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A well-known putative tumor suppressor WW domain–containing oxidoreductase (Wwox) is highly expressed in hormonally regulated tissues and is considered important for the normal development and function of reproductive organs. In this study, we investigated the cellular and subcellular localization of Wwox in normal testes during postnatal days 0–70 using Western blotting and immunohistochemistry. Wwox is expressed in testes at all ages. Immunohistochemistry showed that fetal-type and adult-type Leydig cells, immature and mature Sertoli cells, and germ cells (from gonocytes to step 17 spermatids) expressed Wwox except peritubular myoid cells, step 18–19 spermatids, and mature sperm. Wwox localized diffusely in the cytoplasm with focal intense signals in all testicular cells. These signals gradually condensed in germ cells with their differentiation and colocalized with giantin for cis-Golgi marker and partially with golgin-97 for trans-Golgi marker. Biochemically, Wwox was detected in isolated Golgi-enriched fractions. But Wwox was undetectable in the nucleus. This subcellular localization pattern of Wwox was also confirmed in single-cell suspension. These findings indicate that Wwox is functional in most cell types of testis and might locate into Golgi apparatus via interaction with Golgi proteins. These unique localizations might be related to the function of Wwox in testicular development and spermatogenesis

A congenital reduction in the number of nephrons is a critical risk factor for both onset of chronic kidney disease (CKD) and its progression to end-stage kidney disease (ESKD). Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and show progressive CKD. This study used an immunohistological method to assess glomerular and interstitial pathogenesis in male HPK rats aged 35-210days. CD68 positive-macrophages were found to infiltrate into glomeruli in HPK rats aged 35 and 70days and to infiltrate into interstitial tissue in rats aged 140 and 210days. HPK rats aged 35 and 70days showed glomerular hypertrophy, loss of normal linear immunostaining of podocine, and increased expression of PDGFr-β, TGF-β, collagens, and fibronectin, with all of these alterations gradually deteriorating with age. α-SMA-positive myofibroblasts were rarely detected in glomerular tufts, whereas α-SMA-positive glomerular parietal epithelium (GPE) cells were frequently observed along Bowman's capsular walls. The numbers of PDGFr-β-positive fibroblasts in interstitial tissue were increased in rats aged 35days and older, whereas interstitial fibrosis, characterized by the increased expression of tubular PDGF-BB, the appearance of myofibroblasts doubly positive for PDGFr-β and α-SMA, and increased expression of collagens and fibronectin, were observed in rats aged 70 and older. These results clearly indicate that congenital CKD with only 20% of nephrons cause renal fibrosis in rats.

A variety of animal models of diabetes mellitus (DM) are required to study the genetics and pathophysiology of DM. We established a novel rat strain showing nonobese type 2 diabetes with enlarged kidneys from the LEA.PET-pet congenic strain and named it Diabetes with Enlarged Kidney (DEK). The body growth of DEK affected rats was similar to that of normal rats before the development of DM but was attenuated with the deterioration of DM. There was a marked difference in the etiology of DEK by gender: DM phenotypes including polyuria, polydipsia, and hyperglycemia (nonfasting blood glucose over 300 mg/dl) were found in male rats aged over 10 weeks but not in female rats. The cumulative incidence of DM in DEK males at the age of 30 weeks was 44.8%. Oral glucose tolerance tests showed glucose intolerance and decreased insulin secretion in response to glucose loading in affected males, features which were exacerbated with age. Affected males exhibited disorganized architecture of pancreatic islets, decreased numbers of β cells, and markedly decreased expression of insulin, despite no pathological findings of hemorrhage or infiltration of inflammatory cells in the pancreatic islet. Age-related islet fibrosis appeared similar in normal and affected males. Affected males also showed enlarged kidneys with dilation of renal tubules in both the cortex and medulla, but no obvious glomerular lesions typical of diabetic nephropathy (DN) at the age of 30 weeks. Plasma levels of urea nitrogen and creatinine were normal, but hypoalbuminemia was detected. These pathophysiological features in affected males indicated that their renal function was almost maintained despite severe DM. Taken together, these findings indicate that the affected males of the DEK strain are a novel nonobese type 2 diabetes rat model useful for studying the mechanisms underlying β cell loss and identifying genetic factors protective against DN.

