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Mucopolysaccharidosis type VII (MPS VII) is a fatal, progressive, degenerative lysosomal storage disease caused by the deficiency of β-glucuronidase (GUSB; EC 3.2.1.31) activity necessary for the degradation of glycosaminoglycans by cleaving glucuronide moieties (1). MPS VII is inherited as an autosomal recessive trait and has been described in humans (1), dogs (2), mice (3), and cats (4, 5). The clinical, pathologic, and genetic features of MPS VII have been extensively described in mice (3, 6–10), and the disease is clinically and histopathologically indistinguishable from MPS VII in humans.

The MPS VII mouse is characterized by a smaller stature, skeletal abnormalities, and a pug-nosed appearance due to shortened facial bones (3, 6, 9). On microscopic examination extensive vacuolar storage is present in many tissues, resulting from the accumulation of undegraded glycosaminoglycans (GAGs) in the lysosomes. Affected tissues include cornea, retina, heart, trachea, liver, spleen, skeleton, and the brain. Clinically, the affected mouse does not appear grossly abnormal until about 3 wk of age (3). Histologically, storage is present in most tissues by 3 wk of age (11).

Newborn MPS VII mice are grossly indistinguishable from their normal littermates. However, distended lysosomes can be seen microscopically and the accumulation of GAGs can be found biochemically in the brain at birth (6, 12). Monocyte lineage Kupffer cells in the liver and sinusoidal lining cells in the spleen also contain vacuoles that are indicative of storage (6). Because of the presence of pathology at birth, the MPS VII mouse is an ideal model for the study of in utero treatment modalities, which are aimed at forestalling disease manifestations. One approach involves retroviral vector-mediated correction of affected fetal liver cells, which contain hematopoietic stem cells, and subsequent transplantation into affected fetuses (13, 14). Previous experiments have shown that only a small amount of the functional gene product (enzyme) is capable of correcting major organs, even in adults where the fully developed disease can be reversed (15, 16).

There are numerous reports describing pathologic findings in animals and humans affected with lysosomal storage diseases. Of those that looked at fetuses, however, all but one (17) describe disease lesions at only a single time point during gestation rather than following the progression of disease during fetal development. The knowledge of onset and extent of lesions is important for designing a rationale approach to in utero therapy. Information on the absence or presence of disease at E13.5 is important in the mouse model, since fetal hematopoietic stem cells are transplanted at this time due to immunotolerance of the host (18). To assess success of in utero treatment, it is necessary to know at what fetal age lesions become apparent. This paper describes histopathologic and enzymatic abnormalities found in murine MPS VII fetuses at various gestational ages.

METHODS

The original B6.C-H-2bml/ByBir-gusmps/+ mice (3) were obtained from The Jackson Laboratory (Bar Harbor, Maine) about 10 y ago and have been bred in our colony since then. These mice have a single bp deletion in exon 10 resulting in a frameshift and a premature stop codon (7). Translation of the resulting mRNA would produce a truncated, probably nonfunctional protein, but since the GUSB mRNA levels are reduced by over 200-fold (3), probably little or no truncated protein is produced. Mice were maintained and cared for according to the University of Pennsylvania's Guidelines for the Care and Use of Laboratory Animals, which ensured compliance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act.

Tail clips were obtained for PCR to determine the genetic status of each fetus. Genomic GUSB DNA was amplified using the following primers: CCTGTGTCATTTGCATGTG (forward primer) and GATAACATCCACGTACCGG (reverse primer). The 95-bp product lacks a Nci I restriction site in the mutant allele while the normal allele is cleaved into a 77- and 18-bp product (11).

To determine gestational ages of the fetuses, female and male adult mice were separated for 2 wk before being paired for mating. Thereafter, the female mice were checked daily for the presence of vaginal plugs that were counted as d E0.5. The females that were determined to be pregnant were separated from the males. Whole litters of fetuses were removed at E9.5, E11.5, E13.5, E15.5, and E18.5.

