Reduced Mitotic Activity and Increased Apoptosis of Fetal Sertoli Cells in Rat Hypogonadic (hgn/hgn) Testes

Abstract

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...

Postnatal Development of Hypoplastic Thymus in Semi-Lethal Dwarf pet/pet Males

“…Thymuses were removed from all rats, weighed, fixed in 10% phosphatebuffered formalin, and processed for paraffin sectioning as described previously [4,9,11]. Sections (thickness, 5 µm) were mounted on poly-L-lysine coated slides, stained with hematoxylin and eosin, and examined by light microscopy as described previously [4,9,11]. Differences between normal and pet/pet rats were assessed by unpaired Student ttests, with statistical significance set at P<0.05 (Excel 2008 for Mac, Microsoft Corp, Redmond, WA, U.S.A) [4,11].…”

“…At least 4 pet/ pet and normal males each were euthanized by ether overdoses at 0, 1, 3, 7, 12, and 21 days of age. Thymuses were removed from all rats, weighed, fixed in 10% phosphatebuffered formalin, and processed for paraffin sectioning as described previously [4,9,11]. Sections (thickness, 5 µm) were mounted on poly-L-lysine coated slides, stained with hematoxylin and eosin, and examined by light microscopy as described previously [4,9,11].…”

“…One hour before sacrifice, rats were intraperitoneally injected with 100 mg/kg body weight 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO, U.S.A.). Isolated thymuses were processed for histological section as described above, and BrdU-labeled cells were detected by immunohistochemistry [9,11]. Apoptotic cells were detected with an in situ apoptosis detection kit (TaKaRa Bio, Ohtsu, Japan) [9,11].…”

“…Isolated thymuses were processed for histological section as described above, and BrdU-labeled cells were detected by immunohistochemistry [9,11]. Apoptotic cells were detected with an in situ apoptosis detection kit (TaKaRa Bio, Ohtsu, Japan) [9,11]. All sections were counterstained with hematoxylin.…”

“…All sections were counterstained with hematoxylin. The numbers of BrdUlabeled and TUNEL-positive cells were counted as described [11]. The areas for cell counts were defined by a square field (12421.779 µm 2 ) in the finder of a CCD camera system (Pixcera Co., Osaka, Japan) attached to a light microscope (Olympus Co., Tokyo, Japan).…”