The Harry M. Zweig Memorial Fund for Equine Research

Characterization of Equine Phospholipase C Zeta (PLC Zeta)
as it Relates to Stallion Fertility

Dr. Sylvia Bedford-Guaus and Dr. Mark Roberson

Bedford-GuausDr. Sylvia Bedford-Guaus

RobersonDr. Mark Roberson

Whenever thinking about the importance of stallion fertility evaluation, Cigar comes to mind. The great descendent of Northern Dancer and Seattle Slew, Cigar was voted horse of the year in 1995 and 1996, was unbeaten in 16 consecutive races and was the all time leading money winner at retirement in 1996.  Sadly, Cigar was unable to ever get a single mare pregnant. Using standard breeding soundness evaluations, the reasons for Cigar’s infertility were never determined. More than a decade later, our routine for fertility evaluation in stallions has not changed appreciably. 

Our understanding of Cigar’s infertility might not have altered his outcome since the Jockey Club does not allow assisted reproduction as a mean of producing foals worthy of registration. However, Cigar’s story underscores the importance of developing a battery of new and innovative tests that could specifically assess sperm function.  Given this possibility, we predict that potentially large sums might be saved by the equine breeding industry in the effort involved in ascertaining that a stallion is infertile or subfertile once it books a large number of mares that are found to be open at the end of one or several breeding seasons. In the best scenario, some causes of subfertility might be overcome by reducing the book of mares, reinforcing covers, reducing stallion use and/or controlling the stallion’s environment. 

In the context of novel methods of fertility evaluation in stallions, the immediate aim of this one-year proposal is to characterize the specific activity, localization and expression pattern of equine phospholipase C zeta (PLCζ) within the testis. The long term goal of this research is to use PLCζ as the first molecular probe to assess subfertility and/or infertility in problem stallions.  Present Status in Stallion Fertility Evaluation

The only tests of sperm evaluation presently performed routinely in the laboratory/clinical setting include measures of percent motility and morphologically normal sperm, both of which have been shown to be marginally correlated with pregnancy rates (Jasko et al., 1990, 1992). In one study, the fertility of 99 stallions (n = 43 Standardbreds and 56 Thoroughbreds) was evaluated for two seasons and correlated to sperm evaluation measures. Characteristics most highly correlated with fertility data for both breeds combined were: subjective appraisal of the percentage of motile and progressively motile spermatozoa and, computerized analysis of percentage of motile sperm. However, on the basis of evaluation of a single ejaculate for each stallion, the variation in these parameters only accounted for approximately 20% of the observed variation in fertility rate. 

The sperm chromatin structure assay is another test that is sometimes used to evaluate sperm from problem stallions (Love and Kenney, 1998; reviewed by Neild et al., 2005). This test is based on the fact that DNA from normal sperm is highly compacted and resistant to damage. Therefore, the assay detects sperm that have poorly packaged DNA and presumably decreased ability to produce healthy embryos. In one study, sperm chromatin denaturation was inversely correlated, albeit marginally, with percent pregnancy rates per estrous cycle (Love and Kenney, 1998). The results of the above studies support the notion that routine laboratory tests for sperm evaluation do not provide a highly predictive measure of stallion fertility. Therefore, we are interested in developing tests that directly evaluate sperm function and thus, presumably, the sperm’s ability to fertilize an egg and produce a healthy embryo.  What is phospholipase C zeta (PLCζ)?

In all mammalian species studied thus far, including the horse (Bedford et al., 2003, 2004), the sperm’s contribution at fertilization is not limited to providing the male’s DNA.  Importantly, the sperm is also responsible for initiating and maintaining embryonic development. The way in which the sperm triggers embryonic development is by introducing a protein within the oocyte (egg) at the time of fertilization which induces numerous intracellular calcium ([Ca2+]i) rises. These changes in [Ca2+]i occur with a frequency that is species-specific, lasting for several hours (reviewed by Fissore et al., 1998). Furthermore, from studies in laboratory species, it is known that the maintenance of these [Ca2+]i oscillations is crucial for embryo development beyond the time of implantation (Ozil, 1998;Ozil and Huneau, 2001). 