Male hypogonadism (hgn/hgn) rats show testicular hypoplasia accompanied by dysplastic development of seminiferous tubules due to loss-of-function mutation of the gene encoding Astrin, which is required for mitotic progression in the division cycle of HeLa cells. In the present study, we examined the cytological base leading to the decrease of Sertoli cells in hgn/hgn testes. In hgn/hgn testes on postnatal day 3, anti-phospho-histone H3 (Ser10) (pH3)-positive mitotic phase and TUNEL-positive apoptosis increased in GATA4-positive Sertoli cells. Isolated immature Sertoli cells from hgn/hgn testes showed increased pH3-assessed mitotic index accompanied by decreased 5-bromo-2'-deoxyuridine-incorporation and increased TUNEL-positive apoptosis, suggesting mitotic delay and cell death. In the visualization of mitotic progression by nocodazole (NOC)-mediated cell cycle arrest and subsequent release, hgn/hgn rat-derived Sertoli cells failed to make the transition from prometaphase to metaphase, and the cells with micronuclei and TUNEL-positive cells gradually increased in a time-dependent manner. Western blot analysis detected ≈142 kDa protein expected as Astrin in extracts of +/+ and +/hgn testes and cultured normal Sertoli cells but not in extracts of hgn/hgn testes. CLASP1 was detected in extracts of both normal and hgn/hgn testes, whereas it was localized in kinetochore of normal mitotic Sertoli cells but diffused in cytoplasm of hgn/hgn Sertoli cells. These results indicate that Astrin is required for normal mitotic progression in immature Sertoli cells and that the most severe type of testicullar dysplasia in hgn/hgn rats is caused by mitotic cell death of immature Sertoli cells due to lack of Astrin.

WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.

Renal hypoplasia due to a congenitally reduced number of nephrons progresses to chronic kidney disease and may cause renal anemia, given that the kidneys are a major source of erythropoietin in adults. Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and develop CKD. This study assessed the renal function and hematologic changes in HPK rats from 70 to 210 d of age. HPK rats demonstrated deterioration of renal excretory function, slightly macrocytic erythropenia at all days examined, age-related increases in splenic hemosiderosis accompanied by a tendency toward increased hemolysis, normal plasma erythropoietin levels associated with increased hepatic and decreased renal erythropoietin production, and maintenance of the response for erythropoietin production to hypoxic conditions, with increased interstitial fibrosis at 140 d of age. These results indicate that increases in splenic hemosiderosis and the membrane fragility of RBC might be associated with erythropenia and that hepatic production of erythropoietin might contribute to maintaining the blood Hgb concentration in HPK rats.

The petit rat (pet/pet) is a new semi-lethal dwarf mutant with anomalies in the thymus and testes, defects inherited as a single autosomal recessive trait. At birth, these pet/pet rats show low birth weight and extremely small thymuses; at 140 days of age, their thymuses show abnormal involution. In the present study, we examined early postnatal development of hypoplastic pet/pet thymuses. In addition to being hypoplastic at birth, pet/pet thymus growth was almost completely impaired during the early postnatal period. As shown by cellular incorporation of BrdU, the mitotic activity was lower in pet/pet than in normal thymuses, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that apoptosis occurred more often in pet/pet than in normal thymus cells during the first few days after birth. These results indicate that postnatal development of the hypoplastic pet/pet thymus is defective due to the reduced proliferation and increased apoptosis of thymic cells.

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.

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 3s- hydroxysteroid dehydrogenase-positive adult-type Leydig cells and 11s-hydroxysteroid dehydrogenase-positive mature adult-type Ley- dig cells were also severely retarded in the testes of lde/lde rats. Consistent with these defects, significantly lower plasma follicle- stimulating hormone (FSH), luteinizing hormone (LH), and testos- terone 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 hypo- thalamus-pituitary-testicular axis is established, lde/lde rats would be useful as a model for reproductive disorder with pediatric epilepsy.

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.

Vinclozolin is a systemic dicarboximide fungicide that is used on fruits, vegetables, ornamental plants, and turf grass. Vinclozolin and its metabolites are known to be endocrine disruptors and act as androgen receptor antagonists. The hypothesis tested in the current study is that transient embryonic exposure to an anti-androgenic endocrine disruptor at the time of testis determination alters testis development and subsequently influences adult spermatogenic capacity and male reproduction. The effects of vinclozolin on embryonic testicular cord formation in vitro were examined, as well as the effects of transient in utero vinclozolin exposure on postnatal testis development and function. Embryonic day 13 (E13, sperm-positive vaginal smear day = E0) gonads were cultured in the absence or presence of vinclozolin (50-500microM). Vinclozolin treated gonads had significantly fewer cords (P < 0.05) and the histology of the cords that formed were abnormal as compared to vehicle-treated organs. Pregnant rats were exposed to vinclozolin (100 mg/kg/day) between embryonic days 8 and 14 (E8-E14) of development. Testis morphology and function were analyzed from postnatal day (P) 0, pubertal P20, and adult P60. No significant effect of vinclozolin on testis histology or germ cell viability was observed in P0 testis. The pubertal P20 testis from vinclozolin exposed animals had significantly higher numbers of apoptotic germ cells (P < 0.01), but testis weight was not affected. The adult P60 sperm motility was significantly lower in vinclozolin exposed males (P < 0.01). In addition, apoptotic germ cell number in testis of vinclozolin exposed animals was higher in adult P60 animals. Observations demonstrate that vinclozolin can effect embryonic testicular cord formation in vitro and that transient in utero exposure to vinclozolin increases apoptotic germ cell numbers in the testis of pubertal and adult animals. This correlated to reduced sperm motility in the adult. In conclusion, transient exposure to vinclozolin during the time of testis differentiation (i.e. cord formation) alters testis development and function. Observations indicate that transient exposure to an anti-androgenic endocrine disruptor during embryonic development causes delayed effects later in adult life on spermatogenic capacity.

