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Identification of X monosomy cells from a gonad of mixed gonadal dysgenesis with a 46,XY karyotype: case report.

Nishina-Uchida N, Fukuzawa R, Hasegawa Y, Morison IM - Medicine (Baltimore) (2015)

Bottom Line: Fluorescence in situ hybridization (FISH) for X and Y chromosomes and immunofluorescence for SRY along with testicular and ovarian lineage markers SOX9 and FOXL2, respectively, were performed on paraffin sections from the gonad to ascertain the somatic mosaic state for 45,X monosomy and 46,XY cells.SRY expression was absent in approximately 10% of precursor granulosa cells (FOXL2 positive) and precursor Sertoli/granulosa cells (both SOX9 and FOXL2 positive) within gonadoblastomas, confirming the involvement of 45,X cells.A combination of analysis of FISH and immunofluorescence for SRY in the gonadal tissue could identify 45,X cells in MGD with 46,XY.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular and Developmental Pathology Research Group (NN-U, RF); Division of Endocrinology and Metabolism (NN-U, YH); Department of Pathology and Laboratory Medicine, Tokyo Metropolitan Children's Medical Center, 2-8-29 Musashidai, Fuchu, Tokyo, Japan (RF); and Department of Pathology, Dunedin School of Medicine, University of Otago, P.O. Box 913, Dunedin, New Zealand (RF, IMM).

ABSTRACT
Mixed gonadal dysgenesis (MGD) is a disorder of sexual development that typically has a mosaic 45,X/46,XY karyotype. A 1-year-old infant with 46,XY identified by peripheral blood karyotype demonstrated clinical manifestations and gonadal pathologic features of MGD. Fluorescence in situ hybridization (FISH) for X and Y chromosomes and immunofluorescence for SRY along with testicular and ovarian lineage markers SOX9 and FOXL2, respectively, were performed on paraffin sections from the gonad to ascertain the somatic mosaic state for 45,X monosomy and 46,XY cells. The gonad consisted of cells with X and XY signals, which were further quantified in comparison with a normal control testis by a digital image analysis program. The average percentages of 45,X cells of this patient's gonad and a control testis were 39.0% and 5.7%, respectively (χ2 test, P < 0.001). SRY expression was absent in approximately 10% of precursor granulosa cells (FOXL2 positive) and precursor Sertoli/granulosa cells (both SOX9 and FOXL2 positive) within gonadoblastomas, confirming the involvement of 45,X cells. A combination of analysis of FISH and immunofluorescence for SRY in the gonadal tissue could identify 45,X cells in MGD with 46,XY.