Recent research has shown that the protein introduced by the sperm (referred to as the ‘sperm factor’) responsible for these [Ca2+]i oscillations is the enzyme PLCζ (Saunders et al., 2002). This is a testis-specific protein that has been shown to be the ‘sperm factor’ in all mammalian species studied thus far (Cox et al., 2002; Saunders et al., 2002; Kurokawa et al., 2005; Bedford et al., 2006). PLCζ catalyzes the hydrolysis of phosphoinositides to liberate an important second messenger, inositol tris-phosphate or IP3. IP3 in turn facilitates the release of intracellular calcium within stores via a specific IP3 receptor present on the endoplasmic reticulum. The gene sequence for PLCζ has been fully characterized in humans, cynomolgus monkeys, mice, rats (reviewed by Swann et al., 2006), cattle, pigs and dogs. Although this protein is highly conserved, differences in sequence appear to be critical for species-specific differences in PLCζ activity. Knowing the exact gene and protein sequence, as well as understanding its expression pattern, is crucial for using this protein as a means to evaluate male fertility in the horse. 

PLCζ in the Context of Male Fertility
In human couples seeking in vitro technology to have a baby, it is estimated that about 3% of cases of unexplained infertility in men are characterized by phenotypically ‘normal’ sperm with an inability of the sperm to initiate embryonic development of the egg (Eldar Geva et al., 2003; Mahutte and Arici, 2003). In couples where the men have abnormally shaped sperm, this percentage is much higher (Battaglia et al., 1997; Rybouchkin et al., 1997; Mahutte and Arici, 2003), supporting the possibility that dysfunction in the PLCζ protein may be causal in defining important mechanisms of male infertility. 

Proposed Research
PLCζ is a sperm-delivered protein crucial for activation of the oocyte and thus, fertility. In order to use it as a tool to predict and/or evaluate stallion fertility, its sequence, specific activity and expression pattern must be characterized. Our preliminary studies have resulted in the cloning and sequencing of an equine PLCζ cDNA clone. For these studies, we were able to compare PLCζ sequence information from different species with a partial equine sequence provided by Dr. D. Antczak’s laboratory by virtue of the equine genome project. PCR primers were developed based upon this bioinformatic approach and used to amplify PLCζ cDNA from mature stallion testis. In order to further characterize equine PLCζ we propose three Specific Aims:

  1. To characterize the activity of equine PLCζ in oocytes. Our hypothesis is that equine PLCζ RNA induces [Ca2+]i oscillations in oocytes. To test this, we will produce and purify equine PLCζ RNA in vitro and inject it into oocytes.  Oocytes will then be monitored for [Ca2+]i oscillations as previously described (Bedford et al., 2006). These studies are critical to determine the bioactivity of the novel equine PLCζ clone.
  2. To determine the expression pattern of equine PLCζ throughout the testis, and in immature, freshly ejaculated and capacitated stallion sperm. Our hypothesis is that expression of PLCζ will be observed in more mature cells of the sperm producing lineage and that the expression pattern will change when comparing epididymal, freshly ejaculated and capacitated sperm. This is based upon research in the mouse whereby immature sperm cells are unable to initiate embryonic development when injected into oocytes, presumably due to lack of PLCζ expression or activity. To test this we will use specific anti-PLCζ antibodies to label sections of fixed equine testicular tissue, and epididymal (immature), freshly ejaculated and capacitated sperm using histological and immunohistochemical techniques. 
  3. To determine PLCζ levels in equine sperm from stallions of variable fertility. Our hypothesis is that the innate level of PLCζ protein in equine sperm is functionally correlated with the fertility potential. This is predicated on the notion that the relative amount of PLCζ in sperm from a given species is correlated with the specific [Ca2+]i releasing activity of their sperm (Kurokawa et al., 2005).  Thus, we predict that some stallions of low relative fertility will have markedly reduced levels of PLCζ/sperm compared to stallions of high relative fertility. To test this we will examine equine PLCζ protein levels in sperm samples collected from stallions of low and high fertility based upon breeding records and traditional methods of breeding soundness evaluation described earlier.

In summary, we propose to characterize a novel clone of equine PLCζ and determine its utility as a molecular  marker of fertility in stallions. This research will not only benefit the race-horse breeding industry, but will also enhance our fundamental understanding of comparative mammalian reproductive physiology. Furthermore, for once, assessment of stallion fertility will be at the cutting edge of basic reproductive research.