The PCR product for each target from individual human oocytes was distinguishable from that of cumulus cells, both in absolute amount and in the fraction of samples expressing each target (Table 2), the latter being an indication of the relative abundance of an individual transcript. Specifically, neither (3A- nor (3B-subunit mRNA was detectable in any oocyte, while low levels of a-subunit mRNA were detected in only 11% of human oocytes. FS315 mRNA was detected in 95% of human oocytes, but the signal intensity was relatively low compared to that in cumulus cells. On the other hand, robust signals were detected for activin receptor subtype transcripts in nearly all oocytes (88-100%). ampicillin antibiotic
As expected, high levels of ZP3 and GDF9 mRNAs characterized all oocytes. Cumulus cells, on the other hand, expressed all inhibin/activin system targets examined, including the three inhibin subunits, FS315, and all four activin receptors. In these cells the PCR product signal intensity among FS and the inhibin/activin subunits showed the profile Inh-a > FS315 = pA >> (3B, the latter being undetectable in 20% of the samples.
TABLE 1. Sequence, expected product size, and GenBank accession number for the primers used in PCR (only primers that have not been published elsewhere are shown).
|Transcript||Speciesa||Primer sequence||Size (bp)||Accession #|
|ActRIIA||M||5′ -gaaaatgggagctgctgcaaagttg-3′ 5′-gtaggccatcttgtgatgtctgtac-3′||568||M65287|
|ZP3||H||5′-cacggtgttctcagaggagaa-3′ 5′ -aaggcttgctgaaggaacagg-3′||432||M60504|
a M, mouse; H, human.
b Based on sequence obtained for the present study (see text for more details).