The lde/lde rat is characterized by dwarfism, postnatal lethality, male hypogonadism, a high incidence of epilepsy and many vacuoles in the hippocampus and amygdala. We used a candidate approach to identify the gene responsible for the lde phenotype and assessed the susceptibility of lde/lde rats for audiogenic seizures. Following backcross breeding of lethal dwarfism with epilepsy (LDE) to Brown Norway rats, the lde/lde rats with an altered genetic background showed all pleiotropic phenotypes. The lde locus was mapped to a 1.5-Mbp region on rat chromosome 19 that included the latter half of the Wwox gene. Sequencing of the full-length Wwox transcript identified a 13-bp deletion in exon 9 in lde/lde rats. This mutation causes a frame shift, resulting in aberrant amino acid sequences at the C-terminal. Western blotting showed that both the full-length products of the Wwox gene and its isoform were present in normal testes and hippocampi, whereas both products were undetectable in the testes and hippocampi of lde/lde rats. Sound stimulation induced epileptic seizures in 95% of lde/lde rats, with starting as wild running (WR), sometimes progressing to tonic-clonic convulsions. Electroencephalogram (EEG) analysis showed interictal spikes, fast waves during WR and burst of spikes during clonic phases. The Wwox protein is expressed in the central nervous system (CNS), indicating that abnormal neuronal excitability in lde/lde rats may be because of a lack of Wwox function. The lde/lde rat is not only useful for understanding the multiple functions of Wwox but is also a unique model for studying the physiological function of Wwox in CNS.

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ABSTRACT:The lde/lde rats show a severe dwarf phenotype with early postnatal lethality and a high incidence of epileptic seizure. Seizures are first detected in this model between 16 and 63 days of age, and mostly begin as wild running and progress to generalized tonic-clonic convulsions. Because our histological examination detected many extracellular vacuoles in the hippocampus and amygdaloid bodies of these animals at 28 days of age, these pathological alterations may be related to the epileptogenesis in lde/ lde rats. In addition to these defects, male lde/lde rats have apparently smaller testes with reduced number of germ cells and poorly matured adult-type Leydig cells in comparison with wild-type controls. In the present study, we performed anatomical, histological, and endocrinologic examinations to characterize the testicular phenotype of lde/lde rats at 21, 28, 35, and 56 days of age. Male lde/lde rats showed severely retarded growth of the testes and accessory sex organs. Their seminiferous tubules were significantly smaller and contained markedly fewer germ cells at all time points examined as compared with controls. Significantly fewer Sertoli cells at 21 and 28 days of age, markedly decreased spermatocyte number at 28 days of age, and delayed appearance of spermatids at 56 days of age were observed in the testes of lde/lde rats. More TUNEL (T&T-mediated duTP-biotin nick-end labeling)-positive cells were detected in lde/lde seminiferous tubules, and the largest number of apoptotic cells was recorded at 28 days of age. The increases in 3ß-hydroxysteroid dehydrogenase-positive adult-type Leydig cells and 11ß-hydroxysteroid dehydrogenase-positive mature adult-type Leydig cells were also severely retarded in the testes of lde/lde rats. Consistent with these defects, significantly lower plasma folliclestimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were detected in lde/lde males at 28 days of age, and weak immunostaining for FSH and smaller cytoplasm of LH-positive cells were detected in the anterior pituitary lobes of lde/ lde males. Despite a normal level of plasma LH after 35 days of age, a significantly lower level of plasma testosterone was detected at 56 days of age. These results indicate that the normal lde allele is related to prepubertal elevations of gonadotropins and normal development of adult-type Leydig cells. Because lde/lde rats experience epileptic seizures during the period when the hypothalamus-pituitary-testicular axis is established, lde/lde rats would be useful as a model for reproductive disorder with pediatric epilepsy.

Abstract. Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/ hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes. Key words: Apoptosis, Astrin, Hypogonadism, Mitosis, Rat, Spag5, Sterility (J. Reprod. Dev. 53: [581][582][583][584][585][586][587][588][589] 2007) ale hypogonadic (hgn/hgn) rats are sterile due to a lack of spermatogenesis during adulthood [1]. Growth of the seminiferous tubules in postnatal hgn/hgn testes is severely affected, and the gonocytes degenerate before entering meiosis [ 2 ] .T h e s e a n i m a l s h a v e h i g h p l a s m a concentrations of gonadotropins and low plasma concentrations of testosterone, indicating that the cause of the hypogonadism is present in their testes [3]. Since hypogonadism is accompanied by renal hypoplasia [4], hgn/hgn females can be identified by laparotomy at weaning. Moreover, their ovaries are hypoplastic at birth, and they have reduced fertility [5]. These animals have one quarter the number of nephrons present in the normal kidney, and their individual nephrons are extremely hypertrophied [6]. Thus, hgn/hgn rats can be used a s a n e x p e r i m e n t a l m o d e l o f h u m a n oligomeganephronia, which progresses to renal insufficiency with advancing age [7].Linkage analysis has shown that the gene responsible for the hgn phenotype is located in an 840-kb region of rat chromosome 10 [8, 9]. The most likely candidate gene in this region is Spag5 (astrin/MAP126) [9]. Spag5 has been identified as a protein interacting with the outer dense fiber of sperm tails in rats [10]. Although it has been reported that targeted disruption of mouse Spag5 has no significant effect on spermatogenesis [11], astrin/hMAP126 (human) [12,13] and mastrin (mouse) [14], which are orthologs of rat Spag5, are m i c r o t u b u l e -a s s o c i a t e d p r o t e i n s t h a t a r e considered to be critical for normal distributi...