| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Division of Endocrinology, Departments of Medicine and Pediatrics (P.H.Y.), Harbor-University of California-Los Angeles Medical Center and Research and Education Institute, Torrance, California 90509; and Pathology and Laboratory Medicine, University of California School of Medicine (P.N.R.), Los Angeles, California 90095
Address all correspondence and request for reprints to: Ronald S. Swerdloff, M.D., Division of Endocrinology and Metabolism, Harbor-University of California-Los Angeles Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: swerdloff{at}gcrc.humc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bronson et al. (9) showed previously that male mice generated by mating the germline transmission chimeric female mice with wild-type male mice had a high incidence of sex chromosome aneuploidy, including XXY and XYY. The female chimeras were produced by pseudopregnant recipients bearing the transferred female embryos containing injected male embryonic stem cells. Evidence was provided showing that when chimeric females were bred with wild-type male mice, their female offspring were fertile; however, about 50% of their male offspring appeared to be sterile, resulting from the high frequency of sex chromosome aneuploidy. However, the phenotypic manifestations of XXY and XYY mice were not characterized (9). The objectives of the present study were to characterize an experimental murine model for the human genetic disorder, Klinefelter syndrome, to determine the testicular phenotype of adult XXY mice, and to examine the ontogeny of germ cell loss in juvenile XXY mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To characterize the testicular phenotype of adult XXY mice, 4 adult XXY mice (8 weeks old) and 4 littermate controls (XY) were used in Exp 1. To examine the ontogeny of germ cell loss, 14 juvenile mice (41,XXY), including 3 at 1 day, 4 at 3 days, 4 at 5 days, 2 at 7 days, and 1 at 10 days of age, and their littermate controls (40,XY) were studied in Exp 2.
Sample collection and tissue preparation
Exp 1. Adult XXY mice and their littermate controls were
injected with heparin (130 IU/100 g BW, ip) 15 min before being killed
by a lethal injection of sodium pentobarbital (100 mg/kg BW, ip) to
facilitate testicular perfusion using a whole body perfusion technique
(10, 11). Body weight was recorded at autopsy. Blood
samples were collected from the inferior vena cava of each animal
immediately after death, and plasma was separated and stored at -20 C
for subsequent hormone assays. The testes were then fixed by vascular
perfusion with 5% glutaraldehyde in 0.05 M cacodylate
buffer (pH 7.4) for 30 min, preceded by a brief saline wash. The testes
were removed, cut into small transverse slices, and placed into the
same fixative overnight. One slice from the middle region of the testis
was processed for routine paraffin embedding. The adjacent testicular
slice was further diced into small pieces (1 x 2 x 2 mm),
postfixed in 1% osmium tetroxide/1.25% potassium ferro-cyanide
mixture, dehydrated in a graded series of ethanols, and embedded in
Arialdite. Embedded testicular specimens were sectioned with an
LKB ultramicrotome (Rockville, MD) at 2.05 µm and
stained with 1% toluidine blue for light microscopic examination and
for morphometric studies (12, 13). For electron
microscopic studies, thin sections from the selected tissue blocks
showing pale gold interference color were cut, stained with uranyl
acetate and lead citrate, and examined with a Hitachi
(Tokyo, Japan) 600 electron microscope at 75 kV (14).
Exp 2. Groups of male pups from chimeric female mice were killed by a lethal injection of sodium pentobarbital (100 mg/kg BW, ip) on day 1, 3, 5, 7, or 10 after birth. The testes were dissected out and immersed in Bouin’s solution overnight, then processed for routine paraffin embedding and sectioning for histological examination. In addition, two or three drops of blood were obtained from the jugular vein and smeared on glass slides for fluorescence in situ hybridization (FISH) detection of the sex chromosomes in interphase nuclei of the white blood cells. Livers were removed, snap-frozen in liquid nitrogen, and stored at -70 C for subsequent isolation of genomic DNA and Southern blot analysis.
Hormone assays
The testosterone concentrations in plasma were measured by RIA,
as reported previously (15). The minimal detection limit
in the assay was 0.25 ng/ml. The intra- and interassay coefficients of
variations were 8% and 11%, respectively. Plasma FSH levels were
measured by RIA, using reagents provided by the NIDDK, as previously
described (15). The minimal detection limit in the assay
was 0.4 ng/ml. The intra- and interassay coefficients of variations
were 11% and 15%, respectively. Plasma LH levels were measured by an
immunofluorometric assay (DELFIA Rat LH, Perkin-Elmer Corp., Turku, Finland), as previously described
(16). The minimal detection limit in the assay was 0.03
ng/ml. The intra- and interassay coefficients of variation were 6% and
8%, respectively.
