Reproductive Toxicology 20 (2005) 283–285

Short communication

Aspartoacylase gene knockout in the mouse: Impact on reproduction

Sankar Surendrana,∗, Sylvia Szucsb, Stephen K. Tyringc, Reuben Matalonb

a Department of Internal Medicine, The University of Texas Medical Branch, Galveston, TX 77555, USAb Department of Pediatrics, The University of Texas Medical Branch, Galveston, TX, USA

c Department of Dermatology, University of Texas Health Science Center, Houston, TX, USA

Received 30 November 2004; received in revised form 31 December 2004; accepted 4 February 2005Available online 12 March 2005

Abstract

Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase (ASPA) gene mutations resulting enzyme deficiency.The homozygous knockout mouse for CD showed symptoms similar observed in patients with CD. Canavan disease leads to early death.Therefore, a role of ASPA in reproduction was investigated using the mouse model for CD. Homozygous (KO/KO) pups, produced by matingfemale heterozygous (KO/+) mouse with KO/+ males had approximately 12% death incidence rates in the first 2 months of life. KO/KOm hese datas©

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others mated with KO/+ males showed fetal death. KO/KO mothers produced fewer offspring compared to KO/+ mothers. Tuggest that ASPA is necessary for normal reproduction and postnatal survival.2005 Elsevier Inc. All rights reserved.

eywords:Canavan disease; Knockout mouse; Aspartoacylase; Postnatal death; Fetal death

. Introduction

Canavan disease (CD) is an autosomal recessive leukodys-rophy characterized by spongy degeneration of the whiteatter of the brain[1,2]. CD is an inborn error of aspartoa-

ylase (ASPA), because genetic mutations in this allele[3]esult in enzyme deficiency[4] and the disease. Aspartoa-ylase hydrolyzesN-acetyl aspartate (NAA) to aspartate andcetate[5]. The clinical features of CD include psychomo-

or retardation, megalencephaly and hypotonia[2]. Canavanisease often leads to early death[6,7].

The ASPA gene was cloned and localized on the shortrm of chromosome 17 (17p13-ter)[3]. Human ASPA genepans approximately 30 kb and contains five introns and sixxons coding for 313 amino acids[3] and an enzyme with aolecular weight of approximately 37 kDa[7].The homozygous (KO/KO) knockout mouse for CD[8]

as used to understand molecular mechanisms involved in

∗ Corresponding author. Tel.: +1 409 747 1227; fax: +1 409 747 3084.

patients with CD[9]. While ASPA activity was deficient[10],levels of aspartate aminotransferase[11], glutamate dehydrogenase and�-ketoglutarate dehydrogenase complex[12]were found to be lower in the brain of the KO/KO mouVacuolation was observed in the white matter of the b[7,8] and at all levels of the spinal cord[13]. Since CD leadto early death[6,7], role of ASPA in reproduction was invetigated using the mouse model for CD.

2. Materials and methods

All animal procedures were performed in compliance wthe Institutional Animal Care and Use Committee at The Uversity of Texas Medical Branch. Mouse offspring were pduced by mating KO/KO or heterozygous (KO/+) femawith KO/+ males as shown inTable 1. Nineteen KO/KO an25 KO/+ mothers uterus were examined at 18–19 daypostcoitum in order to investigate fetal death. Pups wereerally weaned at 22 day of age. The progeny were studie

E-mail address:ssurendr@utmb.edu (S. Surendran). to 112 days of age.

890-6238/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2005.02.001

284 S. Surendran et al. / Reproductive Toxicology 20 (2005) 283–285

Table 1Outcome of heterozygous or homozygous females mated with heterozygous male mice

Genotype Pups genotype Pups age (days) Number of pups examined Pups death (%)

Father Mother

(A) Heterozygous females mated with heterozygous malesKO/+ KO/+ WT 1–28 776 0KO/+ KO/+ WT 29–56 694 0.57KO/+ KO/+ WT 57–84 506 0KO/+ KO/+ WT 85–112 239 0KO/+ KO/+ KO/KO 0–28 255 11.76KO/+ KO/+ KO/KO 29–56 220 12.27KO/+ KO/+ KO/KO 57–84 144 2.7KO/+ KO/+ KO/KO 85–112 109 1.8

(B) KO/KO females mated with heterozygous malesKO/+ KO/KO KO/+ 1–28 21 0KO/+ KO/KO KO/+ 29–56 17 0KO/+ KO/KO KO/+ 57–84 17 0KO/+ KO/KO KO/+ 85–112 14 0KO/+ KO/KO KO/KO 1–28 15 6.6KO/+ KO/KO KO/KO 29–56 14 7.14KO/+ KO/KO KO/KO 57–84 13 0KO/+ KO/KO KO/KO 85–112 7 0

Abbreviations:KO/+, heterozygous; WT, wild-type; KO/KO, homozygous.

3. Results and discussion

The average number of pups, obtained from KO/+ mothermated with KO/+ males was 7.74± 1.41 (n= 1233± S.D.)(Table 1). Fetal death was not seen in the uterus of KO/+mothers. Homozygous pups, obtained from KO/+ femalesmated with KO/+ males, showed approximately 12% deathin the first 2 months of life, while wild-type (WT) pups hadless than 0.6% death (Table 1). Thus, pups with homozygosityhave high rate of mortality.

Some female KO/KO mice bred to KO/+ males did notconceive, whereas other females showed symptoms of preg-nancy, including enlarged abdomen and prominent nipples,but either resorbed their litters or else devoured their new-borns after delivery. Among the homozygous pups producedfrom the KO/KO mothers, approximately 7% death was ob-served in the first 2 months of life.

In order to investigate the occurrence of fetal deaths inKO/KO females mated to KO/+ males, the uteri of nine fe-males were examined on day 18 or day 19 postcoitum. Fe-tal death was observed in nine KO/KO mother’s uterus. Theaverage number of offspring obtained from this cross was4± 3.54 (n= 40± S.D.). This implies fetal lethality as oneof the contributing factors for the fewer pups obtained fromKO/KO mother. Intuitively, fewer numbers of fertilized eggsin the KO/KO mouse may also result in fewer pups. There-f er ofe

mblest -g rain.T re-dt nal

intensity in the brain[7,8,14]. We reported ASPA deficiencyin homozygous pups[10]. ASPA deficiency leads to vacuo-lation in the white matter of the brain[7,8] and at all levelsof the spinal cord in the mouse[13]. From these studies, itis likely that ASPA deficiency affecting the central nervoussystem development may contribute to homozygous pups’death seen in the present study.

Our study suggests that ASPA deficiency affects normalreproduction in the mouse and also leads to postnatal death.Aspartoacylase deficient (KO/KO) female mouse produce ap-proximately 50% fewer pups compared to KO/+ mothers;hence, ASPA expression is a key factor in reproduction andpostnatal survival. Whether ASPA gene transfer[15–17]orASPA enzyme therapy prior to mating would rescue normalreproduction and postnatal survival in the mouse remains tobe determined; this may have important implications for hu-man mothers at risk for CD offspring.

References

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