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FISH and immunofluorescence analyses of the gonad. (A) The gonadal tissue is composed of 45,X and 46,XY cells. The inset highlights the presence of 45,X cells. Red and green signals denote X and Y probes, respectively. (B) Triple color immunofluorescence for SOX9 (testicular lineage marker: green), SRY (Y-chromosome marker: red), and FOXL2 (ovarian lineage marker: light blue) in a normal appearing seminiferous tubule, consisting of Sertoli cells (nuclear SOX9 signals) and germ cells (membranous/cytoplasmic SRY signals). FOXL2-positive cells are absent. (C) Dual color immunofluorescence for SOX9 (green) and FOXL2 (light blue) in a gonadoblastoma comprising pre-granulosa cells only expressing FOXL2 (light blue nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (nuclear emerald green signals indicated by arrowheads). (D) Immunofluorescence for SRY (red) showing that nuclear SRY is present in most pre-granulosa cells, and absent in a minority of pre-granulosa cells (surrounded by white dotted lines). Pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (see C) are also devoid of nuclear SRY signals (indicated by arrowheads in C, D, and E). These confirm the presence of 45,X cells. The membranous/cytoplasmic SRY signals indicate the localization of germ cells (indicated by arrows). (E) Triple color immunofluorescence: (C) SOX9 and FOXL2 merged with (D) SRY. The predominant population of sex cord epithelial cells expresses both FOXL2 and SRY (purple nuclei) indicating pre-granulosa cells with a 46,XY karyotype. It is conceivable that a minority of pre-granulosa cells have a 45,X karyotype (surrounded by white dotted lines) because of lack of SRY expression. Some pre-Sertoli/granulosa cells also have a 45,X karyotype (arrow heads), because they express both SOX9 and FOXL2 but lack SRY expression (nuclear emerald green signals). (F) Dual color immunofluorescence for SOX9 and FOXL2 demonstrating abnormally shaped seminiferous tubules composed of mature Sertoli cells only expressing SOX9 (green nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (emerald-green nuclei surrounded by white dashed lines). An aggregate of germ cells is surrounded by a yellow dotted line. (G) Immunofluorescence for SRY reveals that the cells expressing FOXL2 have nuclear SRY positivity (red nuclear signals surrounded by white dashed lines). The membranous/cytoplasmic SRY immunoreactivity clarifies an aggregate of germ cells (surrounded by yellow dotted lines). (H) Triple color immunofluorescence: merged SOX9 and FOXL2 (F) with SRY (G). The presence of Sertoli cells with SOX9 expression (green nuclei) and co-localization of SRY signals in the nuclei of pre-Sertoli/granulosa cells with both SOX9 and FOXL2 expression (nuclear white, pink or light green nuclei surrounded by white dashed lines) indicate that the abnormally shaped seminiferous tubule is a male structure, showing incomplete testicular differentiation. FISH = fluorescence in situ hybridization.
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Figure 2: FISH and immunofluorescence analyses of the gonad. (A) The gonadal tissue is composed of 45,X and 46,XY cells. The inset highlights the presence of 45,X cells. Red and green signals denote X and Y probes, respectively. (B) Triple color immunofluorescence for SOX9 (testicular lineage marker: green), SRY (Y-chromosome marker: red), and FOXL2 (ovarian lineage marker: light blue) in a normal appearing seminiferous tubule, consisting of Sertoli cells (nuclear SOX9 signals) and germ cells (membranous/cytoplasmic SRY signals). FOXL2-positive cells are absent. (C) Dual color immunofluorescence for SOX9 (green) and FOXL2 (light blue) in a gonadoblastoma comprising pre-granulosa cells only expressing FOXL2 (light blue nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (nuclear emerald green signals indicated by arrowheads). (D) Immunofluorescence for SRY (red) showing that nuclear SRY is present in most pre-granulosa cells, and absent in a minority of pre-granulosa cells (surrounded by white dotted lines). Pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (see C) are also devoid of nuclear SRY signals (indicated by arrowheads in C, D, and E). These confirm the presence of 45,X cells. The membranous/cytoplasmic SRY signals indicate the localization of germ cells (indicated by arrows). (E) Triple color immunofluorescence: (C) SOX9 and FOXL2 merged with (D) SRY. The predominant population of sex cord epithelial cells expresses both FOXL2 and SRY (purple nuclei) indicating pre-granulosa cells with a 46,XY karyotype. It is conceivable that a minority of pre-granulosa cells have a 45,X karyotype (surrounded by white dotted lines) because of lack of SRY expression. Some pre-Sertoli/granulosa cells also have a 45,X karyotype (arrow heads), because they express both SOX9 and FOXL2 but lack SRY expression (nuclear emerald green signals). (F) Dual color immunofluorescence for SOX9 and FOXL2 demonstrating abnormally shaped seminiferous tubules composed of mature Sertoli cells only expressing SOX9 (green nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (emerald-green nuclei surrounded by white dashed lines). An aggregate of germ cells is surrounded by a yellow dotted line. (G) Immunofluorescence for SRY reveals that the cells expressing FOXL2 have nuclear SRY positivity (red nuclear signals surrounded by white dashed lines). The membranous/cytoplasmic SRY immunoreactivity clarifies an aggregate of germ cells (surrounded by yellow dotted lines). (H) Triple color immunofluorescence: merged SOX9 and FOXL2 (F) with SRY (G). The presence of Sertoli cells with SOX9 expression (green nuclei) and co-localization of SRY signals in the nuclei of pre-Sertoli/granulosa cells with both SOX9 and FOXL2 expression (nuclear white, pink or light green nuclei surrounded by white dashed lines) indicate that the abnormally shaped seminiferous tubule is a male structure, showing incomplete testicular differentiation. FISH = fluorescence in situ hybridization.