Flutamide, when administered subcutaneously to female rats at doses of 3, 10, or 30 mg/kg/day during late pregnancy (gestational days 16-21), significantly and dose-dependently decreased anogenital distance (AGD) of the male offspring in each dose group compared to controls. Significant delays in preputial separation were found in males at a dose of 30 mg/kg, but body weight gain was not inhibited. Cryptorchidism and absence of the prostate gland and seminal vesicles were found in males at doses > or = 10 mg/kg, and testicular hypoplasia at a dose of 30 mg/kg. Hypospadias was noted in all dose groups and vaginal pouches at doses of > or =10 mg/kg. The effects on the accessory reproductive organs were severe, although the effects on the testes themselves were mild. However, those effects appeared to become more pronounced with growth, as evaluated on Days 30 and 42 and Weeks 16 to 18. Most of these affected animals displayed cryptorchidism. Male offspring exposed to flutamide in utero showed impairments of sexual behavior as adults in a dose-related manner. Number and frequency of mounts with intromissions was markedly decreased in all treated groups as compared to controls. At 10 mg/kg, no mounting with ejaculation was observed, and at a dose of 30 mg/kg, no mounting with intromission or ejaculation was observed. These changes in sexual behavior were closely associated with abnormalities of the external genitalia. Animals with hypospadias did not display mounts with ejaculation. However, F1 males that copulated at a dose of 3 mg/kg had a normal reproductive function. Histological examination of the reproductive organs revealed degeneration of the seminiferous tubules, hypospermatogenesis, and hypoplasia and inflammation of the seminal vesicles and prostate. Serum levels of FSH, LH, and testosterone in these animals were comparable between control and all dose groups. Therefore, the male reproductive dysfunction seen in the present study could not be attributed to abnormal sex hormone levels during maturation, but to possible demasculinization of the brain and progressively delayed dysmorphology of the male genitalia caused by fetal exposure to flutamide.

The current study was designed to examine the effects of a transient embryonic exposure to the pesticide methoxychlor, an endocrine disruptor, on in vivo rat testis development and function. Gestating female rats were transiently administered methoxychlor (MXC) from embryonic day 7 (E7; EO = plug date) through E15. Embryonic testes were collected at E16 and postnatal (PO = day of birth) testes at P4, P10, P17-20, and P60. Seminiferous cords formed in testes from MXC exposed males. However, at E16, there was a decrease in the area of cords and an increase in interstitial area in MXC exposed testes when compared with controls. At all postnatal ages collected, there did not appear to be differences in seminiferous cord/tubule area, interstitial area, or number of seminiferous cords/tubules between untreated controls and males exposed to MXC. Exposure to the endocrine disruptor also had no effect on the postnatal organ weights of a variety of different organs, nor were testosterone levels altered. Interestingly, there were reductions in the number of germ cells in testes from MXC-exposed males at P17-P20 when compared with untreated controls. Furthermore, there was a twofold increase in apoptotic cells in tubules from pubertal P17-P20-MXC exposed males when compared with untreated controls. Testes were collected from adult P60 males to determine if early embryonic and postnatal alterations in germ cell numbers or testis cellular composition had compromised spermatogenesis. In adult P60 MXC exposed testes there were no gross morphological changes in testis structure or cellular composition over that of controls. However, there was an increase in apoptotic cell number in elongating spermatids in MXC exposed testes. Four P60 males that were exposed to MXC during gestation and 4 control males were bred with unexposed females to determine their ability to produce offspring. All MXC exposed males were capable of impregnating females and had normal litter size and pup weights. Combined observations demonstrated that exposure to MXC during gestation at a critical stage of testis development (ie, sex determination) affects embryonic testis cellular composition, germ cell numbers, and germ cell survival. While alterations in these parameters does not affect the ability of males to produce offspring, there appears to be a reduced spermatogenic capacity associated with MXC treatment. Therefore, transient embryonic exposure to an endocrine disruptor (methoxychlor) during gestation can influence the germline and fertility in adult males.

We report on spike discharges in the EEGs of sleeping El mice, which are considered to be homologous to the state seen in human epileptic patients. The discrete wavelet transform (DWT) was used to decompose the EEGs derived from seven electrodes, with the primary spike frequency detected in the 15.6-7.8 Hz detail. The synchronicity of the spikes was evaluated in 98 samples. We succeeded in detecting the foci of the spikes by applying the electric field model, in which the surface potential was expressed as a function of distance between the focus and each electrode based on Gauss' theorem. The foci were obtained in 73 of 91 spikes so evaluated. In 69 of these 73 spikes, the calculated foci occurred within the brain, with none of the foci occurring at identical positions. The distribution occurred in relatively deeper brain regions, suggesting involvement of the paleocortex rather than the hippocampal system. The present study provides reasonable grounds for an explanation of the EEG using the electric field model supported by efficient foci detection of the spikes in a one-to-one correspondence. This paradigm will be applicable to all animal species including humans and will be helpful in the detection of the epileptic focus and in obtaining an insight into the mechanisms of spike genesis.

