Oviductal Epithelial Cells: Can they select the better sperm?
Juan C Samper and Joanna Ellington
Veterinary Reproductive Services LTD, Abbotsford, BC, Canada and
Washington State University, Pullman, Washington, USA

Introduction

Prior to fertilization, mammalian sperm attach to the oviductal epithelial cells (OEC) where they are stored for varying periods of time. The attached sperm are maintained at this site and then sub-populations of sperm are released over time (Samper et al. 1995). The sperm oviductal binding, which appears to be mediated through lectins (Lefebvre et al. 1995), help the sperm maintain motility for prolonged periods of time while maintaining membrane integrity (Thomas et al. 1995). In addition sperm calcium influx, a necessary prerequisite for acrosome reaction, is delayed while sperm are attached to OEC (Dobrinski et al. 1995). Stallion differences exist in the ability of their sperm to form this reservoir in the oviductal isthmus of the mare, with sub fertile stallions much less likely to have motile sperm in the mare’s oviduct after insemination as compared to fertile stallions (Scott et al. 1995).

In vitro studies using sperm and oviductal cell co culture have been used as a bioassay to evaluate different stallion spermatozoal treatments. The objective of this paper is to summarize some of these findings and to stimulate some discussion on sperm parameters that could be evaluated with this bioassay.

Effect of extender and cryopreservation on OEC binding

            Equine semen for artificial insemination is often cooled to 5°C and diluted with skim milk or egg yolk extenders for short-term storage and transport (12-36 hr). Longer sperm preservation usually requires cryopreservation with extenders containing glycerol and egg yolk or milk to protect sperm from cold shock and help maintain membrane integrity. 

Shortly after insemination the sperm aided by uterine contractions will leave the extender in which they are suspended. However several components from the different extenders could have deleterious effects on either membrane function or integrity and/or on sperm chromatin (DNA) structure.

Although there are several reports on the effect of extenders on sperm oviductal cell interaction, it appears that there is not a significant effect on oviductal binding due to extender alone. However handling of these samples by either cooling for 24 hours or by freezing could have a deleterious effect on oviductal attachment of the sperm and/or on sperm chromatin integrity (Samper et al. 1995, Dobrinski et al. 1995).

In experiments conducted by Samper et al. (1993) and by Dobrinski et al. (1995) it has been shown that sperm incubated in skim milk glucose extender for 24 hours as well as cryopreserved sperm will have a reduced binding ability to the oviductal epithelium in vitro. It is important to keep in mind that there could be a strong and significant stallion by extender interaction, as our previous studies have suggested. Studies in humans have shown that the deleterious effect of sperm cryopreservation on binding to OEC is not only due to decrease motility or viability of these sperm since the percentage of motile sperm able to bind is profoundly decreased after cryopreservation (Ellington et al. 1998).

In addition, recent experiments by Samper et al. (2000), indicated that cold-storage of sperm for 24 hrs in a milk-based extender had a detrimental effect on sperm chromatin that was not observed when sperm were cold stored or frozen in an egg yolk extender. Conversely, sperm motility was not negatively affected when sperm were cold stored for 24 hrs in milk vs. egg yolk, but was highly affected when sperm were frozen and thawed. 

Fertility of bound and unbound sperm

Previous studies have shown that sperm that attach to OEC in-vivo and in-vitro represent a selected sperm population with superior morphology and motility (Ellington et al. 1993 and Scott et al. 1995).  In addition it has also been shown that bull sperm that attach to OEC in vitro have a higher percentage of intact chromatin than that found in sperm that are unable to attach to oviductal cells (Ellington et al. 2000).

Bull sperm, which selectively attached to OEC, supported significantly superior embryo cleavage and embryonic development rates versus those for sperm that did not attach to OEC.  These sperm, which did not attach to OEC, also had lower fertilization rates than did control sperm in media alone. Embryo cleavage rates did not differ for sperm, which had been in co culture for 36 hours compared to sperm that were used for IVF shortly after oviductal cell binding. However, fewer of the embryos produced form aged sperm went on to form blastocysts.  A higher percentage of the sperm, which initially did not attach to the OEC (65±7%), had significantly more damaged chromatin as compared to sperm that did attach (39±3%) after two hrs of co culture (Ellington et al. 2000). 

Relationship between fertility and OEC binding

Because cryopreservation of stallion sperm decreases the number of sperm attaching to OEC and the length of time these sperm survive, studies were done to evaluate both fresh and cryopreserved samples from stallions with a wide range of field fertility (Samper et al.1995).

The numbers of sperm diluted in skim milk and cooled for 24 hours that attached to the OEC (out of an initial 12 million added per well), and the length of time these sperm survived in co culture were monitored.  A significant relationship (r> 0.75) was found between first cycle pregnancy rates and both the percentage of viable sperm that attached to the OEC (range 58-82%), and the number of days the attached sperm survived in co culture (range 108-144 hrs).

In another experiment first cycle pregnancy rates resulting from the use of cryopreserved sperm of seven stallions bred to an average of 31 mares each were compared to the number of sperm bound to oviductal cells. As above, sperm function in co-culture (e.g. numbers attaching to OEC and subsequent survival time) was evaluated on multiple ejaculates from these stallions. First cycle pregnancy rates correlated (r ³ 0.71) with the percentage of cryopreserved sperm attaching to OEC (range 17-59%) and the length of time these sperm survived in co-culture (12-96 hrs).

