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Genetic dosage and position effect of small supernumerary marker chromosome (sSMC) in human sperm nuclei in infertile male patient.

Olszewska M, Wanowska E, Kishore A, Huleyuk N, Georgiadis AP, Yatsenko AN, Mikula M, Zastavna D, Wiland E, Kurpisz M - Sci Rep (2015)

Bottom Line: The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat.Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC.This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Polish Academy of Sciences, Department of Reproductive Biology and Stem Cells, Strzeszynska 32, 60-479 Poznan, Poland.

ABSTRACT
Chromosomes occupy specific distinct areas in the nucleus of the sperm cell that may be altered in males with disrupted spermatogenesis. Here, we present alterations in the positioning of the human chromosomes 15, 18, X and Y between spermatozoa with the small supernumerary marker chromosome (sSMC; sSMC(+)) and spermatozoa with normal chromosome complement (sSMC(-)), for the first time described in the same ejaculate of an infertile, phenotypically normal male patient. Using classical and confocal fluorescent microscopy, the nuclear colocalization of chromosomes 15 and sSMC was analyzed. The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat. Analysis of meiotic segregation showed a 1:1 ratio of sSMC(+) to sSMC(-) spermatozoa, while evaluation of sperm aneuploidy status indicated an increased level of chromosome 13, 18, 21 and 22 disomy, up to 7 × (2.7 - 15.1). Sperm chromatin integrity assessment did not reveal any increase in deprotamination in the patient's sperm chromatin. Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC. This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient.

No MeSH data available.


Related in: MedlinePlus

A schematic model of the radial and confocal spatial analyzes.(a) Schematic representation of the radial measurement method of centromere localization, according to the model of Zalenskaya and Zalensky19, including (b) representative FISH image. Green point: centromere; D and H: distances from centromere; L and l: long and short axes of sperm nucleus; pink area: mirror image of the centromeres’ position. Results depicted in a coordinate system as the normalized means D/L ± SE for OX axis and H/L ± SE for OY axis with (c) hypothetically marked chromocenter areas (aggregations of centromeres), including their mirror images. (d) Scheme of distance measurement (μm) method between FISH signals within sperm nucleus. (e–g) Schematic model of confocal sperm nucleus divided into three concentric shells (e) according to the depth of the nucleus: 1 (‘cen’) the most inner/central shell, 2 (‘int’) intermediate area, 3 (‘per’) peripheral area near the nuclear lamina. FISH signals were assigned to a particular shell after analysis of the z-axis stacks (f); (each shell consisted of 1/3 of the total number of stacks obtained for each sperm nucleus) and analysis of the xy-axes view (g) of the sperm nucleus.
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f5: A schematic model of the radial and confocal spatial analyzes.(a) Schematic representation of the radial measurement method of centromere localization, according to the model of Zalenskaya and Zalensky19, including (b) representative FISH image. Green point: centromere; D and H: distances from centromere; L and l: long and short axes of sperm nucleus; pink area: mirror image of the centromeres’ position. Results depicted in a coordinate system as the normalized means D/L ± SE for OX axis and H/L ± SE for OY axis with (c) hypothetically marked chromocenter areas (aggregations of centromeres), including their mirror images. (d) Scheme of distance measurement (μm) method between FISH signals within sperm nucleus. (e–g) Schematic model of confocal sperm nucleus divided into three concentric shells (e) according to the depth of the nucleus: 1 (‘cen’) the most inner/central shell, 2 (‘int’) intermediate area, 3 (‘per’) peripheral area near the nuclear lamina. FISH signals were assigned to a particular shell after analysis of the z-axis stacks (f); (each shell consisted of 1/3 of the total number of stacks obtained for each sperm nucleus) and analysis of the xy-axes view (g) of the sperm nucleus.

Mentions: The localization of centromeres of chromosomes 15, 18, X, Y and sSMC, as well as of SMAD6 gene locus (15q22.31), was estimated with the radial evaluation technique, according to a method previously described19 and presented briefly in Fig. 5a–c. The following parameters were measured: L: length of the long axis (from tail attachment point to acrosome); l: short axis (at the widest part of the nucleus); and L/l: the ellipsoidal shape determining the decondensation ratio of the nucleus. The value of D/L indicated the location of the chromosome centromere with respect to the length of the sperm nucleus (the ‘tail-acrosome’ criterion; D: distance between the FISH signal and the sperm tail attachment). The H/L value indicated the location of the chromosome centromere in the depth of the nucleus (the ‘center-periphery’ criterion; H: distance between FISH signal and the L axis). The results were depicted on a coordinate system as the mean D/L ± SE for the OX axis and H/L ± SE for the OY axis. It is known that, on a microscopic slide, spermatozoa can obtain only two mirror positions, like a flipping coin. For each chromosome, about 100 FISH signals were analyzed, both for sperm cells with (sSMC+) for cells without the sSMC chromosome (sSMC−). A hierarchal Ward cluster analysis was also performed to check the aggregated localization of the chromosomes in the sSMC+ spermatozoa, as compared to the sSMC− cells.


Genetic dosage and position effect of small supernumerary marker chromosome (sSMC) in human sperm nuclei in infertile male patient.

