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Keywords:

  • astrin;
  • hypogonadism;
  • renal hypoplasia;
  • Spag5;
  • spermatogenesis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The establishment of functionally normal organs is dependent on organogenesis, including cellular proliferation, differentiation, apoptosis and migration during the embryonic and early postnatal period. Reduced proliferation and increased apoptosis in developing organs may result in congenital defects, such as dysplasia and hypoplasia. Depending on the severity, these congenital anomalies may cause functional defects after birth and related diseases at advanced age.

Spontaneous mutant hypogonadic (hgn) rats show male sterility caused by testicular dysplasia (Suzuki et al., 2004b; Yagi et al., 2006), reduced female fertility caused by ovarian hypoplasia (Suzuki et al., 1992, 2006a), progressive renal dysfunction caused by bilateral renal hypoplasia (Suzuki et al., 2005, 2006b), and growth retardation beginning in the late embryonic period (Suzuki et al., 1992, 2006c). These pleiotropic defects are controlled by an autosomal single recessive allele (hgn) (Suzuki et al., 1999), and are apparently related to a reduced number of cells resulting from decreased cell proliferation and increased cell death during the late embryonic and early postnatal periods (Suzuki et al., 1992, 2004b, 2005, 2006c; Yagi et al., 2006, 2007). Using fine linkage analysis and candidate gene approach, we found a 25-bp duplicated insertion mutation in the exon 6 of the astrin/spag5 genome sequence in hgn/hgn rats (Suzuki et al., 2004a, 2006d).

Astrin is a microtubule-associated protein, which is localized to the centrosome and mitotic spindle during mitosis and is necessary for the progression of mitosis in HeLa cells (Mack & Compton, 2001; Gruber et al., 2002). Although spag5, an ortholog of astrin, is highly expressed in the rat testis (Shao et al., 2001), targeted deletion of spag5 did not cause any phenotype in male mice (Xue et al., 2002). The reasons for the discrepancy between in vitro cell culture and in vivo knockout mouse experiments remain unclear. As several variant molecules associated with the astrin/spag5 gene were still immunologically detectable in the knockout mice in the study by Xue et al. (2002), it is possible that not all functionality of spage5 was successfully knocked out in these experiments (Fitzgerald et al., 2006; Suzuki et al., 2006d).

Interestingly, in late embryonic and early postnatal hgn/hgn testes, immature Sertoli cells often show abnormal mitotic metaphase with dispersed chromosomes and increased apoptosis (Suzuki et al., 2006d; Yagi et al., 2006, 2007). These defects are suggestive if we take into consideration the putative role of astrin in mitotic cells. Our previous studies suggested that markedly reduced number of Sertoli cells and their dysfunction are the primary causes of the complete failure of spermatogenesis in hgn/hgn rats (Suzuki et al., 2004b). Thus, astrin transgenic rescue may recover spermatogenesis and male fertility in these rats.

To resolve the discrepancy between the data of astrin reported by several different groups using different methodologies, we isolated rat astrin cDNA and generated astrin transgenic (Tg) rats to examine the function of the transgene in hgn/hgn rats. If the functional loss of astrin is truly responsible for the hgn/hgn phenotype, sterility and other phenotypic characters, such as renal dysfunction and growth retardation, may be rescued in Tg-hgn/hgn rats.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Generation of astrin transgenic rats

The entire coding sequence of astrin cDNA (Suzuki et al., 2006d) was amplified by RT-PCR with rat testis RNA, and cloned into the pCAGGS vector (a kind gift from Dr. J. Miyazaki of Osaka University) (Niwa et al., 1991). The astrin transgenic (Tg) rats were generated by pronuclear injection of the pCAGGS-astrin vector (PhoenixBioCo., Tochigi, Japan). Two hundred injections were performed, and the tail DNA of the pups was used for PCR-based detection of partial transgene sequences. Six pups were positive for the gene and used as the founders in mating experiments.