These data suggest that function of fresh or cryopreserved stallion sperm in an OEC co culture system (e.g. sperm numbers attaching and subsequent survival time) could possible be good indicators of the inherent fertility status of stallions as determined by FSC rates in mares.

Possible uses of OEC as a sperm evaluation byioassay

Fertilization is a multi-factorial process that requires several sperm with normal morphological, physical and biochemical characteristics. Unfortunately most of the sperm assays currently available only test for one or two parameters, which in some cases are poorly related to fertility i.e. motility.   

Sperm attachment to OEC in all species studied is selective for viable sperm in a sample with higher percentages of motility, normal morphology and intact DNA chromatin than sperm, which does not attach (Ellington et al. 1993, Thomas et al. 1994, Ellington et al. 1998).  Additionally, sperm that are capacitated or have altered intracellular calcium levels are less able to attach to OEC (Thomas et al. 1995, Dobrinski et al. 1995).  Therefore, the ability of sperm to attach to OEC in vitro is reflective of several aspects of cellular and biochemical integrity.  

If sperm interactions with OEC in coculture are reflective of the sperm behaviour in the mare's oviducts, the oviductal cell co-culture system could be a useful bio-assay that could evaluate several of the prerequisites that sperm must have in order to achieve fertilization. Among these sperm characteristics we could include:

1) Plasma Membrane Integrity: Available sperm from a given sample that are suitable to participate in the formation of the sperm reservoir i.e. number of sperm biochemically suited for oviductal cell attachment in-vivo.

2) Motility and Longevity: How long can sperm be motile in the mare? i.e. Survival time for sperm in the oviductal reservoir.

3) Integrity of Sperm DNA: If the type of sperm attaching to the oviductal has better chromatin structure than those that are unable to attach, the oviductal cells could provide a functional bioassay to diagnose stallion induced early embryonic death.

4) Sperm Selection For IVF: Since sperm processing and handling seems to be a major obstacle in developing a repeatable IVF system in horses, attached and released sperm could serve as a source of sperm for this procedure.

5) Reproductive Management of Subfertile Stallions: By knowing the sperm quality of a given stallion or sample of semen, as well as the behaviour of those sperm in the mares reproductive tract, recommendations could be made to maximize fertility of problem stallions.

References

1.        Dobrinski I, Ball BA, Thomas PGA, Suarez SS 1995. Intracellular calcium concentration in equine spermatozoa attached to oviductal epithelial cells in vitro. Biology of Reproduction 52, Suppl 1:  Abstract 247.

2.        Dobrinski I., Thomas PGA, Ball B 1995.  Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucida in vitro.  Journal of Andrology 16: 536-542.   

3.        Ellington JE, Varner DD, Mathison D, Ignotz GG, Padilla AW, Miller PG, Ball BA 1993. Equine sperm and oviductal epithelial (uterine tubal) interactions in coculture. Molecular Andrology 4: 101-111.

4.        Ellington JE, Evenson DP, Fleming JE, Brisbois RS, Hiss GA, Broder SJ, Wright RW 1998.  Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone.  Fertility and Sterility 69: 643-649.

5.        Ellington JE, Evenson DP, DeAvila J, Jost LK, Wright RW Jr. 2000.  Bull sperm that attach to oviduct cells in vitro support superior embryonic development as compared to sperm that do not attach. Proceedings of the International Congress on Animal Reproduction, Stockholm, Sweden.

6.        Lefebvre R., DeMott RP, Suarez SS, Samper JC 1995.  Specific inhibitation of equine sperm binding to oviduct epithelium.  Biology of Reproduction Monograph 1: 689-696.

7.        Lefebvre R, Samper JC 1993. Preliminary observations on the interaction between stallion sperm and equine oviductal explants in-vitro. Equine Veterinary Journal, Suppl 15: 39-41.  

8.       Leopold S, Samper JC, Curtis E, Buhr MM in press.  Effect of cryopreservation and oviductal cell conditioned media on CA²⁺ flux of equine spermatozoa.  Journal of Reproduction and Fertility, Suppl 56.

9.        Samper JC, Ellington JE, Burnett K, Jones A, Wright RW 1995.  Use of sperm and oviduct cell coculture as a test for stallion field fertility.  American Association of Equine Practitioners Proceedings 41: 3-5.

10.     Samper JC, Ellington JE, Wright RW, Jost LK and Evenson DP, 2000. The effect of extender type, and cold storage or cryopreservation on stallion sperm chromatin structure. Proceedings of the International Congress on Animal Reproduction, Stockholm, Sweden

11.     Scott MA, Liu IK, Overstreet JW 1995.  Sperm transport to the oviducts: Abnormalities and their clinical implications.  Proceedings Annual Meeting American Association Equine Practitioners Proceedings 41: 1-2.

12.     Thomas PGI, Ball BA, Miller PG, Brinsko SP,  Southwood L  1994. A subpopulation of morphologically normal, motile spermatozoa attach to equine oviductal epithelial cell monolayers Biology of Reproduction 51: 303-309

13.     Thomas PGA, Ball BA, Brinsko SP 1995.  Changes associated with induced capacitation influence the interaction between equine spermatozoa and oviduct epithelial cell monolayers.  Biology of Reproduction Monograph 1: 697-705.