Fertility-associated genes: Diagnostic and therapeutic utility (Proceedings)

Development of fertility diagnostics and therapeutics offers enhanced productivity and economic opportunity for the cattle industry through increased reproductive efficiency. A diagnostic test based upon detection of a marker related to fertility is needed to be able to objectively predict fertility of bulls (Foote, 2003). Heparin can capacitate bovine sperm in vitro, and the effects are mediated by binding proteins produced in the accessory sex glands. During sperm transit at ejaculation, sperm are decorated with those heparin binding proteins. The companion paper pointed to differential heparin binding in relation to fertility potential of bulls. When exposed to identical doses of heparin, sperm from higher compared to lower fertility bulls are more susceptible to its capacitating effects (Ax et al., 1985).

This paper summarizes recent advances in detection of seminal proteins related to bull fertility. Additionally, recombinant forms of those seminal proteins can be added to semen to improve quality post-thaw for A.I. The Ax lab at the University of Arizona has studied Fertility-associated antigen (FAA; McCauley et al., 1999) and Type-2 tissue inhibitor of metalloproteinases (TIMP-2; McCauley et al., 2001; Dawson et al., 2002). Presence of either of those proteins detected with Western blots corresponded to a 13-19% increase in fertility of bulls used to breed cows in multiple-sire pastures (Bellin et al., 1994; 1996; 1998).

In the future, it may be useful to measure combinations of these proteins, or other ones not yet identified, to develop a fertility index. Since subtle genetic changes in a gene can drastically alter the amount or structure of a protein produced, it makes sense that genetic screening with modern molecular techniques will become a powerful selection tool. Is there a commercially available test to detect a marker for fertility potential in semen? To date, the only marker that has progressed to the point of commercialization as a fertility diagnostic is FAA, identified and characterized by Dr. Ax's laboratory at the University of Arizona (McCauley, et al., 1999; 2004). That technology was licensed to a company called ReproTec, Inc. The fertility test is immuno-based and consists of analyzing FAA in a semen sample (McCauley et al., 2004).

FAA and Its Relationship to Fertility

Since a test for FAA is on the market, the importance of FAA as a fertility marker will be reviewed here. Obviously, any other fertility marker could be summarized when appropriate. Furthermore, it is known that FAA is also present in semen from boars, rams, dogs and humans. However, no definitive fertility trials have been conducted with any species other than cattle.

In the early 1980's, research in the Ax lab centered on development of in vitro procedures to capacitate bull sperm prior to in vitro fertilization with bovine eggs. Glycosaminoglycans (GAGs), which are constituents of the female reproductive tract secretions, components in follicular fluid, and present in expanded cumulus-oocyte complexes, effectively capacitate bull sperm in a dose-response manner. Heparin, which is a GAG, is the most potent in terms of capacitation-potentiating activity. As mentioned earlier, sperm from higher fertility bulls are more susceptible to capacitation in vitro (Ax et al., 1985), and display a higher binding affinity for heparin in ligand-binding assays (Marks and Ax, 1985). Fractionation of proteins in semen which bound heparin led to the discovery of FAA and TIMP-2 (McCauley et al., 1999; 2001). Therefore, they are heparin-binding proteins, attach to the surface of sperm during ejaculation, and can transduce the capacitating effects of heparin to sperm cells. This occurs even if sperm are harvested from epididymides and subjected to short-term exposure of seminal fluid or fractionated heparin-binding proteins in laboratory assays (Miller et al., 1990).

Purification of FAA with reconstitution in aqueous media results in auto-hydrolysis (unpublished observations). This poses problems to study its interactions with sperm. Therefore, a recombinant FAA was produced, and it potentiated heparin-induced capacitation in a dose-response manner (Lenz et al., 2000; Fig. 1).

Figure 1. Recombinant FAA potentiated capacitation of bovine sperm.

Additionally, it stabilized acrosomal integrity during freezing and thawing of sperm cryopreserved in a commercial extender (Table 1). Collectively, the data above support an important role for FAA in terms of being able to modify sperm function. Does FAA itself correspond to fertility? Yes. In studies, every bull passed a breeding soundness qualification (Chenoweth et al., 1992) prior to being tested for FAA. Therefore, all of the bulls were classified as potential satisfactory breeders prior to biochemical analyses for FAA in semen. Clearly, presence of detectable FAA in semen provided a competitive advantage for those bulls.

Table 1. Relationship between FAA status and pregnancy rates following natural service in multiple sire pastures.

Future Prospects

DNA-based fertility diagnostics potentially offer many advantages over currently available semen or protein/antibody-based procedures. Selection pressure on fertility could be placed at a day of age, or earlier, prior to substantial economic investment into those animals. Moreover, DNA diagnostics would be applicable to both males and females. If a genetic test is developed for FAA or TIMP-2, it could be used to screen for undesirable alleles in the heifer/cow population. Theoretically, since we have not yet applied selection pressure for FAA/TIMP-2 status in cattle, the allelic frequencies in males and females should be identical. Since multiple alleles are already known to exist along those gene sequences, efforts are now underway to evaluate and rank the importance of those mutations to fertility of bulls and cows. The benefit to the cattle industry will ultimately be an automated high-throughput procedure to identify those animals that should be culled as prospective breeders (male or female). The genetic screening is for single nucleotide polymorphisms (SNPs), a common diagnostic procedure in biomedical research (Lander, et al., 2001; Matzuk and Lamb, 2003).

Genetic screening platforms are now commercially available for the polled gene, coat color, parentage, growth and carcass traits and milk composition. As genetic markers for fertility-specific genes become available, they would be logical candidates to incorporate onto a screening panel. Ultimately, screening could be conducted for females as well as males, and combinations of markers could contribute to accuracy of fertility estimates.

References

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Bellin, M.E., H.E. Hawkins and R.L. Ax. 1994. Fertility of range beef bulls grouped according to presence or absence of heparin-binding proteins in sperm membranes and seminal fluid. J. Anim. Sci., 72: 2441-2448.

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