium oscillations, and therefore no fertilization. However, when they increased amounts of PLC-zeta than would be present naturally, normal fertilization began along with calcium oscillations. Again, this finding supports that this form of infertility could be treatable.

"The identification and characterization of another male infertility-linked PLC-zeta mutation necessitates that we begin to consider use of recombinant PLC-zeta protein in a clinical setting, with the aim being to rescue such cases of egg activation failure. We believe that our research can eventually give hope to many infertile couples in the near future; our goal is to help them have a family."

F. Anthony Lai PhD, Professor, College of Biomedical and Life Sciences, School of Biosciences, Cardiff University, Cardiff, UK.

The team also examined the biochemical properties of PLC-zeta mutant protein and compared it with normal PLC-zeta. The key difference between the two appears to be that abnormal protein is less likely to bind to liposomes containing the molecules PI(3)P and PI(5)P, than normal protein. Liposomes are small circular molecules that assist in cell to cell communication.

Researchers are now investigating the exact role and physiological difference between PLC-zeta with PI(3)P or with PI(5)P. It may be possible that the mutation affects interaction between the two PLC-zeta C2 domains and another, unidentified egg protein or 'PLC-zeta receptor', resulting in infertility.

"Identification of such an egg protein will be a major breakthrough in the fertilization field as it potentially may lead to identification of currently unknown causes of female infertility."

Michail Nomikos MSc PhD

"A new life begins when a sperm fuses with an egg to inject an enzyme initiating calcium oscillations occurring every two minutes for two hours — activating egg development. The exact nature of this activating enzyme has been in some doubt. Nomikos and his colleagues show that a mutation in the enzyme, identified in infertile men, fails to initiate the calcium signals that triggers development. This provides definitive evidence this is an enzyme responsible for initiating new life," adds Sir Michael Berridge FRS, Professor, University of Cambridge, a renowned expert in calcium signalling

Abstract
Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.
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Fertilization calcium wave in a sea urchin egg. The calcium wave initiates at the point of
sperm entry and crosses the egg as a tsunami-like wave, traversing the egg in ∼20 seconds.
Calcium levels are represented by warm colors and height in this plot.
Image Credit: Adapted from McDougall et al. (360). APS Physiological Reviews