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Seminal proteins as fertility markers (Proceedings)

Article

Four specific proteins have been identified in bovine semen related to fertility.

Seminal Proteins Related to Fertility

Four specific proteins have been identified in bovine semen related to fertility, and they are summarized in Table 2 below. Research at Pennsylvania State University in the lab of Dr. Gary Killian led to the discovery that relative amounts of osteopontin (OPN) and a lipocalin-Type D prostaglandin synthase (PGD) in semen were reflective of fertility in Holsteins (Killian et al., 1993; Cancel et al., 1997) and Angus. 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).

Table 1. Characteristics of four known fertility related proteins.

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 tools will become a powerful selection tool.

Examples of that type of approach are contained in a companion paper to this manuscript.

Is there a commercially available test to detect a marker for fertility potential in semen? Yes, a company called ReproTec in Tucson, AZ has a lateral-flow cassette on the market that works much like a home pregnancy test (McCauley et al., 2004). A color change indicates presence of FAA, and that color develops within 20 min after a semen sample is applied to the cassette. Details are provided in the last section of this article and in a companion paper at this symposium.

The Relationship Between Aforementioned Proteins and Fertility

As mentioned, Killian and coworkers (1993) identified four seminal plasma proteins in Holstein bull semen that appeared to be associated with fertility. They consisted of a 55 kDa (pI 4.1), 26 kDa (pI 6.2), 16 kDa (pI 6.7) and 16 kDa (pI 4.1) proteins utilizing two-dimensional (2D) SDS-PAGE separation techniques. By analysis of the protein density, the first two, later characterized as osteopontin (55 kDa; Cancel et al., 1997) and lipocalin-type prostaglandin D (PGD) synthase (26 kDa; Gerena et al., 1998) were more prominent in higher fertility bulls while the latter two (currently uncharacterized) were prevalent in low fertility bulls. Based on relative protein density values, OPN was positively correlated (r=0.48) with fertility (Cancel et al., 1997). When bulls are categorized as above average or below average fertility, lipocalin-type PGD synthase is 3.5 times more prevalent in the above average group of bulls (Killian et al., 1993 and Gerena et al., 1998; 2000). The work of Killian and others has led to a regression model to predict the fertility of bulls based on these four fertility-associated proteins with an R value equal to 0.89 (for formula see Killian et al., 1993).

Currently, the cellular roles of OPN and lipocalin-type PGD synthase and their influence on bull fertility remain to be elucidated. However, OPN plays an integral part in a number of signal transduction pathways (for review see Denhardt et al., 2001) including defense mechanisms and inflammatory conditions. In addition, OPN may serve as a functional cell attachment protein to provide transitory stabilization of the sperm plasma membrane prior to fertilization.

Localization studies have identified lipocalin-type PGD synthase to be present within elongating spermatids, Sertoli cells, rete testis and efferent duct epithelial cells along with epididymal epithelial cells (Gerena et al., 2000). In addition, fluorescent microscopy revealed the presence of lipocalin-type PGD synthase on the apical ridge of the acrosome on ejaculated bovine sperm (Gerena et al., 2000). This evidence would suggest lipocalin-type PGD synthase may play an important role in the spermatogenic cycle, as well as maturation of spermatozoa during storage in the epididymis. Lipocalin-type PGD synthase's multifaceted sites of localization and activity lend support to a number of potential roles that may reflect why bulls with above average fertility have 3.5-fold higher concentrations of this protein.

These, as well as other functional roles of OPN and lipocalin-type PGD synthase need to be investigated to give us insight on how they influence bull fertility.

Of the heparin-binding proteins, the 31 kDa protein coined fertility-associated antigen (FAA; Bellin et al., 1998) binds heparin with the greatest affinity (Miller et al., 1990). This protein has been isolated in tissue from all bovine accessory sex glands (Nass et al., 1990; and McCauley et al., 1999), and it is present in seminal secretions and on sperm membranes following ejaculation but not on epididymal sperm (McCauley et al., 1996). Using the M1 antibody previously mentioned, sperm membranes/seminal plasma can be analyzed for the presence or absence of this protein, as well as the 24 kDa tissue inhibitor of metalloproteinases-type 2 (TIMP-2) protein. Table 2 represents data from large field trials when bulls were sorted as FAA-positive or FAA-negative by assaying sperm membrane extracts. The data represent over 10,000 cows and a 17 % difference in fertility when cows were exposed to FAA-positive bulls versus their FAA-negative herdmates.