The rat dominant hairless gene (Ht) of the WBN/Ila-Ht rat causes atrichosis in Ht/ Ht and hypotrichosis in Ht/+. Furthermore the Ht/Ht shows signs of abnormal keratinization and almost all of the Ht/Ht die in an immature stage before weaning in the conventional environment. Ht/+ was affected by dermatitis caused by Staphylococcus aureus, suggesting that the gene Ht might involve defense mechanisms against infection. In this study, we performed the linkage analysis of the gene Ht by outocross with the Brown Norway rat in the SPF environment. Ninety-six backcross progeny of (BN × WBN/Ila-Ht/Ht) F1 × WBN/Ila-Ht/Ht were typed with microsatellite markers and the gene Ht was mapped on chromosome 10 between Asgr1 and Nos2 within the map distance of 6.2 cM.

This report describes an autosomal incomplete dominant pattern of inheritance for osteochondrodysplasia in the Scottish Fold cats. A three-generation pedigree was analysed. Cats with folded ears were mated with cats with normal ears. All cats with folded ears, which were presumably heterozygous for the mutated allele, developed osteochondrodysplasia in distal fore- and hindlimbs but not in other bones, including the tail in which bone deformity had been demonstrated in previous studies. The severity of the skeletal lesions of osteochondrodysplasia was different in each affected cat. Most of the cats with severe osteochondrodysplasia showed some clinical signs, but cats with mild disease were clinically unaffected. All Scottish Fold-related cats with folded-ear phenotype, even if heterozygotes, suffered from some degree of osteochondrodysplasia of the distal limbs.

The male hypogonadic rate (hgn/hgn) is accompanied with bilateral hypoplastic kidney (HPK; hpk/hpk)[34]. In this study, we examined the kidney weight (KW), glomerular number (GN), renal pathohistology at adult (80 days of age), and pathogenesis during the early postnatal stage in the hpk/hpk, +/hpk, and +/+ rat. The GN of adult hpk/hpk was significantly less that that of the +/hpk and +/+. Histologically, there were the decreased number of nephrons and the associated secondary changes in the hpk/hpk kidney. The plasma concentrations of urea nitrogen and creatinine were significantly higher in the hpk/hpk than in the +/hpk and +/+. During early postnatal stage, the KW of the hpk/hpk was significantly smaller than those of the +/hpk and +/+, and the GN was fewer in order of the hpk/hpk, +/hpk and +/+, showing the significant differences between given genotypes. The layer of nephrogenic nephron beneath the renal capsula of the neonatal kidney appeared to be thinner in the hpk/hpk than in the normal (+/hpk and +/+). In the hpk/hpk kidney on 3 days of age, the layer of the nephrogenic nephron was absent in some portions beneath the renal capsula. In the hpk/hpk kidney on 7 days of age, further, the layer of immature type of nephron was still observed under the capsula. These studies suggest that the hypoplastic kidney, which may result from reduced nephrogenesis, could not compensate the real weight, GN and renal function during the postnatal nephrogenic stage.

Male hypogonadic (hgn/hgn) rats show male sterility, reduced female fertility, progressive renal insufficiency and body growth retardation. These defects are associated with loss-of-function mutation of astrin and appear to be related to organ hypoplasia resulting from abnormal cell proliferation and increased cell death during embryonic and early postnatal development. As targeted disruption of mouse spag5 (astrin ortholog) has been reported to show no phenotype, we performed rescue experiments based on the introduction of rat astrin cDNA transgene into hgn/hgn rats to determine whether astrin is actually necessary for the establishment of normal male fertility and renal function. Astrin transgenic (Tg) rats were mated with hgn/+ rats of the HGN strain, and Tg-hgn/+ rats were then crossed to obtain Tg-hgn/hgn. Tg-hgn/hgn males showed recovery of body growth, fertility and renal function. Testis size was smaller in these transgenic animals than normal controls, but showed an increase by 16.5-fold compared with hgn/hgn males. Spermatogenesis occurred in Tg-hgn/hgn testes, and their accessory reproductive organs were of approximately normal size. hgn/hgn males show hypergonadotropic hypogonadism. Increased testosterone and decreased LH levels in Tg-hgn/hgn serum indicated the recovery of Leydig cells' function. Tg-hgn/hgn males showed normal reproductive behaviour, and their mating with Tg-hgn/hgn females produced pups in normal litter size. Their renal sizes and glomerular numbers showed complete recovery, and renal function assayed by biochemical parameters was normal. These results indicated that the transgene is functional in the testis and kidney development as well as body growth. In conclusion, astrin is necessary for the establishment of normal size (cell number) and function of the testis and kidney in rats.

