Methods
Zona-Free Hamster Penetration Assay
Zona-free hamster eggs penetrated by human spermatozoa were a generous gift of Dr. Dolores J.
Lamb, and Lata Murthy, Baylor College of Medicine, Houston Texas. These samples were prepared
essentially as described by Johnson et al., 1985. Briefly, after the initial semen analysis the samples
were mixed with an equal volume of test yolk buffer composed of 211 mM N-tris [hydroxymethyl]-ethyl2-amino-ethane sulfonic acid, 96 mM hydroxymethyl-amino-methane, 11 mM dextrose, 1% penicillinstreptomycin containing 20% fresh hen yolk. The resulting mixture was then incubated for 42 h at 4°C.
Following this incubation the samples were rapidly warmed by adding 37°C Biggers, Whitten and
Whittingham’s buffer containing 3% bovine serum albumin (BWW media, Irvine Scientific; Santa Ana,
CA). The spermatozoa were gently pelleted at 650 x g for 10 minutes, supernatant removed, washed
again in BWW media containing bovine serum albumin then subjected to a swim-up. After 90 min the
motile spermatozoa that migrated from the pellet were collected then incubated for 30 min, 1 h or 3 h
with zona-free hamster oocytes prepared essentially as described (Yanigamichi et al., 1976 and Syms
et al., 1985). After incubation, the penetrated eggs were repeatedly washed to remove loosely bound
sperm then placed in the RNeasy Lysis buffer (Qiagen Inc.; Valencia, CA). Samples were then flash
frozen in liquid nitrogen and stored at -80°C until use.
RNA Extraction
RNA was extracted using the RNeasy kit (Qiagen Inc.; Valencia, CA). In brief, the zona-free hamster
human sperm penetrated eggs were rapidly thawed, homogenized with a 26-gauge needle to shear the
DNA then heated for 30 min at 65ºC followed by a final homogenization. The RNA was then bound to
the column, extensively washed then eluted using two 25 ul aliquots of warm (65°C) dH2O.
Dithiothreitol (DTT) was then added to final concentration of 5 mM along with 40 units of RNase Block
(Strategene; La Jolla, CA) to the eluent. Residual DNA was digested with DNA-Free DNase I (Ambion;
Austin, TX) as recommended by the manufacturer. The samples were then stored at -80ºC until use.
RT-PCR
Forty L Reverse Transcription Polymerase Chain Reactions (RT-PCR) were performed using Oligo-dT
(0.5g/L) and random hexamer (0.2g/L) primers and the Sensis Script enzyme kit (Qiagen Inc;
Valencia, CA). The resulting cDNAs provided templates for REAL-TIME PCR assays.
REAL-TIME PCR
Real-Time PCR assays were carried out using the MJ Research Opticon Monitor (MJ Research;
Waltham, MA) as similarly described (Ostermeier et al., 2003). In these assays, fluorescence of sybergreen (Molecular Probes; Eugene, OR) at a final concentration of 0.25 X was measured at each
amplification step. Thirty-seven cycles of PCR were carried out using the HotStar Taq polymerase
system (Qiagen Inc., Valencia, CA) and the primer pairs shown in Supplementary Table 1, at the
specified annealing temperatures, to amplify the indicated transcript.
References:
Johnson, A., Bassham, B., Lipshultz, L.I. and Lamb, D.J. A quality control system for the optimized
sperm penetration assay. Fertil Steril 64, 832-7 (1995).
Ostermeier, G.C., Liu, Z.D., Martins, R.P., Bharadwaj, R.R., Ellis, J., Draghici, S. and Krawetz S.A.
Nuclear matrix association of the human beta-globin locus utilizing a novel approach to
quantitative Real-Time PCR. Nucleic Acids Res. 31, 3257-66 (2003).
Syms, A.J., Johnson, A.R., Lipshultz, L.I. and Smith R.G. Effect of aging and cold temperature storage
of hamster ova as assessed in the sperm penetration assay. Fertil Steril 43, 766-72 (1985).
Yanagimachi, R., Yanagimachi, H. and Rogers, B.J. The use of zona-free animal ova as a test-system
for the assessment of the fertilizing capacity of human spermatozoa. Biol Reprod. 15, 471-6
(1976).
Supplementary Table 1. REAL-TIME PCR primers and conditions.
Transcript
PRM2 (u)
PRM2 (L)
FOXG1B (u)
FOXG1B (L)
WNT5A (u)
WNT5A (L)
SOX13 (u)
SOX13 (L)
WHSC1 (u)
WHSC1 (L)
Clusterin (u)
Clusterin (L)
AKAP4 (u)
AKAP4 (L)
Oscillin (u)
Oscillin (L)
HSBP1 [CDH13] (u)
HSBP1 [CDH13] (L)
RPL2 (u)
RPL2 (L)
NLVCF (u)
NLVCF (L)
Hamster Actin (u)
Hamster Actin (L)
Primer Sequence
cDNA Size Genomic Size
TAT AGG CGC AGA CAC TGC TCT CGA
148bp
310bp
GCC TTC TGC ATG TTC TCT TCC TGG
CGC GGG CCA GAC CAG TTA CTT TTT
549bp
549bp
TGG AAA TCT GGC GGC TCT TAG AGA
TCA CAG AGG TGT TGC AGC GTA TCA
395bp
395bp
GGT TGC TTC GTC CTG CTC A
CCT TGG ATC AGT ATG GGA ACC CCA
213bp
215bp
AAA TCC CCC GAG TGT AAA CAG GAG
TTG CCC AAA TAT CCT TGA TCC
307bp
307bp
CCC CCA ACT GGC AAG TCT CAA CTC
AGA GCT CTG CAC GTC ACC AAG TAA
110bp
110bp
ATG AGC AGC AGA GTC GAG TGT TAG
CAA TGC CAC AGA ACT ATC AAG ACT
460bp
1656bp
CAG GCA ACT GCT CAA CTG TAT
CCG CTC TCC TGC TTA CCT AGG AGG
334bp
334bp
GGT ACC AAA CAT AGC CCT TAG GCC
ACAGGGCTGAGAACCGTTAGT
581bp
581bp
GCCGAAAGCATAAAGTATTGT
ATG GCA CAG AAA TGG TAT CAA GAA
257bp
1169bp
GGG CAA TGT AGG CAA GTC GAT CGA
AAG GAA GGG CCA CAT TAC ACA CCA
137bp
137bp
GGG CAG CAT GTT AAG GAT AGC AGC
AGG CAT TGC TGA CCG GAT G
207bp
323bp
CTA GAA GCA TTT GCG GTG GAC GAT
cDNA Size- size of cDNA amplicon. Genomic Size- size of genomic DNA amplicon.
TA- Annealing Temperature in °C.
TA
66.7
63.9
61.7
61.5
59.7
64.8
62.8
63.4
58
63.3
62.8
66