Morphometric assessment of Leydig cells in adult mice
The volume and number of Leydig cells on glutaraldehyde fixed,
epoxy-embedded, toluidine blue-stained testicular sections were
determined by an accepted stereological technique, as described
previously (17). In brief, measurements for the Leydig
cell volume were carried out at x1000 magnification. As the Leydig
cell nuclei are nearly spherical in the mouse, the volume of an
individual nucleus (Vn) was calculated from the mean diameter of the
nucleus using the equation Vn = 1/6 
D3.
The mean nuclear diameter (D) was obtained by direct measurement of its
largest cross-sectioned profiles in serial sections. The volume of an
average Leydig cell (Vc) was derived from its nuclear volume (Vn), as
determined above, and the volume density of the nucleus (Vvn) within
the cell using the equation Vc = Vn/Vvn. The Vvn was obtained by
dividing the number of points falling on the nuclei by the total number
of points lying over the Leydig cells (nucleus and cytoplasm). Point
counting was carried out using the same ocular grid containing 121
intersections. The numerical density of Leydig cells (Nv) was collected
by dividing the total volume density of the Leydig cells by the volume
of an individual Leydig cell. The total volume density of Leydig cells
per testis was obtained by point counting at x400 magnification. The
absolute number of Leydig cells per testis was determined by
multiplying the number of Leydig cells per unit volume of the testis by
the testis volume.
Morphometric assessment of germ cells in juvenile mice
The method used for germ cell quantitation was similar to that
described previously (18). In brief, testicular sections
were examined with an American Optical Microscope (Scientific
Instruments, Buffalo, NY) with a x40 objective and a x10 eyepiece. A
square grid fitted within the eyepiece provided a reference area of
62,500 µm2. Germ cells within the frame of grid
were counted. To correct for shrinkage (if any) in the reference area,
the diameters of 10 randomly selected transverse sections of the
seminiferous tubules were measured from each of the XY and XXY animals
across the minor axes of their cross-sectioned profiles
(14). As there was no significant change in tubule
diameter between juvenile XY (49.7 ± 2.08 µm) and XXY (48
± 3.17 µm) mice, no correction factor was used for this study.
Fibroblast culture and karyotype analysis
Standard karyotyping was performed on cultured fibroblasts
obtained from ear-clips. Briefly, a 1- to 2-mm2
section of tissue was dissected from a piece of ear in a sterile
manner. The sample was minced with a scalpel and digested with
collagenase for 1 h. The dispersed cells were suspended in
Amino-max (Oncor, Inc., Gaithersburg, MD) medium supplemented
with 5% FBS. Amino-max supports the growth of anchorage-dependent
fibroblast cells. The cells were placed on glass coverslips and
cultured for 6–10 days at 37 C in a CO2
incubator. The coverslips were harvested following the in
situ method of harvesting after appropriate colony formation was
observed. The coverslips with cells were mounted on clean glass slides,
air-dried, and Giemsa-trypsin G-banded following standard cytogenetics
protocols. Ten to 15 cells from each specimen were analyzed.
FISH
X chromosome painting was carried out in cultured fibroblast in
metaphase. After the harvest of fibroblasts, the slides were aged at 4
C for 2–3 days. FISH with mouse chromosome X-paint probe (Oncor, Inc.)
was performed following the protocol provided by the manufacturer.
After dehydration by serial ethanol washing, air-drying, and denaturing
by incubation in 70% formamide/2 x SSC (standard saline citrate)
at 72 C for 2 min, 10 µl denatured X-paint probe labeled with
digoxygenin were applied to the slides and hybridized overnight at 37 C
in a humidified chamber. Sixty microliters of Texas red-labeled
antidigoxygenin antibody were added to the slides and incubated under a
plastic coverslip for 10 min at 37 C. After washing slides in 1 x
phosphate-buffered detergent (PBD), the chromosomes were stained
with 4,6-diamidino-2-phenylindole (DAPI) red signal from the
probe was visualized on a fluorescence microscope equipped with the
appropriate filters.