Tissues for enzymatic activity were snap frozen in isopentane submersed in liquid nitrogen. Frozen tissues were sectioned at 10 μm for GUSB expression and stained as previously described using a naphthol-AS-BI β-D-glucuronide substrate (11). Tissues for microscopic evaluation of the distended lysosomes were fixed in buffered 18.5% formalin before being embedded in JB4+® (Polysciences, Inc., Warrington, NJ) and sectioned at 1 μm. The sections were covered with several drops of 1% toluidin blue and left to dry on a 60°C hot plate. The slides were then rinsed with dH2O, dipped in xylene, and covered with a coverslip after applying a drop of Permount® (Fisher Scientific, Fair Lawn, NJ).

For electron microscopy, mouse livers were dissected from the fetuses, trimmed into 2-mm blocks, refixed in 2.5% glutaraldehyde for 4 h, and processed according to previously published (19, 20). Briefly, the specimens were washed with 0.2 M sodium cacodylate, postfixed with 2% aqueous osmium tetroxide, stained en bloc with 1% uranyl acetate, dehydrated with ethanol, and embedded in LX-112 medium. Ultrathin sections (∼80 nm) were cut with a diamond knife, mounted on uncoated copper grids, briefly stained with uranyl acetate and lead citrate, and examined with a Philips CM-100 transmission electron microscope operated at 60 kv.

β-glucuronidase and total hexosaminidase enzyme activities in homogenates of fetal livers were determined using a microfluorometric assay with 4-methylumbelliferyl-β-D-glucuronide and 4-methylumbelliferyl-glucosaminide, respectively, as previously described (11, 21). Protein content of the homogenates was measured with the Bio-Rad Protein Assay® (Bio-Rad, Hercules, CA) using BSA as a standard. Cryostat sections of whole fetuses were air dried on glass slides and assayed for GUSB activity using a histochemical stain, which detects the biologically relevant function by enzymatic cleavage of a β-D-glucuronide moiety (11).

RESULTS

Fetuses were genotyped at E9.5, E11.5, E13.5, E15.5, and E18.5 (Table 1). There were significantly fewer affected fetuses in each age group than the 25% expected from Mendellian inheritance. At E13.5, only 20% of the fetuses were affected, which is significantly different from the expected 25% (p < 0.03), but not from the 18.0% of newborn mice (p = 0.25). Although, the number of fetuses analyzed at E9.5, E11.5, and E18.5 was small, the overall percentage of fetuses affected with MPS VII (18.9%) did not differ significantly from the percentage of newborn MPS VII mice (18.0%).

Table 1 Beta-glucuronidase genotypes of fetal and newborn mice * Statistical difference of affected fetuses in each age group compared to the percentage of expected MPS VII mice (25%), determined by Fisher's Exact Test. ND, not determined in groups with fewer than three litters.
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β-glucuronidase enzymatic activities in MPS VII fetal livers were never higher than background (Table 2). In normal and carrier fetal livers, GUSB levels increased from E11.5, reaching higher than adult liver levels at E13.5 and E15.5. At E18.5, the level was about the same as the normal adult level, then increased again at birth. When compared with adult bone marrow which contains the hematopoietic cells, GUSB activities were always much higher in fetal livers. Livers of carrier fetuses had approximately half the activity measured in normal age-matched fetal livers, as expected. The difference between normal fetal livers at every time point measured and those of MPS VII fetuses as well as adult normal liver and bone marrow was always statistically significant (p < 0.005), while the difference between MPS VII fetal livers and adult bone marrow and liver was never significant.

Table 2 Lysosomal enzyme activities in mouse fetal livers during gestation * BM, bone marrow. † (N), number of mice examined. ‡ +/+, Normal; +/−, Carrier; −/−, MPS VII. § ND, not determined.
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Adult MPS VII mice have nonspecific secondary elevations of some normal lysosomal enzymes, which is thought to result from increased lysosomal volume in storage cells (15, 22). To evaluate this in fetal MPS VII mice, total hexosaminidase (HEX) and α-galactosidase (GLA) activities were determined. The HEX activities in normal and carrier fetal livers were lower than in adult mice, except at birth where normal fetal livers reached approximately normal adult levels (Table 2). From E11.5 to birth HEX activities did not differ statistically between normal, carrier, and MPS VII fetal livers of the same age group, even though at birth HEX activities were almost twice as high in neonatal MPS VII livers when compared with normal newborn livers. This is due to the large range of activities measured, which is reflected in the SEM. Thus, secondary elevations in HEX activities were not detected in MPS VII fetuses to the degree that they were seen in the adult MPS VII tissues (Table 2). Interestingly, there were high levels of HEX activity at E11.5, after which they decreased and did not increase to similar levels until E18.5. The values of GLA were so low throughout gestation that differences between normals and affecteds could not be evaluated (data not shown).

