The Harry M. Zweig Memorial Fund for Equine Research

Further Characterization of the Specific Activity and Ultrastructural Localization of Phospholipase C Zeta in Fertile and Subfertile Stallions

- Dr. Sylvia Bedford-Guaus and Dr. Mark Roberson

BedfordDr. Sylvia Bedford-Guaus

RobersonDr. Mark Roberson

Our standard methods for breeding soundness evaluation and routine sperm laboratory tests do not provide a direct assessment of stallion fertility. Measures of sperm motility or sperm morphology are only marginally correlated with pregnancy rates (Jasko et al., 1990, 1992). Fertile sperm must be able to reach the site of fertilization, fertilize the oocyte and trigger the initiation of embryonic development. Only tests that directly evaluate these sperm functions can be directly correlated with actual fertility. In this regard, we have began the characterization of a sperm-specific enzyme, phospholipase C zeta (PLC?), which is responsible for inducing embryonic development at fertilization in all mammalian species studied thus far. Because species specific differences in PLC? activity have been reported, it is essential to characterize PLC? in the stallion prior to its use as a tool for fertility evaluation.

Notably, a subfertile stallion was recently presented for evaluation at our equine breeding facility at Cornell University. Although this stallion had borderline semen quality, he was able to produce an average of 700 million progressively motile, morphologically normal sperm per day, which should be sufficient to get mares in foal. However, this stallion was subfertile with pregnancy rates = 20%. Semiquantitation of PLC? showed that this stallion had relatively less amounts of PLC? when compared to one of our proven fertile stallions and this may explain the reported low fertility rates of this stallion. This example underscores the importance of characterizing PLC? in fertile and subfertile/infertile stallions with the long term goal of using this as part of a battery of tests to evaluate sperm function and thus stallion fertility.

What is phospholipase C zeta (PLC?)?
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 intracellular calcium ([Ca2+]i) rises that are termed ‘oscillations’. These [Ca2+]i oscillations occur with a frequency that is species-specific and last for several hours (reviewed by Fissore et al., 1998).

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 PLC? (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, 2008). PLC? catalyzes the hydrolysis of phosphoinositides to liberate an important second messenger, inositol trisphosphate or IP3. In turn, IP3 facilitates the release of intracellular calcium within stores via a specific IP3 receptor present on the endoplasmic reticulum. The gene sequence for PLC? 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 PLC? activity. We have cloned the sequence for equine PLC?. Understanding how this sequence relates to its specific activity is the next step in the characterization of PLC? for evaluation of stallion fertility.

PLC? 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 (~70%; Battaglia et al., 1997; Rybouchkin et al., 1997; Mahutte and Arici, 2003), supporting the possibility that dysfunction in the PLC? protein may be causal in defining important mechanisms of male infertility.

Our preliminary conclusions in the characterization of equine PLC? thus far can be summarized as: i) the predicted protein sequence for equine PLC? shows highest homology with that of porcine PLC?, with lesser homology observed in the region known to be important for its specific activity (Appendix 1;Fig.6); ii) equine PLC? is expressed in later stages of the spermatogenic sperm cell lineage, namely starting at the round spermatid stage (see progress report Fig.1); and, iii) in all stages of sperm maturation, namely epididymal, ejaculated and capacitated sperm, PLC? is localized to both head and tail regions of the sperm (see progress report Fig. 2). In order to further characterize equine PLC? we propose three Specific Aims:

  1. To characterize the specific [Ca2+]i-releasing activity of immature testicular sperm cells, of different regions of mature sperm, and of equine PLC? in oocytes. Our hypothesis is that round spermatids obtained from equine testicular tissue and both the head and tail of equine sperm have the ability to induce [Ca2+]i oscillations in oocytes and this is correlated to the specific activity of equine PLC?. Our hypothesis is based on preliminary results showing that PLC? expression is observed in round sperm cells in testicular tissue (see progress report Fig.1) and that PLC? localizes to both head and tail regions of stallion sperm (see progress report Figs.2,3). To test our hypothesis, round spermatids, sperm heads or tails will be injected into oocytes to monitor their [Ca2+]i-releasing activity. These studies might reveal fundamental differences in the physiology of PLC? expression from what has been reported in other species. For instance, mice round spermatids do not possess PLC? activity, and thus cannot initiate embryonic development when injected into oocytes (Kimura and Yanagimachi, 1995). Moreover, PLC? activity has not been identified in the tail of any other mammalian species studied thus far (Yoon and Fissore, 2007; Young et al., 2008). In order to test the specific activity of equine PLC?, we will produce and purify equine PLC? RNA in vitro and different concentrations of this product will be injected into oocytes. In these studies, oocytes will be monitored for [Ca2+]i oscillations as previously described (Bedford et al., 2006, 2008). These studies are critical to determine the bioactivity of PLC? expressed in different stages of sperm maturation and/or different regions of the equine sperm, and of the novel equine PLC? clone.
  2. To investigate the ultrastructural localization of PLC? in equine sperm. Our hypothesis is that PLC? with high enzymatic activity is localized to specific membrane fractions known as membrane rafts.
    Previous research suggests that PLC? may be localized in specific membrane regions, in particular that underlying the acrosome (cap over the sperm head; Kurokawa et al., 2005). Immunofluorescence studies in our lab have localized PLC? over areas of the sperm head and tail (see progress report Fig.2). However, these findings do not give us detailed information as to the ultrastructural localization of PLC? and whether it is associated with specific membrane regions. We are interested in investigating whether PLC? is stored in specific regions of enriched enzymatic activity (membrane rafts). For this purpose, sperm domains (membrane rafts, organelles) will be fractionated by differential centrifugation and the presence of PLC? in the different membrane domains identified with the use of PLC?-specific antibodies. Additionally, these fractions will be injected into oocytes to ascertain their ability to trigger [Ca2+]i oscillations.
  3. To determine PLC? levels and regional expression in equine sperm from stallions of variable fertility.
    Our hypothesis is that the innate level of PLC? protein and its localization in equine sperm are functionally correlated with the fertility potential. The relative amount of PLC? 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 PLC?/sperm compared to stallions of high relative fertility, as already observed for one stallion that presented to our clinic with adequate sperm numbers yet low fertility rates. To test this we will examine equine PLC? protein levels in sperm samples collected from stallions of low and high fertility based upon breeding records and traditional methods of breeding soundness evaluation. In stallions displaying decreased PLC? expression we will further investigate its regional localization in sperm. This may yield information not only regarding relative quantity of PLC? but also its pattern of expression and bioavailability as correlated to its ability to induce oocyte activation and thus as it relates to stallion fertility.

In summary, we propose to fully characterize equine PLC? in regards to bioavailability, specific activity, and as a tool to evaluate stallion fertility. This research will greatly benefit the race-horse breeding industry as it relates to using PLC? as a molecular tool to predict and/or evaluate stallion fertility. Moreover, we have already identified some relevant species-specific characteristics of equine PLC?; further in depth study of these differences will greatly enhance our overall knowledge in basic mammalian reproductive physiology using the horses as a model.