Mating experiments

Tg rats (founders) were mated with hgn/+ rats, and then resultant Tg-hgn/+ rats were crossed to obtain Tg-hgn/hgn. All resulting pups were sexed at birth, and body weights were determined once a week. Germline transmission and phenotypic rescue were confirmed. Genotyping for the hgn allele was performed by PCR amplification of the 25-bp insertion into exon 6 of the spag5 gene (Suzuki et al., 2006d).

Animal care

All rats were kept in a clean conventional animal room in our laboratory under a controlled light–dark (14 : 10 h) cycle and supplied with standard pellet diet (CR-LPF; Oriental Yeast Co. Ltd., Itabashi-ku, Tokyo, Japan) and water ad libitum (Katayama et al., 2011). All experimental procedures and animal care were performed in accordance with the Guidelines of the Animal Care and Use Committee of Nippon Veterinary and Life Science University (Yagi et al., 2007).

Macroscopic observation of reproductive organs and organ weight

We euthanized Tg-hgn/+, Tg-hgn/hgn and hgn/hgn male rats at 126 days old by ether overdose and observed their reproductive organs. Normal (+/+, hgn/+,= 4), Tg-hgn/hgn (n = 3) and hgn/hgn (= 4) male rats at 179–265 days old were also euthanized and their organs were weighed on an electric balance. The urogenital organ weight data of rats with unrelated urogenital defects probably because of genetic background variation (less than 5% of all rats examined) and unstable expression of the transgene (less than 5% for all Tg-hgn/hgn rats in initial generations) were excluded.

Histological examination

Testes were fixed in Bouin's fixative for 24 h, rinsed with PBS, dehydrated through a graded ethanol series, embedded in paraffin and cut into sections 5 μm thick. The sections were deparaffinized in xylene, hydrated through a graded ethanol series, immersed in water and then stained with haematoxylin and eosin. Photographs of the sections were taken by optical/fluorescence microscopy (Biozero; Keyence, Osaka, Japan).

Immunohistochemistry for vimentin

Sections of testis were boiled in 0.01 m Citrate buffer (pH 6.0) for 15 min. The sections were immersed in methanol containing 3% H2O2 to inactivate internal peroxidases and incubated in PBS containing 10% BSA for 60 min to block non-specific antigen–antibody reaction. Sections were incubated with antibodies against vimentin (mouse monoclonal, Vimentin Ab-2, clone V9; NeoMarkers, Fremont, CA, USA) overnight at 4°C. After several washes with PBS, the sections were incubated with Histofine Simple Stain Rat MAX-PO (Nichirei Biosciences, Chuo-ku, Tokyo, Japan) for 1 h at room temperature, washed three times with PBS and incubated with 3,3′-diaminobenzidine chromogen for colouring reaction. Counterstaining was performed with haematoxylin.

Fertility testing of male rats

Tg-hgn/+ or Tg-hgn/hgn male rats were cohabited with proestrus female rats. Mating was confirmed with vaginal plug and spermatozoa in the vagina on the next day. Litter sizes from mating of Tg-hgn/+ or Tg-hgn/hgn males with Tg-hgn/hgn females were compared.

Serum LH and testosterone assay

Blood samples were collected from the vena jugularis. Serum concentrations of LH were measured in normal (= 5), Tg-hgn/hgn (= 5) and hgn/hgn (= 4) males at 195–215 day of age by enzyme-linked immunosorbent assay using rodent LH ELISA kit (Endocrine Technologies, Inc., Newark, CA, USA). Serum concentrations of testosterone were measured in normal (= 3), Tg-hgn/hgn (n = 3) and hgn/hgn (= 3) male rats at 128–131 days of age by RIA (Mitsubishi Chemical Medience Corporation, Minato-ku, Tokyo, Japan).

Determination of number of glomeruli

Glomeruli in the right kidney of 255–304-day-old rats (= 2 for +/+, = 3 for Tg-hgn/hgn and hgn/hgn) were counted by the HCl-maceration methods as described previously (Suzuki & Suzuki, 1995; Suzuki et al., 2005). Although one hgn/hgn rat showed hydronephrosis of the left kidney, which is a polygenic trait in the genetic background of the HGN strain (Suzuki et al., 2006d), the right was apparently normal despite compensatory hypertrophy.

