Glycosaminoglycans contribute to a variety of tissue structures and comprise a large portion of the extracellular matrix. One significant glycosaminoglycan is heparan sulfate, which binds to and assembles extracellular matrix proteins contributing to its infrastructure, as well as other processes including tissue remodeling, vascularization, and metastatic events (Vlodavsky et al., 2002). Heparanase (HPSE) is the predominate endoglycosidase that degrades heparan sulfate. Porcine HPSE is located on SSC8 at approximately 116 cM (Miles et al., 2009). Suggested QTL for total number of piglets born (King et al., 2003), number of piglets born alive (King et al., 2003) and significant QTL for weight of ovary (Rohrer et al., 1999) have been found in this region on SSC8 spanning from ∼90 to 135 cM (Hu et al., 2007).

Breakdown of heparan sulfate by HPSE contributes to structural alterations to not only the extracellular matrix, but also to the basement membrane of tissue structures. Temporal levels of HPSE were evident in granulosa cells and macrophages of human and murine ovaries during the luteal phase and luteal regression, supporting a role for HPSE in extracellular matrix and basement membrane remodeling in the ovary (Haimov-Kochman et al., 2005).

Matrix metalloproteinases in association with HPSE have been implicated in endometrial remodeling during gestation in cattle and baboons ((Hashizume, 2007; D'Souza et al., 2008). In the porcine placenta, HPSE expression was elevated and localized to the cuboidal trophoblast cells (Miles et al., 2009). These findings suggest that HPSE may function to alter and modify the uterine environment and placenta thereby potentially impacting litter size and prenatal survivability.

Another endoglycosidase with implications in placental and ovulatory development is hyaluronidase (HYAL). Interaction between hyaluronan and CD44, its primary receptor, in expanding cumulus cells of the ovary affect oocyte maturation (Yokoo et al., 2010). Furthermore, it has been established that degradation of hyaluronan by HYAL is necessary for normal follicular development and maturation (Tempel et al., 2000). Hyaluronidase 1, 2, and 3 were localized to granulosa cells of murine ovaries (Orimoto et al., 2008) and were involved in induction of apoptosis and follicular atresia independent of their catalytic activity upon hyaluronan.

Increased levels of hyaluronan, along with decreased levels of HYAL3, within the decidua of gestating mice was correlated with spontaneous abortion in comparison to day 7.5 normal pregnancy mice (Cordo-Russo et al., 2009). Vallet et al. (2010) reported a putative role of stromal hyaluronidases in the structural development and angiogenesis of developing pig placentas.

The HYAL1–3 gene family resides on SSC13 (Gatphayak et al., 2004) and map to approximately 27 cM as determined by use of comparative radiation hybrid and linkage maps (Meyers et al., 2005). Within proximity to the HYAL genes, several QTL have been identified with implications to reproductive traits in swine (Hu et al., 2007). Two ovulation rate QTL (Rathje et al., 1997; Bidanel et al., 2008), an age at puberty QTL (Bidanel et al., 2008), and most recently a QTL for testosterone levels (Ren et al., 2009) have been reported in the same region on SSC13. Interestingly, a SNP within HYAL2 was associated with levels of testosterone and androstenone in Norwegian Landrace boars (Moe et al., 2009). Furthermore HYAL2 SNP haplotypes had a negative effect on testosterone levels, which may be indicative of reduced fertility.

Heparanse, HYAL1, and HYAL2 play a role in reproduction and production in swine and are located within close proximity to reported QTL for fertility and production. Therefore, the objective of this study was to determine if SNP within HPSE, HYAL1, or HYAL2 were associated with reproduction and production traits within a Landrace–Duroc–Yorkshire (LDY) resource population.

Partial gene sequencing of HPSE, HYAL1, and HYAL2 yielded a total of 35 SNP (Table 2). Three of the identified SNP within HYAL1 (52629_135, 52629_143, and 52629_808) have been previously reported (Gatphayak et al., 2004; Moe et al., 2009) and one SNP within HYAL2 (67637_222) has also been reported (Moe et al., 2009). However, these SNP had not been submitted to the GenBank dbSNP resource therefore all identified SNP were submitted using the nomenclature from the current study. Of the 35 SNPs, 12 were incorporated into an assay using MassArray Assay Design software (Sequenom). The subset of HPSE (8 markers), HYAL1 (2 markers), and HYAL2 (2 markers) SNPs were genotyped in a LDY population and had a minor allele frequency of 0.06 or greater (Table 2).

Summaries of phenotypic data analyzed including mean, SD, ranges, and heritabilities are presented in Table 3. All production and weaning to estrus interval data were collected from females following their first parity. Seven traits were analyzed using single locus analysis. Associations with P-values less than 0.05 prior to Bonferroni corrections are reported in Table 4. Three markers maintained nominal significance (α < 0.10) and one marker maintained significance (α < 0.05) after multiple-test correction (Table 4).

Reproductive traits, age at puberty and ovulation rate, evaluated within the current study had minimal associations (P < 0.03) when initial analyses were performed, but no associations were maintained following correction procedures.