Identifying specific mouse sequences in the X and Y chromosomes in interphase nuclei by FISH was performed using blood smear. Slides with blood smears were air-dried for 4–6 days. Slides were immersed in 0.4% KCl solution for 15 min and then put in fixative (3:1 methanol:acetic acid) for 3 min. The FISH procedure was performed following the protocol provided by the manufacturer. Briefly, 10 µl biotin-labeled mouse chromosome X probe (AGL, Inc., Melbourne, FL) or 10 µl digoxygenin-labeled mouse chromosome Y probe (Oncor) were applied to the slides and hybridized overnight at 37 C in a humidified chamber. Sixty microliters of Texas Red-labeled avidin or 60 µl fluorescein isothiocyanate-labeled antidigoxygenin antibody were applied to the slides and incubated under a plastic coverslip for 10 min at 37 C. After washing slides in 1 x PBD, the chromosomes were stained with DAPI. The red dots (X chromosome) or a green dot (Y chromosome) within the nuclei of white blood cells were visualized on a fluorescence microscope equipped with triple filter (19).
Southern blotting
Genomic DNA was isolated from mouse liver (Easy-DNA Kit/Genomic
DNA isolation, Invitrogen, Carlsbad, CA) and digested with
EcoRI enzyme. Restriction digestion was carried out for 3–6
h in a volume of 50 µl containing 20–30 U EcoRI and 10
µg genomic DNA (20). The DNA fragments were subjected to
electrophoresis on a 1% agarose gel and were then transferred to a
nylon membrane. The nylon membrane was dried under vacuum for 1 h
at 80 C. DNA probes (human ZFY gene coding sequence) were
nick-translated to a specific activity of 2–3 x
108 cpm/µg with
[32P]deoxy-CTP in the reaction mixture. Nylon
membranes were prehybridized for approximately 1 h at 65 C in
5 x SSC, 5 x Denhardt’s reagent, and 250 µg/ml
heat-denatured salmon DNA and then for 3 h in 5 x SSC,
1 x Denhardt’s reagent, 10% dextran sulfate, 250 µg/ml
heat-denatured salmon DNA, and 0.1% SDS.
32P-Labeled probe at 2–3 x
106 cpm/ml was added, and the incubation was
continued overnight. At the end of hybridization, the nylon membrane
was washed four times with 50 mM Tris, 1
mM EDTA, 1 x Denhardt’s solution, 0.1%
SDS, and 0.1% sodium pyrophosphate (pH 8.0) over a 1-h period. The
nylon membrane was dried briefly in a vacuum oven and exposed to
prefogged Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) at -70 C with a DuPont lightning
intensifier screen (DuPont Merck Pharmaceutical Co.,
Wilmington, DE).
Statistical analysis
Statistical analyses were performed using the SigmaStat 2.0
Program (Jandel Corp., San Rafael, CA). Results were tested for
statistical significance using the Student-Newman-Keuls methods test
after one-way repeated measures ANOVA. Differences were considered
significant at P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) using cultured fibroblasts, and this
was further confirmed by X chromosome painting or chromosome X-specific
subcentromere labeling using the FISH technique (Fig. 1B).
|
) and testis weight (Fig. 2B) between
adult XY and XXY mice are shown in Fig. 2. Although no difference was
observed in body weight, the weight of the testis in XXY mice was
reduced to 20% of that in XY mice. Due to the large variation among
animals, there was no statistically significant difference in the mean
levels of plasma testosterone between adult XXY and XY mice. However,
the mean levels of LH and FSH in adult XXY mice were significantly
increased compared with those in the controls (Table 1).
|
|
), the testicular
histological examination of adult XXY mouse showed a complete cessation
of spermatogenesis (Fig. 3B). The seminiferous tubules consist of
Sertoli cells, and there were no germ cells. Leydig cells appeared to
be more abundant. Electron microscopic examination showed Sertoli cells
of XXY males containing scanty amounts of cytoplasm and irregular
nuclei with prominent nucleoli (Fig. 4B)
compared with the controls (Fig. 4A). In some tubules, nests of
apparently degenerating Sertoli cells were found in XXY mice (Fig. 4C).