Mentions: A 1-year-old infant was referred to our hospital for gonadectomy and clitoroplasty. The patient was noted to have ambiguous genitalia with clitorimegaly soon after birth (Figure 1A and B). The right gonad was palpable in the labia majora, and the left one was not. The karyotype of peripheral lymphocytes was 46,XY and fluorescence in situ hybridization (FISH) for the SRY gene was positive. Ultrasound and magnetic resonance imaging studies revealed a hypoplastic uterus and an undescended gonad in the right hand side of the labia majora. At operation, the right gonad was a normal-appearing testis having the epididymis and deferent duct, whereas the left gonad resembled a streak gonad. Thus, gonadectomy was performed only for the right gonad. Histology of the gonad showed a streak testis consisting predominantly of differentiated normal-appearing seminiferous tubules with an area of undifferentiated gonadal tissue (UGT) and abnormally shaped seminiferous tubules at the periphery of the gonad (Figure 1C, Figure 2B–H). Nests of gonadoblastomas were present within the UGT (Figure 2C–E). The abnormally shaped seminiferous tubules consisted of focally proliferating germ cells, Sertoli cells, and pre-Sertoli/granulosa cells occasionally forming a cribriform arrangement, resembling a gonadoblastoma or an intratubular germ cell neoplasm (Figure 2F–H). Tissues derived from both Wolffian and Müllerian ducts were involved (Figure 1C). The clinical and histopathological characteristics were consistent with MGD.


Identification of X monosomy cells from a gonad of mixed gonadal dysgenesis with a 46,XY karyotype: case report.

Nishina-Uchida N, Fukuzawa R, Hasegawa Y, Morison IM - Medicine (Baltimore) (2015)