Blood chemistry analysis

Blood samples were collected from the vena cava or vena jugularis of normal (= 3), Tg-hgn/hgn (= 3) and hgn/hgn (= 5) males at 250–285 days of age with a heparinized plastic syringe under ether anaesthesia. Plasma samples were obtained and stored at −20°C until assay. Plasma concentrations of urea nitrogen (UN), creatinine (CRE), albumin (ALB) and total protein (TP) were measured automatically (Dri-Chem 3500V; Fujifilm Medical Company, Minato-ku Tokyo, Japan) (Suzuki et al., 2007).

Statistical analysis

Organ weights, results of blood chemistry assays and glomerular number are reported as means and standard deviations, and the differences were evaluated using the unpaired Student's t-test with < 0.05 taken to indicate statistical significance.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Phenotype of Tg rats

Tg rats were indistinguishable from normal rats in all aspects examined, including viability, growth, organ weight and fertility in both males and females (data not shown). Therefore, Tg-hgn/+ rats were used as controls in some experiments.

Body growth of Tg-hgn/hgn

The body weight of Tg-hgn/hgn males was intermediate between those of hgn/+ and hgn/hgn males, but significantly greater than that of hgn/hgn males (Fig. 1). This result indicated that growth retardation in hgn/hgn rats was recovered by the presence of the transgene.

image

Figure 1. Growth of hgn/+, hgn/hgn and Tg-hgn/hgn males during nursing (A) (= 12–29 for each genotype) and after weaning (B) (= 5–18 for each genotype). The body weights of Tg-hgn/hgn males were significantly (p < 0.05) greater than those of hgn/hgn males at all ages examined except for 3 days of age, and significantly (p < 0.05) smaller than those of hgn/+ males at 7, 12, 49 and 56 days of age.

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Macroscopic observation of reproductive organs and organ weights

To determine whether the astrin cDNA transgene is able to recover the hypogonadism in hgn/hgn male, we first performed macroscopic observation of reproductive organs in adult rats. As shown in Fig. 2, testis and accessory reproductive organs of Tg-hgn/hgn were markedly larger than those of hgn/hgn, and some accessory reproductive organs appeared to be similar in size to those of normal males. The weights of almost all organs in Tg-hgn/hgn, which were affected in hgn/hgn, were recovered from 52% to similar to those in normal controls (Table 1). Testis weight of Tg-hgn/hgn was 16.5-fold greater than that of hgn/hgn. The weights of all accessory reproductive organs were greater than those of hgn/hgn, and the absolute weights of the deferent duct, coagulating gland, seminal vesicle, dorsal lobe of the prostate gland, bulbourethral gland and levator ani in Tg-hgn/hgn were significantly greater than those of hgn/hgn (Table 1). The kidneys of Tg-hgn/hgn were also similar in weight to those of normal rats (Table 1).

image

Figure 2. Macroscopic observation of whole urogenital system of Tg-hgn/+ (left), Tg-hgn/hgn (centre) and hgn/hgn (right) males at 126 days of age. Testis (arrowheads) and accessory sex organs of Tg-hgn/hgn were markedly greater than those of hgn/hgn. 1: seminal vesicles, 2: epididymis, 3: ventral lobe of the prostate gland, 4: Cowper's gland, 5: penis, 6: preputial gland.

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Table 1. Organ weights of adult male rats
 Absolute (mg)
 Normal (+/+, hgn/+) n = 4Tg-hgn/hgn n = 3hgn/hgn n = 4
  1. a

    Significantly (p < 0.05) less than values for normal.

  2. b

    Significantly (p < 0.05) greater than values for hgn/hgn.

  3. c

    Data from a normal rat with hydronephrosis were excluded.

  4. d

    Mean value from two hgn/hgn rats, since epididymides of two hgn/hgn were not discerned

  5. clearly.

