Assessment of survival and fertilization rates
Ovaries from 440 mature Holstein cows were collected from a local abattoir and immediately used in the IVF experiments. Fertilization of oocytes was as previously described [7, 11]. In brief, oocytes were aspirated from antral follicles (> 2–6 mm), processed in different media and in incubated in maturation medium for 20–24 hours. On Day 2, oocytes were fertilized with frozen-thawed percoll-separated bull semen that had been adjusted to a final concentration of 1 million sperm/ml. Oocytes and sperm were co-incubated for a period of 18–24 h. After the fertilization period, putative zygotes were stripped of their cumulus cells by vortexing for 3 minutes, then washed 3 times in TALP-Hepes. Gametes from a total of 440 cows and eight bulls were used in the IVF experiment. Fertilization rate was calculated as proportion of cleaved embryos 48 h post fertilization out of total number of oocytes exposed to sperm. Survival rate of embryos was calculated as the number of blastocysts on Day 7 of development out of the number of total embryos cultured. Viability of blastocysts was determined as a function of the embryo's ability to attain the morphological stage of blastocyst on Day 7 of development. Embryos that failed to show cellular compaction (morula stage) on day 5 or 6 were considered non viable. Therefore only embryos exhibiting adequate compaction followed by the formation of a blastocoele on Day 7 were considered viable. Embryos were preserved in RNALater RNA Stabilization reagent (Qiagen, Valencia, CA) to avoid RNA degradation.
Polymorphism identification and genotyping
Respect to [7], we extended our search for SNPs to include 8,998 bp upstream of STAT5A, all exons of STAT3, and 3,699 bp upstream sequences of STAT3. Table 1 shows only primers with which SNPs were identified. Genomic DNA was extracted from ovaries by grinding 30–100 mg from each ovary using the AquaPure Genomic DNA kit (Bio-Rad, Hercules, CA). The DNA concentration was measured using a spectrophotometer (Ultraspec 2100; Amersham Biosciences). DNA pools were constructed from 50 different ovary samples to contain 50 ng of DNA from each sample and amplified with the primers listed in Table 1. Amplification was performed in a 25-μl reaction volume, which included 50 ng genomic DNA, 50 ng each primer, 200 μM each dNTP, 2.5 μl 10× PCR buffer (Promega, Madison, WI), and 0.5 u Taq DNA polymerase (Promega). The temperature cycles were as follows: 95°C for 5 min, followed by 32 cycles of 94°C for 45 s, touchdown annealing from 63–50°C for 45 s (-2°C/cycle), 72°C for 45 s, and a final extension at 72°C for 8 min. The PCR products of the pooled DNA samples were sequenced using BigDye terminator (Applied Biosystems, Foster City, CA), and SNPs were identified by visually inspecting sequence traces. Individual cows and bulls were genotyped by sequencing.
Embryo genotyping
Genomic DNA and RNA were extracted from embryos using Ambion kit (Applied Biosystems). Embryos were genotyped for SNP153137 (G/C) in exon 8 of STAT5A using primers STATF1 and STATR1 and for SNP177338 in exon 12 of STAT3 using primers STAT3F12 and STAT3R12 (Table 1). Amplification was performed in a 25 μl reaction volume, which included 3 μl of embryo DNA, 50 ng each primer, 200 μM each dNTP, 5.0 μl 5× PCR buffer, and 1.5 u Taq DNA polymerase (Promega). The temperature cycles were as follows: 95°C for 5 min, followed by 32 cycles of 94°C for 45 s, touchdown annealing from 65–53°C for 45 s, 72°C for 45 s, and a final extension at 72°C for 7 min. The PCR products were amplified in a nested PCR reaction using primers STAT14 and STAT13 for SNP153137 and primers STAT3F12A and STAT3R12A for SNP177338 (Table 1). The nested PCR reaction included 1 μl PCR product, 50 ng each primer, 200 μM each dNTP, 5.0 μl 5× PCR buffer, and 1.5 u Taq DNA polymerase (Promega). The temperature cycles were as described for the first PCR except the total number of cycles was set to 16. Products of the nested PCR were genotyped by digestion with the restriction enzyme BstE II, which allows one to distinguish alleles C and G of SNP153137. For SNP177338, PCR products were digested with the restriction enzyme MspA1 I which allows one to distinguish alleles G and A.
