Progressive methylation of POU5F1 regulatory regions during blastocyst development.

The POU5F1 gene encodes one of the 'core' transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5' regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere with POU5F1 expression and function in in vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends on de novo methylation of its promoter. However, little is known about the epigenetic regulation of POU5F1 expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of the POU5F1 5' upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5' regulatory region and the first exon accompanying differentiation and the gradual repression of POU5F1 Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.

Methylation profile of the promoters of Nanog and Oct4 in ICSI human embryos.

STUDY QUESTION: What is the methylation status of the Nanog and Oct4 promoters in human gametes and ICSI embryos and is abnormal reprogramming of their methylation associated with developmental failure of ICSI embryos? SUMMARY ANSWER: Developmental failure of human ICSI embryos is associated with high methylation of the Oct4 promoter. WHAT IS KNOWN ALREADY: Nanog and Oct4 genes play critical roles in the establishment and maintenance of pluripotency during normal early embryonic development, and both are negatively regulated through the methylation of their promoters. STUDY DESIGN, SIZE AND DURATION: We analysed the methylation profile of Nanog and Oct4 promoters in 5 control sperm from normally fertile men, 70 metaphase II oocytes, 21 4-cell control ICSI embryos, 7 control blastocysts and 45 ICSI embryos arrested at 2- to 8-cell stage following prolonged culture. PARTICIPANTS, MATERIALS, SETTING AND METHODS: Embryos and gametes were donated for research by patients from the Department of Reproductive Medicine at the Hôpital Femme Mère Enfant (Bron, France) and the Clinique du Tonkin (Villeurbanne, France) after giving their informed consent. MAIN RESULTS: For both promoters, high methylation was observed in sperm cells. Although, in general, the promoters were unmethylated in oocytes, the methylation of some alleles was observed, particularly in oocytes from women with known infertility. Both gene promoters were hypomethylated in control blastocyst ICM (inner cell mass) and in control 2-8-cells embryos obtained from 6 out of 8 couples. However, they appeared highly methylated in embryos obtained from the other two couples. In most arrested ICSI embryos, the Nanog promoter was unmethylated while the Oct4 promoter was highly methylated. High methylation of the Oct4 promoter was significantly more pronounced in embryos from couples where a male factor was the only known cause of infertility. When the embryos were heterozygous for a G/A single nucleotide polymorphism, both alleles could be methylated, each likely representing a paternally inherited or a maternally inherited copy. LIMITATIONS AND REASONS FOR CAUTION: The study was done on a limited number of oocytes and embryos and the gametes of the couples were not available. WIDER IMPLICATIONS OF THE FINDINGS: These results provide new insight regarding the roles of epigenetic abnormalities in early developmental failure in humans. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. There was no competing interest.

Dynamic CpG methylation of the KCNQ1OT1 gene during maturation of human oocytes.

BACKGROUND: Imprinted genes, many of which are involved in development, are marked during gametogenesis to allow their parent-of-origin specific expression, and DNA methylation at CpG islands is part of this epigenetic mark. Maternal imprint is apposed on oocyte during growth and maturation. Factors interfering with normal oocyte differentiation such as gonadotrophin stimulation and in vitro maturation (IVM) may possibly alter imprint resetting. METHODS: We examined the methylation of the KCNQ1OT1 differentially methylated region (KvDMR1) in human oocytes at different stages of their development: germinal vesicle (GV), metaphase I (MI) or metaphase II (MII). RESULTS: About 60% of alleles were fully methylated in GV oocytes and that full imprint is acquired in most MII oocytes. Similarly to in vivo, de novo methylation of DNA occurred in vitro during oocyte maturation. Following in vitro culture for 28 h, GV and MI oocytes are significantly more methylated when they are obtained from natural cycles than from patients undergoing gonadotrophin stimulation. CONCLUSION: This observation suggests that hyperstimulation likely recruits young follicles that are unable to acquire imprint at KvDMR1 during the course of the maturing process.

Photoaffinity labeling of human sex hormone-binding globulin using 17alpha-alkylamine derivatives of 3beta-androstanediol substituted with azidonitrophenylamido, azidonitrophenylamino, or trifluoroazidonitrophenylamino chromophores. Localization of Trp-84 in the vicinity of the steroid-binding site.

Purified human SHBG was photoaffinity labeled with 17alpha-aminomethyl (M), 17alpha-aminoethyl (E), and 17alpha-aminopropyl (P) derivatives of [3alpha-(3)H]-5alpha-androstane-3beta,17beta-diol coupled to 5-azido-2-nitrobenzoylamido (ANB), 4-azido-2-nitrophenylamino (ANP), and 5-azido-2-nitro-3,4,6-trifluorophenylamino (ANTFP) chromophores. Successful labeling was achieved in all cases except for the two photoreagents with the shortest side chains, namely, ANP-M and ANTFP-M derivatives. Edman sequencing and mass spectrometry of immunopurified photolabeled tryptic fragments revealed that radioactivity was present either on the sequence of residues 73-94, uniquely at the level of Trp-84 (stable covalent labeling), or on one of the two overlapping sequences of residues 126-134 and 126-135, at the level of Pro-130 (labile labeling) and Lys-134 (either stable or partially labile labeling), respectively. The same Trp-84 was photolabeled with the three ANB derivatives of increasing lengths, and by the ANP-P photoreagent. This residue was the exclusive target for the shortest [(3)H]ANB-M photoreagent but was a minor site for the longest [(3)H]ANB-P photoreagent, essentially recovered at the level of Pro-130. The [(3)H]ANB-E photoreagent of intermediate size also labeled exclusively Trp-84, except in some experiments in which photolabeling was recovered predominantly at the level of Pro-130. The [(3)H]ANP-P photoreagent with an overall length similar to that of the ANB-P photoreagent labeled simultaneously Trp-84 (minor site) and Lys-134. The other [(3)H]ANP-E, [(3)H]ANTFP-E, and [(3)H]ANTFP-P derivatives labeled in all cases Lys-134. These findings indicate that the conserved Trp-84 and the two Pro-130 and Lys-134 residues are all located in the vicinity of the D ring of steroid ligands and remain freely accessible from the C17alpha position, thus providing biochemical data delineating the corresponding region of the steroid-binding site.

Specific photoaffinity-labeling of Tyr-50 on the heavy chain and of Tyr-32 on the light chain in the steroid combining site of a mouse monoclonal anti-estradiol antibody using C3-, C6-, and C7-linked 5-azido-2-nitrobenzoylamidoestradiol photoreagents.

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody 9D3, raised against the same immunogen as that employed for generating the reported anti-estradiol antibody 15H11 [Rousselot, P., et al. (1997) Biochemistry 36, 7860-7868], was found to exhibit an opposite specificity profile with a much stronger recognition of the D-ring than of the A-ring extremity of the steroid, but a similar lack of specificity for both 6- and 7-positions of the B-ring. This antibody was photoaffinity-labeled with five (5-azido-2-nitrobenzoyl)amido (ANBA) derivatives of [17alpha-(3)H]estradiol, synthesized from 3-aminoethyloxy, 3-(aminoethylamido)carboxymethyloxy, 6alpha- and 6beta-amino, and 7-[O-(aminoethylamido)carboxymethyl]oximino precursors. After tryptic digestion, the radioactive peptides on L and H chains were immunopurified with the immobilized antibody 9D3, separated by reversed-phase liquid chromatography, sequenced, and characterized by mass spectrometry, including post-source decay-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The long 3-(ANBA-ethylamido)carboxymethyl ether photoreagent was found to label TyrL-32 (on CDR L1), whereas no labeling was observed with the shorter 3-derivative, a result in agreement with a binding pocket large enough to explain the high cross-reactivity with estradiol 3-conjugates. The two 6alpha- and 6beta-ANBA-estradiol isomers, as well as the 7-[O-(ANBA-ethylamido)carboxymethyl]oximinoestradiol photoreagent derived from the steroid hapten, labeled the same TyrL-32 residue. The 6beta-ANBA epimer also labeled TyrH-50 (at the basis of CDR H2). These experiments indicate that TyrL-32 is freely accessible from the three C3, C6, and C7 positions, all presumed to be exposed to solvent, while TyrH-50 is probably located on the beta-face of estradiol. These results, obtained in solution, provide experimental data useful for molecular modeling of the steroid-antibody complex.

Photoaffinity labeling of homologous Met-133 and Met-139 amino acids of rabbit and sheep sex hormone-binding globulins with the unsubstituted Delta 6-testosterone photoreagent.

Purified rabbit and sheep sex hormone-binding globulins (SHBGs) were photolabeled by Delta 6-testosterone. The maximal levels of specific incorporation were respectively 0.33 and 0.30 mol of label/mol of homodimer. Tryptic cleavage of photolabeled SHBGs gave a single radioactive peptide for rabbit SHBG and two major radioactive peptides S1 and S2 for sheep SHBG. Edman sequencing of the photolabeled peptide of rabbit SHBG revealed a single sequence corresponding to peptidic fragment Leu-118-Lys-134. Subcleavage of this peptide with elastase led to a single radioactive peptidic fragment corresponding to dipeptide Met-133-Lys-134, identified by mass spectrometry, while deletion of the C-terminal residue with carboxypeptidase B showed that all the radioactivity remained on peptide Leu-118-Met-133, thus demonstrating that photolabeling occurred exclusively on Met-133, the only residue common to the two radioactive subcleaved peptides. Edman sequencing of peptides S1 and S2 of sheep SHBG showed a same single sequence corresponding to residues Gln-126-Arg-140 which contained no identifiable phenylthiohydantoin derivative at cycle 14, thus indicating that in both cases the corresponding Met-139 residue is the main site of photolabeling, as confirmed for peptide S1 by the presence at this cycle of a major peak of radioactivity while in peptide S2 the photoattachment of Delta 6-testosterone was found labile in the conditions of sequencing. The photolabeled peptide S1 was characterized by mass spectrometry which showed the covalent fixation of one mole of Delta 6-testosterone and the presence of a biantennary oligosaccharide attached at Asn-133, which suggests that the steroid-binding site is probably not deeply buried in the SHBG homodimer.

Specific photoaffinity labeling of Tyr-49 on the light chain in the steroid-combining site of a mouse monoclonal anti-estradiol antibody using two epimeric 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amidoestradiol photoreagents.

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody was photoaffinity labeled with two cross-reactive 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amido[17alpha-3H]estradiol photoreagents (6alpha- and 6beta-ANBA-[17alpha-3H]estradiol). Covalently bound radioactivity was found exclusively on the light chain. The maximal level of specific incorporation was 0.18 mol of label per mole of antibody for both photoreagents. In both cases, tryptic digestion of the photolabeled light chain, immunopurification with the immobilized antibody, reverse-phase liquid chromatography, and Edman degradation showed the presence of radioactive peptide GLM-([3H]X)-HGNTLEDGIPSR derived from peptide 46-61 of the light chain sequence (determined from cDNA) in which the unidentified amino acid corresponding to X is a Tyr residue. Two other radioactive peptides were also isolated, one corresponding probably to the methionine sulfoxide derivative of the peptide 46-61 photolabeled with the 6beta-reagent and the other to the N-terminal tetrapeptide 46-49 of the peptide 46-61 photolabeled with the 6alpha-reagent. In all cases, the main peak of radioactivity was released at the fourth Edman cycle, thus suggesting that the same Tyr-49 residue on the light chain was photolabeled. This residue is contiguous to the N-terminal amino acid of the second hypervariable complementary determining region 50-56 of light chain. Covalent labeling was confirmed by mass spectrometry of photolabeled peptides which showed molecular ion values corresponding to the addition of the photoactive 6alpha- or 6beta-ANBA-estradiol nitrene derivatives to the peptide.

Identification of Trp-371 as the main site of specific photoaffinity labeling of corticosteroid binding globulin using delta 6 derivatives of cortisol, corticosterone, and progesterone as unsubstituted photoreagents.

Immunopurified human corticosteroid binding globulin (CBG) was photolabeled with delta 6-[3H]cortisol, delta 6-[4-14C]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone. The maximal levels of specific incorporation, as estimated with tritiated photoreagents, were 0.21, 0.14, and 0.08 mol of label/mol of CBG, respectively. Tryptic cleavage of photolabeled CBG gave in all cases a major radioactive peptide that was no longer detectable when a 100-fold molar excess of cortisol was added to the photoreagents. Edman sequencing of tryptic peptides photolabeled with delta 6-[3H]cortisol or delta 6-[3H]corticosterone showed that these peptides correspond to residues 357-378 of the human CBG sequence. The major peak of radioactivity of these peptides was eluted at the 15th cycle (Trp-371). The radioactive tryptic peptides photolabeled with the four steroid photoreagents were subcleaved with alpha-chymotrypsin. The major part of radioactivity was recovered in the T-[*X]-S-S-L-F hexapeptide 370-375 (major peptide) and in the D-H-F-T-[*X]-S-S-L-F nonapeptide 367-375, at the second and fifth Edman cycles, respectively, whereas no PTH derivative could be identified at these cycles, thus suggesting Trp-371 as the main site of photolabeling for all tested photoreagents. Mass spectrometry of tryptic peptides photolabeled with delta 6-[3H]cortisol and delta 6-[3H]corticosterone and of chymotryptic peptides photolabeled with delta 6-[3H]cortisol, delta 6-[3H]corticosterone, and delta 6-[3H]progesterone showed molecular masses corresponding to the addition of delta 6-steroid photoreagents to the peptide.

Characterization of Met-139 as the photolabeled amino acid residue in the steroid binding site of sex hormone binding globulin using delta 6 derivatives of either testosterone or estradiol as unsubstituted photoaffinity labeling reagents.

Immunopurified human sex hormone binding globulin (SHBG) was photoinactivated and photolabeled by radioinert and radioactive photoaffinity labeling steroids delta 6-testosterone (delta 6-T) and delta 6-estradiol (delta 6-E2). The maximal levels of specific incorporation of these two reagents were 0.50 and 0.33 mol of label/mol of SHBG, respectively. Covalently labeled SHBG fractions were citraconylated, reduced, carboxymethylated, and cleaved by trypsin. Separation of tryptic digests by reverse-phase liquid chromatography gave single radioactive peaks at the same retention times with both steroid reagents. However, the two labeled peptidic fractions could be distinguished by capillary electrophoresis and immunodetection with anti-steroid antibodies, whereas the covalent attachment of radioactivity was confirmed by thin-layer chromatography on silica gel. Edman degradation of the two labeled peptides showed a single sequence His-Pro-Ile-([3H]X)-Arg corresponding to the pentapeptide His-Pro-Ile-Met-Arg 136-140 of SHBG sequence. The coincidence, in both cases, of the absence of an identifiable amino acid residue and of the elution of the most intense peak of radioactivity at the fourth cycle of Edman degradation suggests that the same Met-139 residue was labeled by delta 6-[1,2-3H2]T or by delta 6-[17 alpha-3H]E2. Liquid secondary ion mass spectrometry of the two peptides showed [M+H]+ ions at m/z 939.8 or 923.8, corresponding respectively to the addition of delta 6-T or delta 6-E2 to the pentapeptide. The presence of the steroid molecule in the delta 6-[3H]T-pentapeptide conjugate was confirmed by the difference of 2 mass units with the [M+H]+ peak of the delta 6-[4-14C]T-pentapeptide conjugate.

[Identification of a photolabelled site of the plasma binding protein for testosterone and estradiol (SBP) using tritiated 17 beta-hydroxy- 4,6-androstadien-3-one]. / Identification d'un site de photomarquage de la protéine plasmatique de liaison de la testostérone et de l'oestradiol (SBP) par l'hydroxy-17 beta oxo-3 androstadiène-4,6 tritié.

The testosterone-estradiol binding protein (sex binding protein = SBP), immunopurified from human placental blood, was photolabelled by irradiation at lambda greater than 300 mm in the presence of tritiated 17 beta-hydroxy-androsta-4,6-dien-3-one. High-performance reverse-phase liquid chromatography of tryptic peptides, showed two main peaks of radioactivity. Sequence determination of these two fractions indicated that the radioactivity was associated with an undetectable amino-acid preceded either by the sequence His-Pro-Ile (major peak) or Arg-His-Pro-Ile at the N-terminal site and bearing Arg as C-terminal amino-acid. Comparison with the sequence reported for human SBP (K.A. Walsh et al., Biochemistry, 25, 1986, pp. 7584-7590) suggested that radioactive labelling was localized on the Met-139 residue of the hexapeptide Arg-His-Pro-Ile-Met-Arg (fragment 135-140).