  6. Relative weight : organ weight (mg) × 100 /body weight (g).

Testis1491.7 ± 81.4775.8 ± 114.0a,b46.8 ± 15.9a
Kidneyc1717.4 ± 183.91446.2 ± 145.3b1051.4 ± 107.4a
Epididymides607.3.4 ± 51.2389.9 ± 50.1227.3d
Preputial gland79.9 ± 23.252.9 ± 9.342.4 ± 14.6a
Deferent duct138.1 ± 24.6104.5 ± 6.9b79.5 ± 12.7a
Coagulating gland136.0 ± 18.895.4 ± 27.9b53.0 ± 10.0a
Seminal vesicle915.9 ± 239.3424.2 ± 78.5a,b267.4 ± 24.7a
Ventral lobe of prostate gland676.8 ± 212.8415.6 ± 111.0260.3 ± 52.4a
Dorsal lobe of prostate gland447.5 ± 171.0287.5 ± 84.1b117.3 ± 45.7a
Bladder157.7 ± 32.4114.9 ± 5.0137.3 ± 43.2
Penis400.7 ± 45.4337.6 ± 62.1282.4 ± 28.8a
Bulbourethral gland53.7 ± 6.847.9 ± 9.0b27.3 ± 7.0a
Bulbourethralis1115.2 ± 119.0805.2 ± 208.6536.5 ± 104.5a
Levator ani357.0 ± 35.6305.5 ± 57.0b197.8 ± 46.8a
Adrenal gland27.9 ± 2.423.4 ± 2.726.9 ± 7.4
Brain2186.5 ± 78.32099.3 ± 75.5b1957.3 ± 34.4a
Pituitary12.0 ± 0.512.9 ± 3.210.7 ± 1.1
Spleen1119.8 ± 47.9734.9 ± 281.2832.1 ± 87.0a
Pancreas1679.6 ± 266.21146.3 ± 248.5a1400.4 ± 234.4
Submaxillary gland399.9 ± 49.7279.4 ± 44.2a303.5 ± 51.8a
Salivary gland60.4 ± 5.753.3 ± 7.258.3 ± 8.7
Extraorbital lacrimal gland135.3 ± 12.1121.2 ± 34.0129.6 ± 14.6
Liver21024.4 ± 3440.418863.5 ± 1705.115090.3 ± 2564.0a
Thymus309.3 ± 58.0190.5 ± 77.1165.6 ± 69.7a
Thyroid26.1 ± 6.823.2 ± 10.318.6 ± 5.1
Heart1538.7 ± 125.31421.8 ± 114.91168.3 ± 152.3a
Lung1944.4 ± 290.71946.1 ± 66.6b1646.9 ± 157.6
Perirenal fat5850.5 ± 1360.45247.0 ± 308.54550.6 ± 1422.0
Body weight (g)595.0 ± 36.3550.3 ± 56.8429.5 ± 54.6

Histological examination of the testis

The macroscopic appearances and the testis and accessory reproductive organ weights in Tg-hgn/hgn suggested that the male hypogonadism was rescued by the transgene and spermatozoa was produced. We performed histological examination of the testis in Tg-hgn/hgn rats at 126 days of age. In the hgn/hgn testis, a few abnormal tubules were observed in the fibrous interstitial tissue, and no spermatogenesis was observed (Fig. 3). Although sections of seminiferous tubules of Tg-hgn/hgn were slightly smaller in size than in the Tg-hgn/+ testis, spermatogenesis was observed as normal. Sertoli cell, Leydig cells, myoid cells and germ cells at all differential stages were detected and appeared normal in both Tg-hgn/+ and Tg-hgn/hgn testes (Fig. 3).

image

Figure 3. Testicular histology of Tg-hgn/+ (left), Tg-hgn/hgn (centre) and hgn/hgn (right) males at 126 days of age. Although the diameters of seminiferous tubular sections were narrower in Tg-hgn/hgn than Tg-hgn/+ testes, spermatogenesis was observed in the seminiferous tubules in Tg-hgn/hgn. No spermatogenesis was observed in abnormal seminiferous tubules of hgn/hgn. Islet distribution of Leydig cells surrounding the tubules was also observed in hgn/hgn testis. Scar bars: 50 μm.

