A study by Hyne and Boettcher ( 31 ) reported the existence of specific, low-affinity binding sites for 17β-E 2 on human spermatozoa for which other steroids could compete. A subsequent autoradiographic study observed that the binding of[ 3 H]17β-E 2 to spermatozoa can be reduced by coincubation with other steroids, such as progestagens, which can strongly compete for the 17β-E 2 receptor ( 32 ). The presence of high-affinity (K D × 10 −10 m ) binding sites for 17β-E 2 on human sperm has been further confirmed: after fractionation of 17β-E 2 -bound spermatozoa, they are detectable mainly in the membrane fraction (75–85%), whereas the nuclear fractions shows only about 10% of the total bound radioactivity, and no radioactivity is detected in the cytosolic fraction ( 33 ). Other autoradiographic data confirm that the plasma membrane is the site of receptors with specificity for 17β-E 2 , which are mostly concentrated in the central part of the sperm tail ( 34 ). In competition experiments, the binding sites for [ 3 H]17β-E 2 appear to be specific, the labeled steroid being displaced by cold 17β-E 2 . Specific binding sites for [ 3 H]17β-E 2 are not detectable in the sperm cytosol and in sperm nuclei isolated after homogenization, detergent treatment, and centrifugation ( 35 ).
RNA extraction and real-time polymerase chain reaction (PCR) . Ishikawa/vec and Ishikawa/ERα cells were cultured in starve medium for 2 days and then treated with 10 nM E 2 , 100 nM EDCs, or EtOH vehicle (control) for 18 hr. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). First-strand cDNA synthesis was performed using Superscript reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. The mRNA levels of ER target genes were measured using SYBR green assays (Applied Biosystems, Carlsbad, CA). The Genbank accession numbers ( http:///genbank/ ) and sequences of primers used for real-time PCR were as follows: human PR (NM_): forward 5´-GACGTGGAGGGCGCATAT-3´, reverse 5´-GCAGTCCGCTGTCCTTTTCT-3´; human pS2/TFF1 (NM_): forward 5´-GCCCTCCCAGTCTGCAAATA-3´, reverse 5´-CTGGAGGGACGTCGATGGTA-3´; human GREB1 (NM_014668): forward 5´-CAAAGAATAACCTGTTGGCCC-3´, reverse 5´-GACATGCCTGCGCTCTCATAC-3´; human SPUVE (NM_007173): forward 5´-ATGCCCGAGCAGATGAAATT-3´, reverse 5´-CCAACCCTTGGGCACATG-3´; human WISP2 (NM_003881): forward 5´-TGAGCGGCACACCGAAGAC-3´, reverse 5´ACAGCCATCCAGCACCAG-3´; human SDF-1 (NM_000609): forward 5´-GTGGTCGTGCTGGTCCTC-3´, reverse 5´-GATGCTTGACGTTGGCTCTG-3´. Cycle threshold (Ct) values were obtained using the ABI PRISM 7900 Sequence Detection System and analysis software (Applied Biosystems, Foster City, CA). Each sample was normalized to its β-actin transcript content: forward 5´-GACAGGATGCAGAAGGAGATCAC-3´, reverse 5´-GCTTCATACTCCAGCAGG-3´. The experiments were repeated three times, and results are presented as the mean fold change (± SE; n = 3) relative to control (vehicle-treated) Ishikawa/vec cells.