Table 2. Fertility of range beef bulls following a 60 d exposure period and AI beef bulls after three projected services (equivalent to 60 d exposure) when categorized as FAA-positive or FAA-negative.

Table 3 summarizes results from field trials recently completed to compare pregnancy outcomes of beef cows exposed to bulls after their spermatozoa had been tested for presence or absence of TIMP-2. Bulls were pastured for 60 days at a ratio of 25 cows per bull in multiple-sire pastures. Fertility was 13% higher for bulls whose sperm was qualified as TIMP-2 positive compared to their herdmates categorized as TIMP-2 negative.

Table 3. Fertility of range beef bulls after a 60 d exposure period following characterization of sperm as either TIMP-2 positive or TIMP-2 negative.

Testing for these fertility-related HBPs is commercially available by contacting ReproTec, Inc., (520) 888-0294. Details on collecting and mailing samples can be obtained by contacting ReproTec directly.

References

Bellin, M. E., J. N. Oyarzo, H. E. Hawkins, R. G. Smith, D. W. Forrest, L.R. Sprott and R. L. Ax. 1998. Fertility associated antigen (FAA) on bull sperm indicates fertility potential. J. Anim. Sci. 76:2032-2039.

Bellin, M.E., H.E. Hawkins, J.N. Oyarzo, R.J. Vanderboom, and R.L. Ax, 1996. Monoclonal antibody detection of heparin-binding proteins on sperm corresponds to increased fertility of bulls. J. Anim. Sci. 74:173-182.

Bellin, M.E., H.E. Hawkins, 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.

Cancel, A.M., D.A. Chapman and G.J. Killian. 1999. Osteopontin localization in the Holstein bull reproductive tract. Biol. Reprod. 60:454-460.

Cancel, A.M., D.A. Chapman and G.J. Killian. 1997. Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma. Biol. Reprod. 57:1293-1301.

Dawson, G.R., M.E. Bellin, J.N. Oyarzo, H.E. Hawkins, M.J. Arns and R.L. Ax. 2002. Presence of TIMP-2 on sperm corresponds to fertility of range beef bulls. 27th American Society of Andrology Meeting, Seattle, WA. (Abstr. #121)

Gerena, R.L., D. Irikura, N. Eguchi, Y. Urade and G.J. Killian. 2000. Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm. Biol. Reprod. 62:547-556.

Gerena, R.L., D. Irikura, Y. Urade, N. Eguchi, D.A. Chapman and G.J. Killian. 1998.

Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. Biol. Reprod. 58:826-833.

Killian, G.J., D.A. Chapman and L.A. Rogowski. 1993. Fertility-associated proteins in Holstein bull seminal plasma. Biol. Reprod. 49:1202-1207.

McCauley, T.C., M.E. Bellin and R.L. Ax. 1996. Localization of a heparin-binding protein to distinct regions of bovine sperm. J. Anim. Sci. 74:429-438.

McCauley, T.C., H. M. Zhang, M.E. Bellin, and R.L. Ax, 1999. Purification and characterization of Fertility-Associated Antigen (FAA) in bovine seminal fluid. Mol Reprod Dev. 54:145-153.

McCauley, T.C., H.M. Zhang, M.E. Bellin, and R.L. Ax. 2001. Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2. Mol Reprod Dev. 58:336-341.

Miller, D. J., M. A. Winer, and R. L. Ax. 1990. Heparin-binding proteins from seminal plasma bind to bovine spermatozoa and modulate capacitation by heparin. Biol. Reprod. 42:899-915.

Nass, S.J., D.J. Miller, M.A. Winer, and R.L. Ax. 1990. Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent. Mol. Reprod. Dev. 25:237-246.

Sprott, L.R., M.D. Harris, D.W. Forrest, J. Young, H.M. Zhang, J.N. Oyarzo, M.E. Bellin, and R.L. Ax. 2000. Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen. J. Anim. Sci. 78:795-798.

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