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Immunohistochemistry for vimentin

To characterize the testis phenotype of Tg-hgn/hgn in more detail, we performed immunohistochemistry for vimentin (marker of Sertoli cell in seminiferous tubule) (Suzuki et al., 2004b; Yagi et al., 2006). Vimentin-positive Sertoli cells were arranged in seminiferous tubules in normal rat testis. In hgn/hgn testis, internal structures of the tubules were disrupted, and the indistinctive cells remained in the tubules were not germ cells, but somatic cells with abnormal vimentin filaments. Although the number of Sertoli cells was less and the thickness of the seminiferous epithelium in Tg-hgn/hgn was shorter than that of normal rat, seminiferous tubules of Tg-hgn/hgn contained the arranged Sertoli cells (Fig. 4).

image

Figure 4. Immunohistochemistry for vimentin in adult rat testis. Arranged sertoli cells were observed in seminiferous tubules of normal and Tg-hgn/hgn rat testis. Internal structures of tubules were disturbed and arranged sertoli cells were not found in hgn/hgn. Germ cells (negative cells for vimentin) were seen in normal and Tg-hgn/hgn seminiferous tubules, but not seen in hgn/hgn. Scale bar: 50 μm.

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Serum concentration of LH and testosterone

Serum LH concentration of Tg-hgn/hgn was significantly reduced as almost normal level, compared with that of hgn/hgn males (Table 2). Serum concentrations of testosterone in hgn/hgn were significantly less than that of normal rats (p < 0.05) as described previously (Hakamata et al., 1988). Tg-hgn/hgn showed the recovery of serum testosterone concentration and the significantly higher value of the serum testosterone than those of normal and hgn/hgn rats (p < 0.05) (Table 3).

Table 2. Serum concentration of LH (ng/mL)
Normal (= 5)Tg-hgn/hgn (= 5)hgn/hgn (n = 4)
  1. a

    Significantly (p < 0.05) greater than values for normal and Tg(astrin)- hgn/hgn.

  2. b

    Significantly (p < 0.05) less than values for hgn/hgn.

2.48 ± 0.533.13 ± 0.90b5.83 ± 1.81a
Table 3. Serum concentaration of teststerone (ng/mL)
Normal (= 3)Tg-hgn/hgn (n = 3)hgn/hgn (= 3)
  1. a

    Significantly (p < 0.05) greater than values for hgn/hgn and normal.

  2. b

    Significantly (p < 0.05) less than values for normal and Tg- hgn/hgn.

0.98 ± 0.071.15 ± 0.05a0.11 ± 0.03b

Fertility in Tg-hgn/hgn males

The above data indicated that the astrin cDNA transgene markedly rescued hypogonadism in hgn/hgn. Next, we examined whether the fertility of hgn/hgn was also restored by the astrin cDNA transgene. The breeding data clearly indicated the rescue of fertility in male Tg-hgn/hgn rats. Overnight, cohabitation of 14 Tg-hgn/+ males 24 times with females led to 23 matings and 22 parturitions. Cohabitation of seven Tg-hgn/hgn males 14 times with females led to 12 matings and parturitions. As shown in Table 4, mating between Tg-hgn/hgn males and Tg-hgn/hgn females produced 13.3 ± 4.9 pups. There was no significant difference in litter size between Tg-hgn/+ males and Tg-hgn/hgn males. Therefore, fertility of hgn/hgn was restored by the astrin cDNA transgene.

Table 4. Litter size in Tg-hgn/+ and Tg-hgn/hgn males
MaleFemaleLitter numberLitter size
  1. There was no significant difference in litter size.

Tg-hgn/+Tg-hgn/hgn312.7 ± 1.5
Tg-hgn/hgnTg-hgn/hgn813.3 ± 4.9

Glomerular number

Homozygotes for hgn also exhibit kidney hypoplasia with reduced numbers of nephrons. The organ weight data showed that the size of the kidney was restored in Tg-hgn/hgn males. We examined whether the glomerular number was also recovered in Tg-hgn/hgn males. As shown in Table 5, the number of glomeruli in the right kidneys of male Tg-hgn/hgn rats was similar to that of +/+ rats, and significantly greater than that of hgn/hgn males (< 0.05).