FISH and immunofluorescence analyses of the gonad. (A) The gonadal tissue is composed of 45,X and 46,XY cells. The inset highlights the presence of 45,X cells. Red and green signals denote X and Y probes, respectively. (B) Triple color immunofluorescence for SOX9 (testicular lineage marker: green), SRY (Y-chromosome marker: red), and FOXL2 (ovarian lineage marker: light blue) in a normal appearing seminiferous tubule, consisting of Sertoli cells (nuclear SOX9 signals) and germ cells (membranous/cytoplasmic SRY signals). FOXL2-positive cells are absent. (C) Dual color immunofluorescence for SOX9 (green) and FOXL2 (light blue) in a gonadoblastoma comprising pre-granulosa cells only expressing FOXL2 (light blue nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (nuclear emerald green signals indicated by arrowheads). (D) Immunofluorescence for SRY (red) showing that nuclear SRY is present in most pre-granulosa cells, and absent in a minority of pre-granulosa cells (surrounded by white dotted lines). Pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (see C) are also devoid of nuclear SRY signals (indicated by arrowheads in C, D, and E). These confirm the presence of 45,X cells. The membranous/cytoplasmic SRY signals indicate the localization of germ cells (indicated by arrows). (E) Triple color immunofluorescence: (C) SOX9 and FOXL2 merged with (D) SRY. The predominant population of sex cord epithelial cells expresses both FOXL2 and SRY (purple nuclei) indicating pre-granulosa cells with a 46,XY karyotype. It is conceivable that a minority of pre-granulosa cells have a 45,X karyotype (surrounded by white dotted lines) because of lack of SRY expression. Some pre-Sertoli/granulosa cells also have a 45,X karyotype (arrow heads), because they express both SOX9 and FOXL2 but lack SRY expression (nuclear emerald green signals). (F) Dual color immunofluorescence for SOX9 and FOXL2 demonstrating abnormally shaped seminiferous tubules composed of mature Sertoli cells only expressing SOX9 (green nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (emerald-green nuclei surrounded by white dashed lines). An aggregate of germ cells is surrounded by a yellow dotted line. (G) Immunofluorescence for SRY reveals that the cells expressing FOXL2 have nuclear SRY positivity (red nuclear signals surrounded by white dashed lines). The membranous/cytoplasmic SRY immunoreactivity clarifies an aggregate of germ cells (surrounded by yellow dotted lines). (H) Triple color immunofluorescence: merged SOX9 and FOXL2 (F) with SRY (G). The presence of Sertoli cells with SOX9 expression (green nuclei) and co-localization of SRY signals in the nuclei of pre-Sertoli/granulosa cells with both SOX9 and FOXL2 expression (nuclear white, pink or light green nuclei surrounded by white dashed lines) indicate that the abnormally shaped seminiferous tubule is a male structure, showing incomplete testicular differentiation. FISH = fluorescence in situ hybridization.
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Figure 2: FISH and immunofluorescence analyses of the gonad. (A) The gonadal tissue is composed of 45,X and 46,XY cells. The inset highlights the presence of 45,X cells. Red and green signals denote X and Y probes, respectively. (B) Triple color immunofluorescence for SOX9 (testicular lineage marker: green), SRY (Y-chromosome marker: red), and FOXL2 (ovarian lineage marker: light blue) in a normal appearing seminiferous tubule, consisting of Sertoli cells (nuclear SOX9 signals) and germ cells (membranous/cytoplasmic SRY signals). FOXL2-positive cells are absent. (C) Dual color immunofluorescence for SOX9 (green) and FOXL2 (light blue) in a gonadoblastoma comprising pre-granulosa cells only expressing FOXL2 (light blue nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (nuclear emerald green signals indicated by arrowheads). (D) Immunofluorescence for SRY (red) showing that nuclear SRY is present in most pre-granulosa cells, and absent in a minority of pre-granulosa cells (surrounded by white dotted lines). Pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (see C) are also devoid of nuclear SRY signals (indicated by arrowheads in C, D, and E). These confirm the presence of 45,X cells. The membranous/cytoplasmic SRY signals indicate the localization of germ cells (indicated by arrows). (E) Triple color immunofluorescence: (C) SOX9 and FOXL2 merged with (D) SRY. The predominant population of sex cord epithelial cells expresses both FOXL2 and SRY (purple nuclei) indicating pre-granulosa cells with a 46,XY karyotype. It is conceivable that a minority of pre-granulosa cells have a 45,X karyotype (surrounded by white dotted lines) because of lack of SRY expression. Some pre-Sertoli/granulosa cells also have a 45,X karyotype (arrow heads), because they express both SOX9 and FOXL2 but lack SRY expression (nuclear emerald green signals). (F) Dual color immunofluorescence for SOX9 and FOXL2 demonstrating abnormally shaped seminiferous tubules composed of mature Sertoli cells only expressing SOX9 (green nuclei) and pre-Sertoli/granulosa cells expressing both SOX9 and FOXL2 (emerald-green nuclei surrounded by white dashed lines). An aggregate of germ cells is surrounded by a yellow dotted line. (G) Immunofluorescence for SRY reveals that the cells expressing FOXL2 have nuclear SRY positivity (red nuclear signals surrounded by white dashed lines). The membranous/cytoplasmic SRY immunoreactivity clarifies an aggregate of germ cells (surrounded by yellow dotted lines). (H) Triple color immunofluorescence: merged SOX9 and FOXL2 (F) with SRY (G). The presence of Sertoli cells with SOX9 expression (green nuclei) and co-localization of SRY signals in the nuclei of pre-Sertoli/granulosa cells with both SOX9 and FOXL2 expression (nuclear white, pink or light green nuclei surrounded by white dashed lines) indicate that the abnormally shaped seminiferous tubule is a male structure, showing incomplete testicular differentiation. FISH = fluorescence in situ hybridization.
Mentions: A 1-year-old infant was referred to our hospital for gonadectomy and clitoroplasty. The patient was noted to have ambiguous genitalia with clitorimegaly soon after birth (Figure 1A and B). The right gonad was palpable in the labia majora, and the left one was not. The karyotype of peripheral lymphocytes was 46,XY and fluorescence in situ hybridization (FISH) for the SRY gene was positive. Ultrasound and magnetic resonance imaging studies revealed a hypoplastic uterus and an undescended gonad in the right hand side of the labia majora. At operation, the right gonad was a normal-appearing testis having the epididymis and deferent duct, whereas the left gonad resembled a streak gonad. Thus, gonadectomy was performed only for the right gonad. Histology of the gonad showed a streak testis consisting predominantly of differentiated normal-appearing seminiferous tubules with an area of undifferentiated gonadal tissue (UGT) and abnormally shaped seminiferous tubules at the periphery of the gonad (Figure 1C, Figure 2B–H). Nests of gonadoblastomas were present within the UGT (Figure 2C–E). The abnormally shaped seminiferous tubules consisted of focally proliferating germ cells, Sertoli cells, and pre-Sertoli/granulosa cells occasionally forming a cribriform arrangement, resembling a gonadoblastoma or an intratubular germ cell neoplasm (Figure 2F–H). Tissues derived from both Wolffian and Müllerian ducts were involved (Figure 1C). The clinical and histopathological characteristics were consistent with MGD.