We established an inbred rat strain with unilateral urogenital anomalies from an incidentally identified male rat with unilateral renal agenesis and an undescended left testis. These rats were characterized by unilateral renal agenesis in both sexes, undescended testes with agenesis and hypoplasia of the accessory sex organs in male rats, and complete and partial agenesis of the uterine horn in female rats. All of these urogenital anomalies were unilateral and restricted to the left side; we named this phenotype unilateral urogenital anomalies (UUA). Breeding tests showed that these abnormalities were inherited as polygenic traits. The weight of right kidneys of affected rats was 1.7-fold higher than that of normal rats; histologically, glomerulosclerosis, tubular dilations, and tubular casts were detected at 30 wk of age. These alterations may have resulted from compensatory renal adaptation to the lack of 1 kidney. The cryptorchid left testes of affected male rats showed atrophy of seminiferous tubules and degeneration of spermatocytes and spermatids. These results indicate that the UUA rat may be a good model to study the etiology of unilateral renal agenesis accompanied by agenesis of the reproductive tract and to study compensatory alterations resulting from the congenital loss of 1 kidney.

We have characterized the phenotype of spontaneously mutated rats, found during experimental inbreeding in a closed colony of Wistar Imamichi rats. Mutant rats showed severe dwarfism, short lifespan (early postnatal lethality), and high incidence of epileptic seizures. Mutant rats showed growth retardation after 3 d of age, and at 21 d their weight was about 56% that of normal rats. Most mutant rats died without reaching maturity, and 95% of the mutant rats had an ataxic gait. About 34% of the dwarf rats experienced epileptic seizures, most of which started as 'wild running' convulsions, progressing to generalized tonic-clonic convulsions. At age 28 d, the relative weight of the testes was significantly lower, and the relative weight of the brain was significantly higher, in mutant than in normal rats. Histologically, increased apoptotic germ cells, lack of spermatocytes, and immature Leydig cells were found in the mutant testes, and extracellular vacuoles of various sizes were present in the hippocampus and amygdala of the mutant brain. Mutant rats had significantly increased concentrations of plasma urea nitrogen, creatinine, and inorganic phosphate, as well as decreased concentrations of plasma growth hormone. Hereditary analysis showed that the defects were inherited as a single recessive trait. We have named the hypothetically mutated gene as lde (lethal dwarfism with epilepsy).

Congenital hypoplasia and dysplasia affect the postnatal development of organs, their physiological functioning in adulthood and the incidence of related diseases at an advanced age. Hypogonadic (hgn/hgn) rats are characterized by male sterility, reduced female fertility, progressive renal insufficiency and growth retardation, all controlled by a single recessive allele (hgn) located on chromosome 10. Since our previous studies indicated that the hypoplasia (dysplasia) of the affected organs was present at birth, we examined the embryonic pathogenesis. We mated hgn/hgn females to Brown Norway males and backcrossed F(1) males to hgn/hgn females. The resulting N(1) fetuses were genotyped using a hgn-linked microsatellite. Both sexes of hgn/hgn fetuses showed low body weight after embryonic day (ED) 15.5 and renal hypoplasia after ED 17.5. Their kidneys contained a reduced number of nephrons in a poorly formed nephrogenic zone and renal cortex. The hgn/hgn ovaries contained a small number of oogonia at ED 15.5 and oocytes after ED 17.5. Testicular growth defects were obvious after ED 17.5, and reduced numbers of Sertoli cells were detected at ED 19.5 and 21.5. The seminiferous cords in hgn/hgn testes contained more apoptotic and mitotic cells than those in +/hgn testes. These findings suggest that the phenotypes described in adult hgn/hgn rats result from embryonic hypogenesis, which continues to early postnatal stage and causes a reduction in functional tissues and cells. Since hgn/hgn rats have an insertion mutation in the microtubule-associated protein Spag5 gene, the embryonic hypogenesis described in hgn/hgn rats might result from defective cell proliferation.

Male rats with hypogonadism (hgn/hgn) experience sterility from testicular dysplasia, which is controlled by a single recessive gene, hgn. The postnatal growth of the seminiferous tubules was severely affected. In this study, we localized thehgnlocus to a 320 kb region on rat chromosome 10 and detected the insertion of a 25 bp duplication into the sixth exon of the sperm-associated antigen 5 (Spag5/astrin/MAP126) gene, which codes for a microtubule-associated protein. This mutation results in a truncatedSpag5protein lacking the primary spindle-targeting domain at the C terminus. Immunological staining with antibodies to markers for Sertoli and germ cells during the early postnatal period indicated that the abnormal mitosis with dispersed chromosomes inhgn/hgntestes occurs in proliferating Sertoli cells. Therefore, apoptotic Sertoli cell death would result from the disorganization of the spindle apparatus caused by defectiveSpag5. These findings suggested that theSpag5is essential for testis development in rats and that thehgn/hgnrat is a unique animal model for studying the function ofSpag5.

Flutamide is a drug with antiandrogen effects that are mediated through androgen receptors (ARs). In this study, flutamide was subcutaneously administered to female rats (3, 10 or 30 mg/kg/day) on gestation Days 16-21 to evaluate effects on memory and learning performance in F1 offspring. Brain sexual differentiation was also evaluated by measuring the volume of the sexual dimorphic nucleus of the preoptic area (SDN-POA) and analyzing levels of androgen receptor (AR) mRNA expression in the prostate, hypothalamus and hippocampus. In F1 offspring exposed in utero to flutamide, evaluation of motor activity, learning performance and spatial perception showed that flutamide tended to exert a dose-dependent increase on the motor activity in F1 males, but no significant differences were identified in the other measurements. Prominent changes in development of the SDN-POA were apparent in males after maturation. Doses of > or =3 mg/kg/day resulted in significantly decreased length and volume of the SDN-POA compared to controls. These differences tended to become more marked at higher doses. Volumes of the SDN-POA did not differ significantly between F1 males and females exposed to flutamide at 30 mg/kg/day. AR mRNA was assayed using the dot-blotting method in F1 animals. In flutamide dose groups, AR mRNA expression tended to be increased in the prostate gland and decreased in the hippocampus. These results might suggest that exposure to flutamide in utero might affect controlling AR expression on a hormonal signal transduction system mediated by testosterone. However, these changes were not clearly correlated to learning performance in male offspring other than motor activity.

Rat male hypogonadism (hgn/hgn) is accompanied by oligomeganephronic hypoplastic kidney (HPK), in which each kidney contains one quarter the number of nephrons present in a normal kidney. The nephrons of the HPK are extremely hypertrophied. These characters were apparently common to human oligomeganephronia (OMN). To determine the age-related changes in renal pathophysiology in HPK rats, we measured several parameters of renal function at 70 days, 140 days, 210 days, and 280 days of age. At all time points, relative kidney weight was significantly smaller in HPK rats than in their normal litter mates. In HPK rats, both polyuria and polydipsia became more severe with advancing age. Although creatinine clearance (Ccr) and urinary nitrogen clearance (Cun) were significantly lower in HPK than in normal rats, the values did not decrease with age. A severe form of glomerulosclerosis, as well as interstitial infiltration and cystic dilation of tubules with proteinaceous luminal casts, was observed in the inner cortex and medulla of HPK rats at advanced age. In these animals the surface glomeruli retained their functional architecture but were hypertrophied. Both mean blood pressure (MBP) and proteinuria became more elevated with age in HPK rats, and their urine samples included considerable amounts of high molecular weight protein. These results suggest that the HPK rat is a suitable model of human OMN.

Background Rat male hypogonadisim (hgn/hgn) is accompanied by bilateral hypoplastic kidney (HPK). The HPK contains a reduced number of nephrons that progress to chronic renal failure. In this study, we describe the renal pathophysiology in adult HPK rats as a potential model of oligomeganephronic disease. Methods Urine and blood samples were collected from adult male HPK rats and phenotypically normal littermates at 70 days of age for measurements of urea-nitrogen and creatinine clearances (Cun and Ccr). Glomerular number (GN) and glomerular projective area were determined using the maceration method. Blood pressure was measured. Urinary protein and renal histology were examined. Urinary albumin concentration was determined at early postnatal ages. Results Renal weight was significantly smaller in adult HPK males than in normal males. Polyuria and polydipsia were observed in HPK rats. Ccr and Cun were low in HPK rats compared with those in normal rats. The HPK contained only 20% of the nephrons present in normal kidneys. The Cun and Ccr divided by GN (average values of single nephron Cun and Ccr) of HPK rats were about two and four times greater than normal levels, respectively. This hyperfiltration was not accompanied by systemic hypertension, but was associated with marked glomerular hypertrophy and glomerulosclerosis, which were observed mainly in the inner cortex. A considerable heterogeneity of glomerular size was found in HPK and most glomeruli of surface nephrons retained normal size and histology. A remarkable leakage of albumin into urine was found at 35 and 70 days of age. Conclusions The HPK rat is a useful model for studying the pathophysiology of oligomeganephronic disease as well as glomerular hyperfiltration and hypertrophy induced by severe congenital reduction of nephrons.

The hypogonadic rat (hgn/hgn) shows male sterility, reduced female fertility, and renal hypoplasia, controlled by a single recessive gene located on rat chromosome 10. We developed a fine map around the hgn locus using 565 rat backcross progeny and a Rat/Hamster radiation hybrid panel. The hgn locus was linked to Aldoc (aldolase c) and whn (winged helix of nude), and located in a 0.34-cM region between D10Rat30 and D10Rat68. The distance of the region was approximately 840-kb on rat physical map. Neither loci responsible for male sterility nor renal hypoplasia has been mapped on the homologous regions of mouse chromosome 11 and human chromosome 17. Identification of the gene responsible for the hgn mutation would provide important information on urogenital development.

A short period of exposure of pregnant mice to a strong static magnetic field of 400 mT -- 8000 times that of the earth -- in a dorso-ventral direction had teratogenic effects on developing fetuses. Fetuses were exposed to the static magnetic field in utero for 6 min on 1 day from 7.5 to 14.5 days of pregnancy. Exposed and control groups consisted of 10 pregnant mice each; thus 160 animals were used in total. Various malformations were observed in 15.1%, 13.4%, 15.8%, 16.7%, 20.8%, 24.3%, 24.4%, and 14.1% of fetuses exposed on days 7.5, 8.5, 9.5, I0.5, 11.5, 12.5, 13.5, and 14.5 of pregnancy, respectively. Types of malformations were polydactylism, abdominal fissure, fused rib, vestigial 13th rib, lumbar rib, brain hernia, and curled tail, while only a low incidence (up to 2.8%) of curled tail was detected in control group. These deformations apparently caused by SMF exposure but the effect did not reflect so-called exposure period specificity.

The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.

Hypogonadic rats of the HGN inbred strain show male sterility controlled by an autosomal single recessive allele (hgn). The testis weight of adult hgn/hgn rats is about 1% that of normal rats, and only a few thin seminiferous tubules surrounded by islet-like conformations of Leydig cells are present in the fibrous interstitial tissue of the testis (Suzuki et al. 1988). Primordial germ cells do exist in the neonatal hgn/hgn testis, but cannot differentiate into spermatogonia and degenerate before entering meiosis (Suzuki et al, 1998). hgn/hgn females have reduced fertility, including a small litter size, a reduced number of oocytes, and early reproductive senescence (Suzuki et al. 1992). hgn/hgn rats of both sexes also show bilateral hypoplastic kidney (HPK) with a reduced number (only one-quarter) of glomeruli (Suzuki et al. 1991; Suzuki and Suzuki 1995). Whether the phenotypes of hypogonadism and HPK are caused by a mutation in a single gene or defects in different genes located close to each other remains unclear. No other mutant animal or human disease showing a phenotype similar to that of the hgn/hgn rat has been reported. The hgn/hgn rat would, therefore, be a useful model for investigating the development of mammalian reproductive and urinary organs. In this study, linkage analysis was performed to determine the chromosome location of the hgn locus and to obtain information about candidate genes for hgn. Thirteen hgn/hgn females were selected from the population of the HGN inbred strain by laparotomy for detection of HPK at weaning (Suzuki et al. 1991). One hgn/hgn female was mated with a BN male rat (+/+), and the resulting F1 males were mated with the remaining 12 hgn/hgn females to produce backcross progeny. After the mating, the hgn/hgn females were autopsied to confirm the hgn/hgn phenotype (Suzuki et al. 1992). All male backcross progeny were autopsied at the age of 5 days to avoid postnatal loss, and their phenotypes were determined by macroscopic observation of testicular size. Relative testis weight of the affected rats (0.49 ± 0.06g, average ± S.D.) was about half that of normal rats (1.16 ± 0.09g). The histological appearance of the testes in the affected rats was similar to that of hgn/hgn rats of the HGN strain (Suzuki et al. 1993). Since it was difficult to clearly categorize females of the backcross progeny into affected and normal phenotypes by comparison of renal size, we did not use female rats for linkage analysis. Consequently, 29 affected and 26 normal males were obtained. The segregation ratio of the affected to normal rats did not deviate significantly from the expected 1 : 1 ratio (x 4 0.16, P > 0.20). Because of physical limitations of the PCR machine with a block of 48 samples, high-molecular-weight genomic DNA was isolated from livers of 24 affected and 24 normal rats by phenol extraction. The samples of remaining seven progeny were saved for the next mapping experiment. The total of 48 progeny were initially typed for 33 microsatellite markers on rat chromosomes. Primers for the microsatellite markers were purchased from Research Genetics Inc. (Huntsville, Ala.). The PCR was carried out in 10 m1 of a reaction mixture containing 25 ng of the genomic DNA, 1.0 pmol of each of the oligonucleotide primers, 2.0 nmol of each dNTP, and 0.26 U of Taq polymerase (TaKaRa) in the reaction buffer recommended by the manufacturer. After initial denaturation for 180 s at 92°C, 35 cycles of amplification, each consisting of denaturation for 15 s at 92°C, annealing 60 s at 55°C, and extension for 120 s at 72°C, were carried out with a Thermal Cycler TP2000 (TaKaRa). The PCR products were separated by nondenatured 10% polyacrylamide gel electrophoresis and visualized by silver staining (Silver Stain Plus Kit, Bio-Rad). The recombination fractions and the lod scores among the hgn and the other loci were calculated with Map Manager ver. 2.6.5 (distributed via the World Wide Web, URL: http://mcbio.med.buffalo.edu/mapmagr.html). The order of loci was determined so that the number of recombination events was minimized. By typing the 33 rat microsatellite marker loci in the backcross progeny, significant linkage was observed between the hgn locus and the D10Mgh10 locus on rat Chromosome (Chr) 10 with a lod score (z) of 5.1 and a recombination fraction (u) of 0.167. The D10Mgh3 locus also was linked to the hgn locus (z42.7, u40.25). No significant linkage was observed between the hgn locus and the other 31 loci. Therefore, the 48 progeny were further typed for the following seven loci located on rat Chr 10: D10Mit2, D10Mgh6, D10Mgh8 (Myh3), D10Wox14 (Asgr1), D10Wox6, D10Wox16 (Ngfr), and D10Wox12 (Abp). The highest lod score (z 4 14.4) was obtained for the D10Mit2 locus with a u value of 0. Segregation of alleles of the nine microsatellite loci as well as the hgn locus in the 48 progeny (Fig. 1) and a linkage map of rat Chr 10 with those loci (Fig. 2) are shown. The order and distances of the nine loci on the linkage map are basically identical with linkage maps reported previously (Jacob et al. 1995; Bihoreau et al. 1997). We therefore concluded that the hgn locus is located in the region close to the D10Mit2 locus on rat Chr 10. The Abp gene (Shbg in mouse) encodes androgen binding proteins expressed in Sertoli cells (Sallivan et al. 1991), which are apparently affected in the testis of hgn/hgn rats, suggesting that the gene is a candidate for hgh. As shown in Fig. 1, the presence of recombinations between the hgn and Abp loci, however, ruled out the Abp gene as a candidate. As shown in Fig. 2, the order of the functional genes (Myh3/Myhse, Abp/Shbg, Asgr1, and Ngfr) on rat Chr 10 is identical to that of the corresponding region of mouse Chr 11. Thus, the mouse gene corresponding to the hgn locus might be located in the region between map positions 37 (Asgr1) and 56 (Ngfr) of mouse Chr 11 (derived from the Mouse Genome Database; MGD). Possible candidate genes for the hgn mutation, including the Tex4, Fert2, Ube2b-rs2, Gsg2, and Lhx1 genes, have been mapped to this region. The Ube2b gene, which is implicated in postreplication repair, is located on either Chr 13 (Ube2b-rs1) or Chr 11 (Ube2brs2). Inactivation of the Ube2b gene of the mouse causes male sterility associated with chromatin modification, although homoCorrespondence to: H. Suzuki Mammalian Genome 10, 1106–1107 (1999).

In rats with genetically hypoplastic kidneys (hpk/hpk) and associated hypogonadism (hgn/hgn), their kidneys contain only one-quarter the number of nephrons that are found in those of normal rats [26]. Not surprisingly, therefore, renal excretive function has been shown to be depressed in hpk/hpk rats [26]. In the study presented here, we have examined the process of the progression of renal failure and the development of renal secondary disease in hpk/hpk rats. The plasma concentrations of urea-nitrogen and creatinine were significantly higher in adult hpk/hpk rats than in normal rats. These values elevated gradually and the degree of renal histological damage also progressed with advancing age in the hpk/hpk rats. In addition, renal anemia appeared at 140 days of age or later in these rats, and hyperplasia of the parathyroid glands was visible macroscopically at 280 days of age. In the hpk/hpk rats plasma levels of calcium and phosphorus were significantly lower and higher than in normal rats, respectively, at 280 days of age. Pathologically, the left femora of hpk/hpk rats exhibited fibrous osteodystrophy at 280 days of age and the calcium content of the right femora (as a percentage of the dry weight of bone) was significantly lower than in normal rats at both 210 and 280 days of age. These results indicate that the reduced nephrogenesis of the hpk/hpk rats causes progressive renal failure, secondarily inducing anemia, hyperparathyroidism, and osteodystrophy.

In male hypogonadic mutant rat (hgn/hgn), gonocytes degenerate and peritubular cells form multiple layers around seminiferous tubules during early postnatal testicular development. Alkaline phosphatase (AP) activity has been used as not only a tracer for the primordial germ cells (PGCs) but also a histochemical marker for the peritubular myoid cells. In the present study, we examined the localization of AP activity during the postnatal testicular development in the hgn/hgn and phenotypically normal (+/+ or +/hgn) rat. In the normal testis, high AP activity was located in the surface of the PGCs on 3 days of age. As the PGCs differentiated into spermatogonia, the AP activity drastically decreased in intensity on 7 days and was completely lost by 12 days. In the hgn/hgn, the PGCs showing high AP activity occupied the inside of dilated seminiferous tubules on 3 and 7 days of age. The luminal AP activity declined gradually by 18 days and disappeared on 21 days, when the germ cells completely degenerated in the hgn/hgn testis. In the normal, high AP activity emerged in a layer of the peritubular cells surrounding the tubules on 7 days and afterwards, indicating that the peritubular cells were differentiating into myoid cells. In the hgn/hgn, the peritubular cells formed multiple layers around the tubules and showed weak AP activity on 3 to 18 days of age. On 21 days of age, high AP activity emerged in a single layer of the peritubular cells directly attached to the basement membrane in the hgn/hgn. These results indicate that the PGCs showing AP activity kept remained at later stage in the hgn/ hgn and that the single layer of mature myoid cells showing high AP activity appeared much later in the hgn/hgn testis than in normal.