These cells were rounded in appearance and detached from the basal
lamina. Their nuclei were highly irregular and contained abnormal
chromatin clumps along the nuclear envelope. Such nests of apparently
degenerating Sertoli cells have been reported in mice after ethanol
treatment (21) and in the highly involuted tubules of sys
(symplastic spermatids) mice (22). Leydig cells from XXY
mouse displayed ultrastructural evidence of cellular hypertrophy (Fig. 4D), including the appearance of large systems of concentric cisternae
of smooth endoplasmic reticulum similar to those described previously
in hypertrophied murine Leydig cells (23).
|
|
. Comparing to the control (XY)
value (217.2 ± 7.17 µm), the diameter of seminiferous tubules
was dramatically decreased in the adult XXY mice (135.2 ± 0.33
µm). On the contrary, in XXY mice, the volume of individual Leydig
cells was increased by 1.4-fold (Fig. 5A), and the number of Leydig
cells was increased by 7-fold compared with the values measured in XY
males (Fig. 5B), respectively.
|
). In normal male mice, which have one
pair of chromosome 10 and a single X chromosome, one dark up-band (Zfa)
and one light lower band (Zfx) were observed. As expected, two bands of
similar intensity were observed in normal XX female mice. As all the
animals used in this study are phenotypic males as determined by the
presence of testes, mice that showed two bands with similar intensity
on the Southern blots apparently had a XXY karyotype. The XXY mice
identified by Southern blot were further confirmed by FISH analysis
using mouse X and Y chromosome- specific probes.
|
shows the morphological appearance of the seminiferous tubules of XY
(Fig. 7, A and C) and XXY (Fig. 7, B and D) mice at 7 and 10 days of
age. Compared with wild-type males, there were more degenerating germ
cells observed in the seminiferous tubules of 5- and 7-day-old XXY
mice. However, in 10-day-old mice, the number of germ cells was
significantly reduced compared with that in controls. Leydig cells
seemed to be normal in appearance in both XY and XXY mice at 10 days of
age.
|
. There was no significant difference in
the number of germ cells among 1-, 3-, and 5-day-old XY and XXY mice.
In contrast, there was a significant decrease in the number of germ
cells beginning with 7-day-old XXY mice compared with their littermate
controls. A further decrease in germ cell number was observed in
10-day-old XXY mice.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The testicular histology of patients with Klinefelter syndrome (47,XXY) appeared to be near normal during infancy, after which degenerative changes began to occur (6). We observed a similar progressive loss of germ cells in XXY mice beginning at 7 days of age. The progressive loss of germ cells ultimately results in the absence of germ cells in adult XXY mice. The exact cellular and molecular mechanisms that trigger germ cell loss in XXY mice remain to be defined. It is possible that the altered dosage of X-encoded genes in germ cells may accelerate germ cell loss during the postnatal period. The germ cell loss in XXY mice begins at a time when germ cells in normal mice reinitiate mitosis and before the onset of germ cell meiosis (30, 31). During this period spermatogonia are required to migrate from the central location to reach the periphery of the seminiferous cord to reinitiate proliferation. At the new location, spermatogonia encounter extracellular matrix factors in the basal lamina and are thought to mature into the first generation of type A spermatogonia (32, 33). Any germ cells that fail to move away from the central region of the seminiferous cord eventually degenerate (34). In this study we observed that most of the degenerated germ cells were located near the central region of the seminiferous cord, suggesting the defect in spermatogonial migration during the postnatal period in XXY mice. We do not know at present whether the defect in the XXY testis is intrinsic to germ cells or is due to the inability of the Sertoli cells in XXY testis to support normal germ cell development. Sertoli cells are known to support and nurture different classes of germ cells by producing a variety of paracrine factors that regulate germ cell proliferation, differentiation, and death. For example, Sertoli cell dysfunction could lead to increased germ cell degeneration in the testis (35). Although the relationship between germ cells and Sertoli cells in postnatal XXY testis is unknown, Hunt et al. (36) demonstrated that prenatal germ cell proliferation is impaired in vivo, but not in vitro, suggesting a defect in Sertoli cells and germ cell communication in the differentiating XXY testis. Using a different breeding scheme to generate XXY mice, these investigators have also shown that although defects in germ cell behavior are apparent during the prenatal period, the actual demise of XXY germ cells appears to occur in the first few days after birth (37, 38). Ongoing studies in our laboratory, using spermatogonia transplant technology (38, 39, 40), will provide evidence illuminating the interactions between XXY Sertoli cells and XY germ cells, or vice versa.