Bottom Line: Fluorescence in situ hybridization (FISH) for X and Y chromosomes and immunofluorescence for SRY along with testicular and ovarian lineage markers SOX9 and FOXL2, respectively, were performed on paraffin sections from the gonad to ascertain the somatic mosaic state for 45,X monosomy and 46,XY cells.SRY expression was absent in approximately 10% of precursor granulosa cells (FOXL2 positive) and precursor Sertoli/granulosa cells (both SOX9 and FOXL2 positive) within gonadoblastomas, confirming the involvement of 45,X cells.A combination of analysis of FISH and immunofluorescence for SRY in the gonadal tissue could identify 45,X cells in MGD with 46,XY.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular and Developmental Pathology Research Group (NN-U, RF); Division of Endocrinology and Metabolism (NN-U, YH); Department of Pathology and Laboratory Medicine, Tokyo Metropolitan Children's Medical Center, 2-8-29 Musashidai, Fuchu, Tokyo, Japan (RF); and Department of Pathology, Dunedin School of Medicine, University of Otago, P.O. Box 913, Dunedin, New Zealand (RF, IMM).

ABSTRACT
Mixed gonadal dysgenesis (MGD) is a disorder of sexual development that typically has a mosaic 45,X/46,XY karyotype. A 1-year-old infant with 46,XY identified by peripheral blood karyotype demonstrated clinical manifestations and gonadal pathologic features of MGD. Fluorescence in situ hybridization (FISH) for X and Y chromosomes and immunofluorescence for SRY along with testicular and ovarian lineage markers SOX9 and FOXL2, respectively, were performed on paraffin sections from the gonad to ascertain the somatic mosaic state for 45,X monosomy and 46,XY cells. The gonad consisted of cells with X and XY signals, which were further quantified in comparison with a normal control testis by a digital image analysis program. The average percentages of 45,X cells of this patient's gonad and a control testis were 39.0% and 5.7%, respectively (χ2 test, P < 0.001). SRY expression was absent in approximately 10% of precursor granulosa cells (FOXL2 positive) and precursor Sertoli/granulosa cells (both SOX9 and FOXL2 positive) within gonadoblastomas, confirming the involvement of 45,X cells. A combination of analysis of FISH and immunofluorescence for SRY in the gonadal tissue could identify 45,X cells in MGD with 46,XY.

Show MeSH
Related in: MedlinePlus