Reproduction in Domestic Animals

370 JP Kastelic and JC Thundathilsperm–oocyte interactions. Following capacitation,sperm interact with the zona pellucida (ZP). For thisinteraction, galactosyl transferase (Larson and Miller2000), p47 (Ensslin et al. 1998), sp56 (Cheng et al. 1994)and zonadhesin (Hardy and Garbers 1995) are apparentlysperm receptors, whereas zona glycoprotein (ZP3)is the ligand (Yanagimachi 1994). This interaction leadsto an acrosome reaction (Yanagimachi 1994); the inneracrosomal membrane interacts with a second glycoprotein,ZP2, facilitating the secondary binding of sperm tothe zona matrix during penetration (Yanagimachi 1994;Aitken 2006).There are several reports regarding the associationbetween sperm–zona binding and fertility. There weredifferences among bulls (with known fertility) in therelative number of sperm bound to bovine oocytes(Fazeli et al. 1993). However, large numbers of oocytesare needed and differences among bulls are detectableonly if they exceed differences among oocytes (Zhanget al. 1995). Alternatively, a hemizona assay (Fazeliet al. 1997) uses both halves of a ZP to compare bindingability of control sperm (known fertility) and test sperm(unknown fertility). Zona binding and hemizona bindingassays and non-return rates were significantlycorrelated. In vitro zona penetration assays have beenused to predict in vivo fertility (Puglisi et al. 2004).However, sperm penetration varies according to sperm–oocyte ratio, duration of incubation and heparin concentration,limiting the value of this test.Sperm Oolemma Fusion and Sperm DNADecondensationHenault et al. (1995) used a homologous zona-freeoocyte penetration assay to demonstrate the effects ofaccessory gland fluid from low- vs high-fertility bulls.Moreover, competitive penetration of zona-free bovineoocytes by fluorochrome-labelled bull sperm was relatedto in vivo fertility (Henault and Killian 1995) andevaluated the ability of sperm to undergo chromatindecondensation and pronucleus formation. In theabsence of a ZP, there is an equal opportunity for allsperm to fuse with the oocyte membrane, undergo DNAdecondensation and pronuclei formation (Thundathilet al. 2001a).Sperm chromatin integrity is an important determinantof the ability of sperm to form normal pronuclei.Sperm DNA is associated with histone nucleoproteinsand organized into classical nucleosome core particlesduring early spermatogenesis. However, these histonenucleoproteins are replaced by transition proteins,which are subsequently replaced by protamines (Yanagimachi1994). Ultimately, chromatin of mature spermhas a compact toroidal structure that resists denaturation.Lewis and Aitken (2005) suggested oxidativestress was a major cause of sperm DNA damage, withreduced pre-implantation embryo development andpregnancy rates. Increased testicular temperature reducedthe stability of sperm DNA (Karabinus et al.1997) and impaired the ability of these sperm to undergoDNA decondensation and pronuclei formation (Walterset al. 2006). However, links among elevated testiculartemperature, oxidative stress and sperm DNA damagein bulls remain unclear. The sperm cell structure assay(SCSA) uses flow cytometry to determine chromatinintegrity, based on resistance to acid denaturation.Sperm are exposed to low pH and stained with acridineorange, which emits green or red fluorescence when itbinds to double- (intact) or single-stranded DNA(denatured), respectively. The ratio of red to (red + -green) fluorescence measures chromatin denaturation,which is significantly correlated with fertility (Ballacheyet al. 1987, 1988; Januskauskas et al. 2001; Waterhouseet al. 2006). In brief, flow cytometry-based approachesprovide a quantitative measure of the structural integrityof sperm chromatin, based on a large number ofsperm, whereas IVF-based tests evaluate the ability ofsperm to undergo DNA decondensation and pronuclearformation during fertilization.Association Between In Vitro Fertilization andFertilityMarquant-Le Guienne et al. (1990) reported that pronuclearformation can be used to predict field fertility ofbulls. Zhang et al. (1997) reported that fertilizationbased on cleavage rate was highly correlated with nonreturnrates, but blastocyst production varied amongtest dates. However, fertility predictions were moreaccurate when based on several laboratory assays vs asingle assay (Truelson et al. 1996). In this regard, aseven-variable model (post-thaw total motility, postthawsperm with a linear motile pattern, sperm concentration,concentration of motile sperm after swim-up,sperm ZP-binding, cleavage rate of total oocytes andblastocyst rate of total oocytes) accounted for 84.6% ofthe variation in non-return rates (Zhang et al. 1999).However, this approach may not be sensitive enough todiscriminate among highly fertile bulls. Similarly, amodel with 30 post-thaw sperm characteristics (includingcleavage rate) accurately predicted fertility (based onconception and non-return rates) of both high- and lowfertilitybulls (Phillips et al. 2004).Future DirectionsThe cell biology approaches described earlier may serveas supplementary tests to a standard breeding soundnessevaluation and improve the reliability of fertility predictions.However, a better understanding of the regulationof sperm function and its contributions to earlyembryo development at the molecular level may lead toreliable molecular markers of fertility, with implicationsfor predicting fertility variations in bulls used for AI.As DNA is transcriptionally inactive in ejaculatedsperm, physiological functions are regulated by structuraland functional proteins and identifying theseproteins may have implications for predicting fertility.The role of heat-shock proteins in sperm capacitation(Kamaruddin et al. 2004) and their potential role insperm–oocyte binding have been reported (Matweeet al. 2001). Specific sperm proteins involved in theinteraction between sperm and ZP (Larson and Miller2000; Inoue et al. 2005) and sperm–oocyte adhesion andegg activation (Evans and Florman 2002) have beenidentified, and the role of angiotensin converting enzymeÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag