Immunofluorescence Staining for Cytosine Modifications Like 5-Methylcytosine and Its Oxidative Derivatives and FISH

Pendina, Anna A.; Efimova, Olga A.; Tikhonov, Andrei V.; Chiryaeva, Olga G.; Федорова, И. М.; Koltsova, Alla S.; Krapivin, Mikhail I.; Parfenyev, Sergey; Kuznetzova, T. V.; Baranov, Vladislav S

doi:10.1007/978-3-662-52959-1_35

Cited by 12 publications

(12 citation statements)

References 13 publications

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“…To determine nuclei ploidy, FISH was performed on the testicular cytogenetic preparations according to standard procedures with minor modifications [ 65 ]. After aging at +55 °C overnight, the slides were dehydrated in ethanol series (70 %, 80 % and 95 %), washed in 4xSSC at +37 °C for 3 min, incubated in 0.01 % pepsin solution at +37 °C for 25 min and washed again in 4xSSC at +37 °C for 3 min.…”

Section: Methodsmentioning

confidence: 99%

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality

et al. 2017

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We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality – normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) – and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.

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Section: Methodsmentioning

confidence: 99%

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality

et al. 2017

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“…The peripheral blood lymphocytes were donated for the study by 11 healthy karyotypically normal volunteers aged 18–54 years. The lymphocytes were cultured for 72 h at +37 °C in the presence of phytohemagglutinin (PHA) (PanEco, Moscow, Russia) according to the standard protocol with minor modifications used in our laboratory [ 73 ].…”

Section: Methodsmentioning

confidence: 99%

“…For chromosome preparations from the tripronucleate zygotes, 5 µL of 0.1% colchicine (Merck) was added to the culture medium at the point of visualisation of three pronuclei. After the pronuclei had dissolved (i.e., in 6–12 h), the zygotes were fixed on glass slides as described previously [ 74 ], with minor modifications [ 65 , 73 ].…”

Section: Methodsmentioning

confidence: 99%

Telomere Length in Metaphase Chromosomes of Human Triploid Zygotes

Pendina

Krapivin

Efimova

et al. 2021

IJMS

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The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote—when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes’ parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived (paternal) chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived (maternal) ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain “imprinting” of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent’s age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual’s gametes with those in chromosomes inherited from different individuals’ gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual’s reaction norm.

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“…Prior to the start of an IVF/PGT-SR cycle, cytogenetic diagnoses in all of the translocation carriers were verified by conventional karyotyping followed by fluorescence in situ hybridization (FISH) if required. All conventional karyotyping and FISH procedures including PHA-stimulation of peripheral blood lymphocytes, preparation of metaphase chromosomes, QFH/AcD staining and hybridization were performed according to standard protocols with modifications repeatedly used in our previous studies [ 6 , 7 , 8 , 9 , 10 ]. The final cytogenetic diagnoses are summarized in Table A1 .…”

Section: Methodsmentioning

confidence: 99%

Age and Serum AMH and FSH Levels as Predictors of the Number of Oocytes Retrieved from Chromosomal Translocation Carriers after Controlled Ovarian Hyperstimulation: Applicability and Limitations

Shilenkova

Pendina

Mekina

et al. 2020

Genes

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We studied the impact of age and the serum anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH) levels on the number of cumulus–oocyte complexes (COCs) retrieved from female reciprocal and Robertsonian translocation carriers after controlled ovarian hyperstimulation (COH). The number of COCs retrieved after COH was retrospectively analyzed in female translocation carriers and 46,XX partners of male translocation carriers from 100 couples. The median number of COCs varied from nine to 16 and did not differ among subgroups of women categorized by age, presence and type of a translocation. The number of COCs correlated negatively with the woman’s age in both the reciprocal and the Robertsonian translocation carriers, while in 46,XX women no correlation was detected. The number of COCs did not differ between the reciprocal and the Robertsonian translocation carriers aged either <35 or ≥35 years. In translocation carriers, the number of COCs correlated with the serum AMH level only in the younger-age subgroups; the correlation was strong positive in reciprocal and moderate positive in Robertsonian translocation carriers. The 46,XX women aged both <35 and ≥35 years showed similar moderate positive correlations. Across all subgroups, the number of COCs correlated moderately negatively with the serum FSH level only in Robertsonian translocation carriers aged <35 years. Our results suggest that chromosomal translocations per se do not increase the risk of poor oocyte retrieval outcome after COH. In translocation carriers, oocyte retrieval outcome depends to a large extent on their age. The serum AMH level strongly predicts oocyte retrieval outcomes only in young reciprocal translocation carriers, while the serum FSH level has a moderate predictive value in young Robertsonian translocation carriers.

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Immunofluorescence Staining for Cytosine Modifications Like 5-Methylcytosine and Its Oxidative Derivatives and FISH

Cited by 12 publications

References 13 publications

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality

Telomere Length in Metaphase Chromosomes of Human Triploid Zygotes

Age and Serum AMH and FSH Levels as Predictors of the Number of Oocytes Retrieved from Chromosomal Translocation Carriers after Controlled Ovarian Hyperstimulation: Applicability and Limitations

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