It is possible to detect small numbers of individual GUSB positive cells in tissues, by using a histochemical staining reaction, when the total GUSB enzymatic activity of a tissue is not above background within the variance of the assay (23). When MPS VII fetuses were examined microscopically, no positive cells were found in any of the affected fetal tissues (Fig. 1). The GUSB reaction product was about half as strong in carrier as in normal fetuses (not shown), reflecting their biochemically measured GUSB activity levels (24, 25). In normal and carrier fetuses, minimal GUSB activity can be detected at E11.5. At E13.5 and E15.5 the intensity of staining increased in the fetal liver, while it decreased again by E18.5. In the intestines and bone anlagen (cartilage), positive cells were first detected at E13.5 and the intensity of staining increased steadily until birth. The kidneys were not positive until E18.5. Interestingly, GUSB was seen in the eyes only at E13.5. Very little GUSB activity was demonstrated in the developing brain, except at E15.5, when the neural tube stained bright red indicating increased GUSB activity.

Figure 1
figure 1

Absence of β-glucuronidase in MPS VII fetuses. Frozen sections (10 μm) of whole fetuses (normal and MPS VII) stained using naphthol-AS-BI β-D-glucuronide substrate. The red color indicates the presence of biologically active β-glucuronidase. The background color has been changed by computer to highlight the areas of intense red staining. +/+: Normal; +/−: Carrier; −/−: MPS VII.

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Histopathology was evaluated after fixing tissues in formalin and embedding them in plastic before sectioning. At E13.5 there was no evidence of vacuolation in any tissues by light microscopy, but at E15.5 a few small vacuoles were present in some cells that lined sinusoids of the MPS VII livers (Fig. 2, B and D). The tissue specimens were too small to perform quantitative biochemical analyses for glycosaminoglycans, as on adult tissues (10). Therefore, the tissues were evaluated by electron microscopy to determine whether typical storage lesions could be seen in livers at E13.5. Despite a thorough electron microscopic examination of numerous areas of E13.5 fetal livers, no storage vacuoles were found (Fig. 2B). In contrast, at E15.5 electron microscopy showed vacuolated reticulo-endothelial cells, which were identified by the presence of gap junctions (Fig. 2D). However, the number of vacuolated cells was low and the vacuoles were small in size and few per cell. Vacuolation was not seen in any other tissue at E15.5. At E18.5 vacuolation was present in most endothelial and Kupffer cells in all sections of fetal liver (not shown). Storage lesions were also seen in perivascular areas near the meninges of the brain at E18.5, but the vacuoles were few in number and were only detected in a small number of cells (not shown). Vacuolation was more prominent in the brain by birth, as previously described (8, 12).

Figure 2
figure 2

Fetal liver pathology. Photomicrographs of toluidin blue (light microscopy) and uranyl acetate and lead citrate (electron microscopy) stained livers of normal (A, C) and MPS VII (B, D) mice at E13.5 (A, B) and E15.5 (C, D). The arrowheads point to the areas of vacuolation (reticulo-endothelial cells). The white bar indicates 10 μm in the light micrograph (top portion of each panel) and the black bar 1 μm in the electron micrograph (bottom portion of each panel).