Table 5. Right kidney weight and glomerular number of +/+, Tg-hgn/hgn and hgn/hgn at 255–304 days of age
GenotypeKW (mg)GNGN/KW (mg)
  1. a

    Significantly greater than values for hgn/hgn (< 0.05).

  2. KW: right kidney weight, GN: glomerular number.

  3. Values of Tg-hgn/hgn and hgn/hgn represent means ± SD. Values of +/+ are averages of two rats.

+/+ (= 2)1703.82678015.7
Tg-hgn/hgn (= 3)1576.7 ± 329.728485 ± 4698a18.1 ± 2.3a
hgn/hgn (= 3)1277.8 ± 409.912067 ± 264010.0 ± 3.3

Plasma concentrations of UN, CRE, TP and ALB

In hgn/hgn rats, the plasma concentrations of UN and CRE were gradually increased, but that of ALB was decreased with the advance of renal dysfunction (Suzuki et al., 2005, 2006b). The plasma concentrations of UN and CRE of Tg-hgn/hgn males were almost comparable with those of normal controls and were significantly lower than those of hgn/hgn males (< 0.05). The plasma ALB concentration was also comparable between normal and Tg-hgn/hgn males, and was significantly higher in Tg-hgn/hgn than in hgn/hgn males (< 0.05) (Table 6). These data strongly suggested that the number of glomeruli and renal function were completely restored in Tg-hgn/hgn males.

Table 6. Plasma concentrations of biochemical markers related with renal function in 250–285-day-old rats
 Normal (n = 3)Tg-hgn/hgn (n = 3)hgn/hgn (n = 5)
  1. a

    Significantly less than value for hgn/hgn (< 0.05).

  2. b

    Significantly greater than value for hgn/hgn (< 0.05).

UN (mg/dL)14.9 ± 1.1a14.5 ± 1.2a41.2 ± 14.2
CRE (mg/dL)0.47 ± 0.06a0.47 ± 0.06a1.20 ± 0.37
ALB (g/dL)3.3 ± 0.1a3.3 ± 0.1b2.4 ± 0.1
TP (g/dL)5.9 ± 0.46.0 ± 0.15.7 ± 0.5

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The hgn/hgn rat was identified as a spontaneous mutation originally found in our laboratory and established as a mutant strain. Our previous study suggested that the hgn/hgn pleiotropic phenotype is associated with a 25-bp duplicated insertion mutation in the astrin gene (Suzuki et al., 2006d). This mutation is considered to cause a frame shift leading to the introduction of a stop codon, producing a truncated product lacking almost all of the coiled-coil domain (Suzuki et al., 2006d), which is needed for spindle localization of astrin (Mack & Compton, 2001). This finding, however, was inconsistent with an early report indicating no phenotype in mice with targeted deletion of spag5 (astrin ortholog) (Xue et al., 2002). On the other hand, a study using HeLa cells indicated that astrin was localized in centrosomes and the mitotic spindle in mitotic cells, and silencing of astrin in HeLa cells by RNA interference resulted in mitotic arrest and apoptosis with formation of multipolar and highly disordered spindles (Gruber et al., 2002). This prompted us to perform the astrin rescue experiment in hgn/hgn rats. As the function of astrin in mitosis has only been demonstrated in human HeLa cells (Gruber et al., 2002) and mouse oocytes (Yuan et al., 2009), the actual function of astrin in vivo was unclear. In this study, we clearly demonstrated that astrin is functional in vivo because the transgene recovered male sterility, renal dysfunction and body growth retardation in hgn/hgn rats.