Expression analysis of STAT5A and STAT3
To analyze the expression patterns of STAT5A and STAT3, SNPs identified in heterozygous individuals were employed to distinguish between monoallelic and biallelic expression. Dams and sires of heterozygous embryos were genotyped in order to determine parental origin of monoallelically-expressed alleles. Primers were designed to amplify fragments spanning more than one exon to exclude the possibility of mistyping due to genomic DNA contamination in the RT-PCR reactions. Primers STAT14 and STAT11 were designed in exons 8 and 11, respectively to amplify a 360 bp fragment which includes SNP153137 from the STAT5A cDNA (Table 1). Primers STAT3-1 and STAT3-2 were designed in exons 13 and 9, respectively to amplify a 253 bp of cDNA fragment of STAT3 which includes SNP177338. Primers b-actin F/b-actin R (Table 1) were used to amplify 191 bp from the housekeeping gene b-actin (GenBank accession number NM_173979) cDNA as a positive control.
In order to test monoallelic versus biallelic expression in fetal tissues, organs from five fetuses at ages 68 to 90 days of age were obtained from a local slaughterhouse. All specimens were preserved in RNALater RNA Stabilization reagent (Qiagen) to avoid RNA degradation. Organs were ground with a mortar and pestle in liquid nitrogen into a fine powder, which then was used for either RNA or DNA extraction. For fetuses heterozygous for SNP153137 (n = 5), the expression pattern of STAT5A was analyzed in a wide range of organs: brain, ovary, liver, pituitary, adrenal gland, lung, skeletal muscle, heart, spleen, teste, cotyledon, mammary gland, rib, kidney, eye, and intestine. The RT-PCR was performed using Qiagen OneStep RT-PCR Kit (Qiagen). The RT-PCR cycling conditions included incubation at 50°C for 30 min, 95°C for 15 min, and then touchdown PCR conditions, as described for genomic DNA PCR amplifications. The RT-PCR products for SNP153137 and SNP177338 were genotyped by digestion with the restriction enzymes BstE II and MspA1 I, respectively, as described for embryo genotyping.
Statistical analysis
Differences in monoallelic expression of STAT5A and in parent-of-origin for heterozygous degenerative embryos vs. blastocysts were tested through a Pearson's Chi-squared test. Given the low number of counts in some of the cells, p-values obtained from a Monte Carlo test with 2000 replicates and those obtained through Yates' continuity correction were reported. All analyses were performed with the stat package of Rsoftware v. 2.5.1 http://www.r-project.org.
Association between SNPs and fertilization and survival rate at Day 7 were analyzed using the following mixed linear model:
y
ijk
= μ + oi +s
j
+ SNP
ijk
+ e
ijk
where y
ijk
represents in turn, the survival or fertilization rate of a batch of ova k from ovary i fertilized with semen from sire j; μ represents a general constant (mean) for the trait considered; o
i
represents the random effect of the individual ovary from which ova were harvested; s
j
represents the random effect of sire used in the fertilization; SNP
ijk
represents the fixed effect of the genotype for the SNP considered; and e
ijk
represent the residuals, assumed normal and independent with mean 0 and variance I σ2
e
. Ovaries and sires were both assumed uncorrelated in the analysis, with variance structures I σ2
o
and I σ2
s
respectively. After data editing, ovaries from which fewer than 4 eggs were harvested were excluded from the analysis. All analyses were performed with the function lmer of the lme4 package of R software v. 2.5.1 http://www.r-project.org.