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DISCUSSION

Studies in human fetuses with MPS VII have been limited to prenatal diagnoses by chorionic villus sampling or by examination of the fetuses after termination of pregnancy (26–31). Findings included fetal hydrops, accumulation of GAGs in the amniotic fluid and the lack of GUSB activity in the chorionic villi and in fetal tissues. In human fetuses with other types of mucopolysaccharidoses, findings have included:1) little or no structural abnormalities in fetuses with Morquio and San Filippo A (32, 33);2) increased storage of GAGs and secondary accumulation of GM1- and GM2-gangliosidoses in the brains of fetuses affected with Hunter and Hurler syndromes (33); and 3) lysosomal storage in spleen, liver, and the peripheral and central nervous systems in Hurler disease (34, 35). More information is available on the fetal pathology of some of the more common lysosomal storage diseases such as Tay-Sachs (36) and globoid cell leukodystrophy (37–42).

Animal models have been used to study the prenatal onset of disease in goats with β-mannosidosis (43, 44) and sheep with GM1-gangliosidosis (45), but only at single time points during gestation. The only other study describing the detailed progression of a lysosomal storage disease during embryonic life was performed in Japanese quail with acid α-glucosidase deficiency (Pompe's disease) (17). Storage of glycogen was demonstrated in the heart at a very early age (incubation d 3) by electron microscopy before it was visible by light microscopy.

In this study of MPS VII in fetal mice, fewer than the expected 25% MPS VII pups were born from matings between carriers. Similar percentages of the fetuses were affected with MPS VII as newborn mouse pups (17–18%), indicating primarily a fertilization problem or early intra-uterine losses (e.g. failure to implant). These results are in contrast to recently published findings, where the expected 25% MPS VII fetuses were found during late gestation (46). It is possible that the difference is related to environmental factors or, most likely, to the fact that the colonies have been separated for over ten years. Thus, other modifying mutations may have occurred leading to differences in intrauterine losses. In fact, when the MPS VII carrier mice described here were bred to a different strain, the percentage of affected fetuses and pups increased, reaching the expected 25% (10). Nevertheless, both the aforementioned study and the previous study from our laboratory (10) found an additional 30% loss by weaning age. This probably reflects a combination of maternal neglect, the lack of the ability to produce enough milk, and neonatal weakness.

A complete absence of GUSB activity in MPS VII fetuses was demonstrated by both histochemical and quantitative biochemical assays. This showed that there was no transfer of maternal enzyme to fetuses, thus it is unlikely that the disease could be treated by giving the mother large amounts of GUSB. Another study found that there was no difference in pathology in weanling pups from heterozygous dams and MPS VII dams that had been treated by enzyme injections when they were younger (46). Since the MPS VII dams do not produce any endogenous enzyme and it can be safely assumed that there is no recombinant enzyme remaining in the dams' tissues, there can be no transfer of GUSB to the pups. Thus, the pathology being the same in both groups of MPS VII pups, the results also suggest that there is little to no transfer of GUSB from a heterozygous dam to affected fetuses in utero.

Affected fetuses did not have significant secondary elevations of HEX or GLA, which are elevated in MPS VII adults (15). This coincided with the minimal amount of lysosomal distention that was observed in the fetal tissues. Although HEX activities were higher in neonatal MPS VII pups when compared with normal littermates, the difference was not statistically significant. In addition, since the levels of GLA activity were much lower in normal fetuses than in adults, secondary elevations may not be apparent until well after birth. Although secondary elevations of other lysosomal enzymes have been reported in one human fetus, this was not seen in either its newborn affected sibling (30) or in an unrelated affected fetus (31). The mechanisms of secondary elevations of normal lysosomal enzymes in affected tissues are not clear (15, 22), but they are concordant with the increase in vacuolation observed as the disease progresses postnatally.

Grossly, the external appearance and internal organs of the fetuses affected with MPS VII were unremarkable. Newborn MPS VII mice do not show any macroscopic signs of disease either. In contrast, by 3 wk of age the disease is clearly recognizable by the blunt facies, joint deformities, and small stature (3, 10). Microscopic vacuoles indicating the presence of lysosomal storage were not detected until E15.5, well after the normal liver begins producing high levels of GUSB. The appearance of vacuoles correlates with the turnover of proteoglycans and marked growth of the fetus, as opposed to cell division (39). Since clinical signs of MPS VII do not appear until well after histologic signs, and since vacuolation was not detectable at E13.5, treatment at this fetal age may result in forestalling the development of the disease altogether.