Our RT-PCR experiments (data not shown) and gene expression data from NCBI (GEO Profiles, NCBI, http://www.ncbi.nlm.nih.gov/geoprofiles) demonstrated the ubiquitous expression of astrin/spag5 in various organs during development. In hgn/hgn rats, body growth retardation began during the embryonic stages (Suzuki et al., 2006c), suggesting that the cause of dwarfism in this mutant strain is not hormonal. Considering the expression pattern of astrin and body growth retardation, we used a CAG vector expected to yield a wide range of expression in all of organs. Our strategy successfully rescued the body growth deficit in hgn/hgn rats. Our previous study indicated that the primary cause of testicular dysplasia is a decreased number of Sertoli cells owing to increased apoptosis and abnormal mitosis (Yagi et al., 2006, 2007), and that most of the cell types in the testis appeared to be secondarily defective in hgn/hgn males (Suzuki et al., 2004b). In this study, both spermatogenesis and spermiogenesis occurred successfully in Tg-hgn/hgn rats, although the testes were still smaller in Tg-hgn/hgn rats than in wild-type controls. The smaller testis size appeared to be accompanied by a smaller number of Sertoli cells. This may be related to dysregulation of spatial and temporal expression of the transgene and/or lack of other variant molecules of astrin.

In Tg-hgn/hgn rats, astrin 132 kDa peptide (homologue for human 134 kDa astrin) is produced from the transgene, but spag5 200 kDa peptide is absent (Suzuki et al., 2006d) because of the mutation of hgn. In a previous study, two peptides of ca. 125 kDa (probably consistent with mouse astrin) and 150 kDa were still immunologically detected in knockout mice lacking 200 kDa spag5 (Xue et al., 2002). These results indicated that while spag5 200 kDa peptide is not required, astrin 132 kDa peptide is necessary for occurrence of both spermatogenesis and spermiogenesis.

Our previous study revealed that plasma testosterone is low and levels of gonadotropins are high in adult hgn/hgn males, and that their hypothalamus and accessory reproductive organs are response to exogenous testosterone (Hakamata et al., 1988). Pathological study during postnatal testicular development indicated that hgn/hgn males show abnormal islet distribution of adult type-Leydig cells surrounding the seminiferous tubules and decrease in testicular testosterone content (Suzuki et al., 1993, 2004b). These results suggested that the hypogonadism is involved in the dysfunction of Leydig cells. In Tg-hgn/hgn males, histological distribution of Leydig cells and their function were rescued. A series of recoveries in accessory reproductive organs, serum LH concentration and male mating behaviour are probably caused by increased testosterone level in serum.

The chronic renal failure in hgn/hgn rats is considered to be caused by a reduced number of nephrons. Associated renal diseases, such as anaemia, hyperparathyroidism and fibrous osteodystrophy, appeared to be secondarily caused by renal dysfunction. Embryonic and early postnatal nephrogenesis are delayed in hgn/hgn rats (Suzuki & Suzuki, 1995; Suzuki et al., 2006c). In Tg-hgn/hgn, however, nephron number was increased to the same level as in normal rats, and renal function was also completely recovered by this increased number of nephrons. These results clearly indicated that astrin is necessary for the establishment of renal function, including the normal number of nephrons.

The results of an in vitro culture experiment using mouse oocytes indicated that astrin is critical for the progression of meiosis and oocyte maturation (Yuan et al., 2009). Female hgn/hgn are fertile but show reduced litter size and early reproductive senescence. These defects may be related to the reduced number of oocytes in the ovary at birth (Suzuki et al., 1992). In addition to the rescue of body growth and renal size (data not shown), Tg-hgn/hgn females showed normal litter size in mating experiments (Table 4). Although we have not yet examined female reproduction in detail in these transgenic animals, reproductive function also seemed to be rescued in Tg-hgn/hgn females. Further investigations are required to determine the role of astrin in ovary development.

This study yielded several important results. First, mutation of the astrin gene is apparently responsible for the hgn phenotype, and our transgene, astrin cDNA, is functional in the developing testis and kidneys, although several variants of astrin/spag5 have been reported to date (Xue et al., 2002; Fitzgerald et al., 2006). Second, in contrast with the results in HeLa cells, our results clearly demonstrated that most of in vivo cell mitosis progresses normally in the absence of astrin, and that the cells in the developing testis and kidney are severely affected by lack of astrin.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to Dr. Jun-ichi Miyazaki (Osaka University) for his generous gift of pCAGGS vector. This work was supported in part by a Grant-in-Aid for Scientific Research to H Suzuki (no. 19580350 and no. 22580341) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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