Olszewska M, Wanowska E, Kishore A, Huleyuk N, Georgiadis AP, Yatsenko AN, Mikula M, Zastavna D, Wiland E, Kurpisz M - Sci Rep (2015)

A schematic model of the radial and confocal spatial analyzes.(a) Schematic representation of the radial measurement method of centromere localization, according to the model of Zalenskaya and Zalensky19, including (b) representative FISH image. Green point: centromere; D and H: distances from centromere; L and l: long and short axes of sperm nucleus; pink area: mirror image of the centromeres’ position. Results depicted in a coordinate system as the normalized means D/L ± SE for OX axis and H/L ± SE for OY axis with (c) hypothetically marked chromocenter areas (aggregations of centromeres), including their mirror images. (d) Scheme of distance measurement (μm) method between FISH signals within sperm nucleus. (e–g) Schematic model of confocal sperm nucleus divided into three concentric shells (e) according to the depth of the nucleus: 1 (‘cen’) the most inner/central shell, 2 (‘int’) intermediate area, 3 (‘per’) peripheral area near the nuclear lamina. FISH signals were assigned to a particular shell after analysis of the z-axis stacks (f); (each shell consisted of 1/3 of the total number of stacks obtained for each sperm nucleus) and analysis of the xy-axes view (g) of the sperm nucleus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663790&req=5

f5: A schematic model of the radial and confocal spatial analyzes.(a) Schematic representation of the radial measurement method of centromere localization, according to the model of Zalenskaya and Zalensky19, including (b) representative FISH image. Green point: centromere; D and H: distances from centromere; L and l: long and short axes of sperm nucleus; pink area: mirror image of the centromeres’ position. Results depicted in a coordinate system as the normalized means D/L ± SE for OX axis and H/L ± SE for OY axis with (c) hypothetically marked chromocenter areas (aggregations of centromeres), including their mirror images. (d) Scheme of distance measurement (μm) method between FISH signals within sperm nucleus. (e–g) Schematic model of confocal sperm nucleus divided into three concentric shells (e) according to the depth of the nucleus: 1 (‘cen’) the most inner/central shell, 2 (‘int’) intermediate area, 3 (‘per’) peripheral area near the nuclear lamina. FISH signals were assigned to a particular shell after analysis of the z-axis stacks (f); (each shell consisted of 1/3 of the total number of stacks obtained for each sperm nucleus) and analysis of the xy-axes view (g) of the sperm nucleus.
Mentions: The localization of centromeres of chromosomes 15, 18, X, Y and sSMC, as well as of SMAD6 gene locus (15q22.31), was estimated with the radial evaluation technique, according to a method previously described19 and presented briefly in Fig. 5a–c. The following parameters were measured: L: length of the long axis (from tail attachment point to acrosome); l: short axis (at the widest part of the nucleus); and L/l: the ellipsoidal shape determining the decondensation ratio of the nucleus. The value of D/L indicated the location of the chromosome centromere with respect to the length of the sperm nucleus (the ‘tail-acrosome’ criterion; D: distance between the FISH signal and the sperm tail attachment). The H/L value indicated the location of the chromosome centromere in the depth of the nucleus (the ‘center-periphery’ criterion; H: distance between FISH signal and the L axis). The results were depicted on a coordinate system as the mean D/L ± SE for the OX axis and H/L ± SE for the OY axis. It is known that, on a microscopic slide, spermatozoa can obtain only two mirror positions, like a flipping coin. For each chromosome, about 100 FISH signals were analyzed, both for sperm cells with (sSMC+) for cells without the sSMC chromosome (sSMC−). A hierarchal Ward cluster analysis was also performed to check the aggregated localization of the chromosomes in the sSMC+ spermatozoa, as compared to the sSMC− cells.

Bottom Line: The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat.Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC.This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Polish Academy of Sciences, Department of Reproductive Biology and Stem Cells, Strzeszynska 32, 60-479 Poznan, Poland.

ABSTRACT
Chromosomes occupy specific distinct areas in the nucleus of the sperm cell that may be altered in males with disrupted spermatogenesis. Here, we present alterations in the positioning of the human chromosomes 15, 18, X and Y between spermatozoa with the small supernumerary marker chromosome (sSMC; sSMC(+)) and spermatozoa with normal chromosome complement (sSMC(-)), for the first time described in the same ejaculate of an infertile, phenotypically normal male patient. Using classical and confocal fluorescent microscopy, the nuclear colocalization of chromosomes 15 and sSMC was analyzed. The molecular cytogenetic characteristics of sSMC delineated the karyotype as 47,XY,+der(15)(pter->p11.2::q11.1->q11.2::p11.2->pter)mat. Analysis of meiotic segregation showed a 1:1 ratio of sSMC(+) to sSMC(-) spermatozoa, while evaluation of sperm aneuploidy status indicated an increased level of chromosome 13, 18, 21 and 22 disomy, up to 7 × (2.7 - 15.1). Sperm chromatin integrity assessment did not reveal any increase in deprotamination in the patient's sperm chromatin. Importantly, we found significant repositioning of chromosomes X and Y towards the nuclear periphery, where both chromosomes were localized in close proximity to the sSMC. This suggests the possible influence of sSMC/XY colocalization on meiotic chromosome division, resulting in abnormal chromosome segregation, and leading to male infertility in the patient.

No MeSH data available.


Related in: MedlinePlus