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RHOXF2 gene, a new candidate gene for spermatogenesis failure.

ABSTRACT: INTRODUCTION:Genes involved in testicular differentiation, spermatogenesis, proliferation and apoptosis of germ cells have been shown to evolve rapidly and display rapid DNA changes. These genes are therefore good candidates for explaining impairments in spermatogenesis. Initial studies of some of these genes appear to confirm this hypothesis. The RHOXF2 candidate gene belongs to the RHOX family clustered in Xq24 and is specifically expressed in the testis. It contains four exons and codes for a 288 amino acid (aa) transcription factor. It has a high degree of homology (>99.9%) with its paralogue RHOXF2B, which is also preferentially expressed in the testis. OBJECTIVES:To sequence RHOXF2 and RHOXF2B in intracytoplasmic sperm injection (ICSI) patients and identify any single-nucleotide polymorphisms (SNPs) associated with impaired spermatogenesis. MATERIALS:A cohort of 327 patients in ICSI programmes at Poissy and Bichat hospitals. All patients gave their written, informed consent to participation. One hundred patients had unaffected spermatogenesis and 227 displayed impaired spermatogenesis. METHODS:The four exons in each of RHOXF2 and RHOXF2B were sequenced in 47 patients with oligospermia or non-obstructive azoospermia. Given that exons 2 and 3 were found to harbour most of the SNPs, only these two exons were sequenced in the remaining 280 subjects. RESULTS:Due to the extremely high degree of sequence identity between RHOXF2 and RHOXF2B, we were not able to distinguish between the sequences of these two genes. Although 9 SNPs were identified, there were no significant frequency differences between ICSI patients with normal vs. impaired spermatogenesis. Two insertions were identified: a 21-nucleotide insertion was retrieved in both groups and a guanine insertion (inducing a premature stop codon) only found in two patients with impaired spermatogenesis. CONCLUSION/OUTLOOK:RHOXF2 is a good candidate for rapid evolution by positive selection. Analysis of the polymorphism frequency in exons 2 and 3 did not allow us to correlate the identified SNPs with male infertility. However, a single nucleotide insertion was identified only in men with impaired spermatogenesis. Further work will be needed to establish whether genetic changes in RHOXF2 can give rise to defects in spermatogenesis.

SUBMITTER: Frainais C

PROVIDER: S-EPMC4349744 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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RHOXF2 gene, a new candidate gene for spermatogenesis failure.

Frainais Christophe C Kannengiesser Caroline C Albert Martine M Molina-Gomes Denise D Boitrelle Florence F Bailly Marc M Grandchamp Bernard B Selva Jacqueline J Vialard François F

Basic and clinical andrology 20140210

<h4>Introduction</h4>Genes involved in testicular differentiation, spermatogenesis, proliferation and apoptosis of germ cells have been shown to evolve rapidly and display rapid DNA changes. These genes are therefore good candidates for explaining impairments in spermatogenesis. Initial studies of some of these genes appear to confirm this hypothesis. The RHOXF2 candidate gene belongs to the RHOX family clustered in Xq24 and is specifically expressed in the testis. It contains four exons and cod ...[more]

PMID: 25780578

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Project description:Spermathecal fluid proteins were cleaned up via acetone precipitation overnight at 20 C. The precipitated protein was pelleted and washed twice with 500 ul of 80% acetone, then the pellet was allowed to air dry (ca. 5 min) prior to solubilization in 50 ul of digestion buffer (6 M urea, 2 M thiourea). Approximately 25 ug of protein were reduced (0.5 ug dithiothreitol, 20 min), alkylated (2.5 ug iodoacetamide, 30 min, dark), and digested (0.5 ug Lys-C for 3 h, then 0.5 ug trypsin overnight). Digested peptides were acidified with one volume of 1% trifluoroacetic acid and desalted with high-capacity STAGE tips. Eluted samples were dried (SpeedVac, Eppendorf, 45 min) and resuspended in Buffer A (0.1% formic acid). Peptide concentrations were determined using a NanoDrop (Thermo, 280 nm) and sample orders were randomized for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Peptides (0.5 ug for each sample) were injected on an EASY-nLC 1000 liquid chromatography system (Thermo) coupled to an Impact II Q-TOF mass spectrometer (Bruker). The LC system included a fused-silica (5 um Aqua C18 particles (Phenomenex)) fritted 2 cm trap column connected to a 50 cm analytical column packed with ReproSil C18 (3 um C18 particles (Dr. Maisch)). The separation gradient ran from 5% to 35% Buffer B (80% acetonitrile, 0.1% formic acid) over 120 min, followed by a 15 min wash at 95% Buffer B (flow rate: 250 uL/min). The instrument parameters were: scan from 150 to 2200 m/z, 100 us transient time, 10 us prepulse storage, 7 eV collision energy, 1500 Vpp Collision RF, a +2 default charge state, 18 Hz spectral acquisition rate, 3.0 s cycle time, and the intensity threshold was 250 counts. Mass spectrometry data were searched using MaxQuant (v1.6.1.0) using default parameters, except match between runs was enabled. Peptide spectral matches, peptide identifications and protein identifications were controlled at 1% false discovery rates (FDRs). Data were analyzed in Perseus (v1.6.1.1). Proteins differentially expressed between heat-shocked and non-heat-shocked samples, as well as among tissues (semen, virgin spermathecae, and mated spermathecae), were identified using t-tests and significant results were corrected to either 5% (heat-shock and cold-shock) or 10% FDR (pesticide stress) (Banjamini-Hochberg method).

Project description:Mammalian spermatogenesis involves formation of haploid cells from the male germline and then a complex morphological transformation to generate motile sperm. Focusing on meiotic prophase, some tissue-specific transcription factors are known (A-MYB) or suspected (RFX2) to play important roles in modulating gene expression in pachytene spermatocytes. The current work was initiated to identify both downstream and upstream regulatory connections for Rfx2.Searches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes) in promoter regions of several new genes: Adam5, Pdcl2, and Spag6. We confirmed a strong promoter-region X-box for Alf, a germ cell-specific variant of general transcription factor TFIIA. Using Alf as an example of a target gene, we showed that its promoter is stimulated by RFX2 in transfected cells and used ChIP analysis to show that the promoter is occupied by RFX2 in vivo. Turning to upstream regulation of the Rfx2 promoter, we identified a cluster of three binding sites (MBS) for the MYB family of transcription factors. Because testis is one of the few sites of A-myb expression, and because spermatogenesis arrests in pachytene in A-myb knockout mice, the MBS cluster implicates Rfx2 as an A-myb target. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites in Rfx2 expression. Further, Rfx2 expression was virtually eliminated in A-myb knockout testes. Immunohistology on testis sections showed that A-MYB expression is up-regulated only after pachytene spermatocytes have clearly moved away from the tubule wall, which correlates with onset of RFX2 expression, whereas B-MYB expression, by contrast, is prevalent only in earlier spermatocytes and spermatogonia.With an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes. Rfx2 itself is a good candidate to be regulated by A-MYB, which is essential for meiotic progression. If Alf is a genuine RFX2 target, then A-myb, Rfx2, and Alf may form part of a transcriptional network that is vital for completion of meiosis and preparation for post-meiotic differentiation.

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RHOXF2 gene, a new candidate gene for spermatogenesis failure.

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RHOXF2 gene, a new candidate gene for spermatogenesis failure.

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