Initial marker analyses identified putative associations for production traits including number of piglets born alive, litter birth weight, and number of piglets weaned for HPSE, HYAL1, and HYAL2. However associations maintained after correction for multiple-testing were restricted to number of piglets born alive and number of piglets weaned. A single HYAL1 marker, 52629_808, was nominally (P = 0.0049) associated with number of piglets born alive having a dominant effect of 1.19 ± 0.422 piglets. HYAL2 marker 67637_222 was associated with number of piglets born alive with an additive effect (P = 0.0063) of 1.02 ± 0.373 piglets and a dominant effect (P = 0.0009) of 1.44 ± 0.433 piglets. Similarly, the same HYAL2 marker had a dominant effect (P = 0.0043) of 1.16 ± 0.407 piglets for number of piglets weaned.

In the current study, several SNPs were identified from three candidate genes, HPSE, HYAL1, and HYAL2. Biological investigations have shown a link among these genes and reproductive and production traits in swine. Therefore, we investigated the relationship between SNPs of these genes and reproductive and production traits.

Two individual HPSE markers had moderate associations with age at puberty or ovulation rate respectively, prior to correction for multiple-testing. Others (Cassady et al., 2001; Holl et al., 2004) have reported putative QTL for age at puberty on SSC8 within close proximity to the position of HPSE. Likewise a QTL for weight of ovary was also described in a Meishan–White composite population between 116 and 137 cM on SSC8 (Rohrer et al., 1999). An age at puberty study using a White Duroc–Erhualian population detected a peak at 77 cM on SSC8 using a 5% chromosome-wide threshold, but a secondary peak just below the threshold was notable at approximately 125 cM within the region of HPSE (Yang et al., 2008). It is also worth noting that Buske et al. (2006) compiled several fertility-related QTL studies and found a predominant clustering of QTL across SSC8 for reproductive traits in swine. Likely the markers we have genotyped within HPSE were not in strong linkage disequilibrium with the causative mutation. But rather we may infer that our proximity on chromosome 8 was relevant based on our moderate association results and supporting literature from others. Another possible candidate gene is secreted phosphoprotein 1 (SPP1), which has been shown to have an impact on conceptus–embryo interactions during implantation in several mammalian species including swine (Ross et al., 2007; Erikson et al., 2009; Kim et al., 2010) and is located at approximately 112 cM on SSC8. Furthermore polymorphisms within SPP1 have been associated with male fertility traits, sperm motility, and abnormal spermatozoa rate, within Pietrain and Pietrain × Hampshire boars (Lin et al., 2006). Secreted phosphoprotein 1 may also harbor mutations linked to fertility traits, but to date no associations for age at puberty or ovulation rate have been reported for SNP within the SPP1 gene either.

An SNP within HYAL2 had moderate associations with number of piglets born alive and associated with number of piglets weaned following multiple-test corrections within the current study. Moe et al. (2009) identified the same SNP within Duroc and Norwegian Landrace breeds. The minor allele frequency in Duroc was less than 1% and nearly 49% in Norwegian Landrace. The HYAL2 SNP was associated independently and within a haplotype analysis with various androgen levels in the plasma of Norwegian Landrace boars, suggesting a relationship of this SNP with fertility-related traits (Moe et al., 2009). Our genotyping efforts identified a minor allele frequency of 7% for the same SNP within the composite Duroc, Yorkshire, and double Landrace-derived population. Enough genetic discrepancy may exist among the breeds used within these two reports. Nonetheless, the HYAL2 marker, 67637_222, was modestly associated with number of piglets born alive and maintained an association with number of piglets weaned following multiple-testing correction in the current study. The HYAL gene family resides near 27 cM on SSC13. No QTL for number of piglets weaned has been reported on SSC13. However, others (Noguera et al., 2009) have reported a single-dimensional model QTL for number of piglets born alive and total number of piglets born within a Meishan × Iberian population on SSC13 (50 cM) and further described separate epistatic interacting QTLs on SSC13 (66–82 cM) and SSC9 (1–7 cM) for number of piglets born alive using a bi-dimensional modeling system. Both of these reported QTL lie distal to HYAL1–3 on SSC13. In the current study, SNP were analyzed under single trait conditions, but in the future bi-dimensional analyses using the Illumina 60K BeadChip data along with additional independently genotyped SNP data may provide guidance and insight into regions with independent as well as epistatic interactions. These putative studies may confirm involvement of the region where the HYAL gene family resides or provide a more refined region on SSC13 for number of piglets born alive and the highly correlated trait of number weaned within close proximity to the HYAL1–3 genes.

The complexity of the reproductive and production traits evaluated within this study increase the difficulty in ascertaining the impact of single candidate gene markers for HPSE, HYAL1, and HYAL2. However, previous reports identified the importance of these two genes for both ovarian and uterine/placental function, which can have an influence on litter size and piglet survivability. HYAL1 and HYAL2 SNP had putative effects on number of piglets born alive as well as number of piglets weaned based upon limited association levels in the current study. These candidate SNP likely have minimal, if any, linkage disequilibrium with the mutation that affects these traits, however linkage was not assessed in this study due to inherent reduction in statistical power. It is also plausible that these intricate traits are influenced by multiple genes working in a coordinated effort. Future use of genome-wide association analyses for swine production and reproduction traits will further refine regions of interest on SSC8 and SSC13 for greater interrogation that will more than likely lead to discovery of causative nucleotides or responsible genes or gene networks. However the use of SNP within HPSE, HYAL1, and HYAL2 do not appear to be suitable for marker assisted selection for reproduction and production traits within the population tested.

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