In a previous study, we found SOHLH1 (spermatogenesis and oogenesis-specific basic helix-loop-helix 1) as the first testis-specific basic helix-loop-helix transcription factor essential for spermatogonial differentiation. SOHLH1 therefore represents an excellent candidate gene for testicular failure such as nonobstructive azoospermia (NOA). We analyzed whether there were mutations in the SOHLH1 gene in 96 Korean patients with NOA. The sequence analysis discovered three novel variations: one intronic variant (c.346-1G>A), and two nonsynonymous exonic variants (c.91T>C and c.529C>A) with known single nucleotide polymorphisms (SNPs), which included six intronic variants, two synonymous, and two nonsynonymous variants. We examined the consequences of mutations in SOHLH1 using in vivo and in vitro assays. Analysis of transcripts from minigenes carrying the c.346-1G>A revealed that splicing site variation leads to the partial deletion at a cryptic splicing site within exon 4. This deletion results in SOHLH1 with a truncated bHLH domain. Transient transfection assay showed that the SOHLH1 mutant with the truncated domain disrupted the transcriptional activity of KIT promoter, whereas two missense mutations harboring either p.Arg37Gln or p.Pro269Ser did not have a significant effect on its transactivation. Our findings indicate that a splice-acceptor site mutation that probably causes a nonfunctional SOHLH1 protein results in nonobstructive azoospermia by the lack of normal spermatogenesis.

a b s t r a c tNobox is an oocyte-specific transcriptional regulator. Nobox deficiency disrupts early folliculogenesis and the expression of oocyte-specific genes in mice. In the present study, we found that peptidylarginine deiminase 6 (Pad6) was downregulated in Nobox-null ovaries. Pad6 is preferentially expressed in oocytes and its transcript is detectable at embryonic day 16.5. In addition, we identified one Nobox DNA-binding element (NBE) within the mouse Pad6 promoter. The NBE includes a core sequence TAATTA. Sequence-specific binding of Nobox to the TAATTA motif was confirmed. Nobox overexpression augmented transcriptional activity of a luciferase reporter driven by mouse Pad6. Our findings indicate that Nobox is a critical regulator that orchestrates oocyte-specific genes such as Pad6 during folliculogenesis.

Lhx8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian folliculogenesis. However, Lhx8 DNA binding sequences are not characterized yet. We aimed to identify and characterize a cis-acting sequence of germ-cell specific transcriptional factor, Lhx8. To identify Lhx8 DNA binding element, Cyclic Amplification of Sequence Target (CAST) Analysis was performed. Electrophoretic Mobility Shift Assay (EMSA) was processed for the binding specificity of Lhx8. Luciferase assay was for the transcriptional activity of Lhx8 through identified DNA binding site. We identified a putative cis-acting sequence, TGATTG as Lhx8 DNA binding element (LBE). In addition, Lhx8 binds to the LBE with high affinity and augments transcriptional activity of luciferase reporter driven by artificial promoter containing the Lhx8 binding element. These findings indicate that Lhx8 directly regulates the transcription of genes containing Lhx8 binding element in oocytes during early folliculogenesis.

LHX8 (LIM homeobox 8) is a germ cell-specific transcription factor that is crucial for folliculogenesis in the ovary and represents a candidate gene for premature ovarian insufficiency. We analyzed whether mutations in the LHX8 gene in 96 Korean women with premature ovarian insufficiency. The sequence analysis identified four known SNPs included one synonymous and three intronic variants and discovered three novel singlenucleotide polymorphisms in intron 4 (c.114+99C>A), intron 4 (c.114+100C>A) and intron 6 (c.390+77C>G) of the LHX8 gene. These polymorphisms were also found in controls at frequencies that were not statistically significant. Functional mutations in LHX8 gene are rare in Korean women with premature ovarian insufficiency.

CorrigendumCorrigendum to ''The oocyte-specific transcription factor, Nobox, regulates the expression of Pad6, a peptidylarginine deiminase in the oocyte" [FEBS Lett. 584 (2010) 3629-3634]