In this study we provided additional evidence to support the idea that in mammals, males with two X chromosomes are sterile due to degeneration of the germ cells before the onset of meiosis (36, 41). The molecular mechanisms induced by the extra X chromosome that lead to male sterility remain elusive. We hypothesize that aberrant X chromosome gene dosage may be responsible for both the progressive loss of germ cells and the hormonal abnormalities in XXY mice. There are 46 chromosomes in humans, but only 40 chromosomes in mice. In addition, there are differences in X chromosome structure between humans and mice (42, 43). In humans, the evolutionary makeup of X chromosome seems to be relatively simple, with 4 evolutionary strata preserved in order from the extremity of the long arm to the distal short arm where the pseudoautosomal region 1 is located (44, 45). In contrast, the mouse X chromosome has been scrambled, with its centromere now located at 1 extremity and regions of conservation intermixed with added regions (46). Comparative analysis of the human and mouse X chromosomes in sex chromosome aneuploidy (XXY) may provide interesting clues about the genes responsible for male sterility, as the majority of the genes on the human X chromosome are preserved or have their homologs on the mouse X chromosome. The XXY mice in this study were derived from female chimeras that had been injected with male ES cells at the blastocyst stage of early embryonic development. It is therefore feasible to modify the X chromosome of ES cells in vitro, such as deleting specific genes or regions by homologous recombination, and study its effects on the phenotype of the resultant XXY mice. Therefore, this mouse model initiates a powerful approach to examine the molecular mechanisms responsible for Klinefelter syndrome, the most common human sex chromosome aneuploidy. However, it is worth noting that there have been multiple evolutionary rearrangements between mouse and human X chromosomes; thus, any conclusions obtained from comparisons between species should be made with caution.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Supported by Grant RO1-28009 from the NICHHD, NIH.
Received September 8, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-leydigism and increased excretion of follicle-stimulating hormone.
J Clin Endocrinol 2:615–627
This article has been cited by other articles:
 |
|
 |
|
  Y. Lue, K. Erkkila, P. Y. Liu, K. Ma, C. Wang, A. S. Hikim, and R. S. Swerdloff Fate of Bone Marrow Stem Cells Transplanted into the Testis: Potential Implication for Men with Testicular Failure Am. J. Pathol., March 1, 2007; 170(3): 899 - 908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ishii, T. Tachiwana, A. Hoshino, N. Tsunekawa, R. Hiramatsu, S. Matoba, M. Kanai-Azuma, H. Kawakami, M. Kurohmaru, and Y. Kanai Potency of testicular somatic environment to support spermatogenesis in XX/Sry transgenic male mice Development, February 1, 2007; 134(3): 449 - 454. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Aksglaede, A. M. Wikstrom, E. R.-D. Meyts, L. Dunkel, N. E. Skakkebaek, and A. Juul Natural history of seminiferous tubule degeneration in Klinefelter syndrome Hum. Reprod. Update, January 1, 2006; 12(1): 39 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Lue, J. D. Jentsch, C. Wang, P. N. Rao, A. P. Sinha Hikim, W. Salameh, and R. S. Swerdloff XXY Mice Exhibit Gonadal and Behavioral Phenotypes Similar to Klinefelter Syndrome Endocrinology, September 1, 2005; 146(9): 4148 - 4154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Gonsalves, P. J Turek, P. N Schlegel, C. V Hopps, J. F. Weier, and R. A R. Pera Recombination in men with Klinefelter syndrome Reproduction, August 1, 2005; 130(2): 223 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Isotani, T. Nakanishi, S. Kobayashi, J. Lee, S. Chuma, N. Nakatsuji, F. Ishino, and M. Okabe Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX{leftrightarrow}XY chimeric testes PNAS, March 15, 2005; 102(11): 4039 - 4044. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Yan, L. Ma, K. H. Burns, and M. M. Matzuk Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice PNAS, May 18, 2004; 101(20): 7793 - 7798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Tachdjian, N. Frydman, N. Morichon-Delvallez, A. L. Du, R. Fanchin, M. Vekemans, and R. Frydman Reproductive genetic counselling in non-mosaic 47,XXY patients: implications for preimplantation or prenatal diagnosis: Case report and review Hum. Reprod., February 1, 2003; 18(2): 271 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-H. Lue, B. L. Lasley, L. S. Laughlin, R. S. Swerdloff, A. P. S. Hikim, A. Leung, J. W. Overstreet, and C. Wang Mild Testicular Hyperthermia Induces Profound Transitional Spermatogenic Suppression Through Increased Germ Cell Apoptosis in Adult Cynomolgus Monkeys (Macaca fascicularis)( J Androl, November 1, 2002; 23(6): 799 - 805. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
