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- Original Article -

Apoptotic Sertoli cell death in hypogonadic(hgn/hgn) rat testes during early postnatal development

Mio Yagi, Katsushi Suzuki, Hirotesu Suzuki

Department of Veterinary Physiology, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan

Abstract

Aim: To determine the involvement of apoptotic cell death in postnatal pathogenesis in mutant strain of hypogonadic (hgn/hgn) rats testes. We evaluated the numbers and types of cells undergoing apoptotic cell death. Methods: Tissue sections were stained by the TUNEL method for in situ detection of apoptotic cells, with specific antibodies used as markers of testicular somatic and germ cells.  Results: We found that apoptosis in the hgn/hgn testes during the early postnatal period occurred primarily in Sertoli cells, which should actively proliferate during this stage of differentiation. These findings strongly suggest that the normal allele of hgn is involved in the direct or indirect control of differentiation and proliferation of Sertoli cells. Conclusion: To our knowledge, this is the first report demonstrating early postnatal apoptosis of Sertoli cells, suggesting that the hgn/hgn rat is a unique model for the study of Sertoli cell deficiency. (Asian J Androl 2006 Sep; 8: 535_541)

Keywords: apoptosis; hgn/hgn; male hypogonadism; Sertili cell; testis; TUNEL; vimentin

Correspondence to: Prof. Hiroetsu Suzuki, Department of Veterinary Physiology, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan.
Tel: +81-422-31-4131, Fax: +81-422-33-2094
E-mail: hiroetsu@nvlu.ac.jp
Received 2006-01-11 Accepted 2006-02-27
DOI: 10.1111/j.1745-7262.2006.00181.x

1 Introduction

Sertoli cells are important for both the development and function of the testes. In embryonic gonads, precursor Sertoli cells express the sex-determining gene Sry, which induces formation of testes cords by activating expression of male specific genes. In adult testes, Sertoli cells support germ cells at various stages of their differentiation and carry out spermatogenesis in cooperation with other testicular cell types and under hormonal control. Therefore, defects in the proliferation and differentiation of Sertoli cells result in testicular dysfunction [1], and the establishment of a normal number of Sertoli cells is critical for a normal level of sperm production during adulthood [2]. Postnatal Sertoli cell proliferation, however, occurs only during a short postnatal period, which, in rats, occurs only 2 weeks after birth [3].

To study testicular development, we have used a mutant strain of hypogonadic (hgn/hgn) rats. Male hgn/hgn rats are sterile because of dysplastic testes development [4], female hgn/hgn rats show reduced fertility as a result of ovarian hypoplasia [5], and rats of both sexes show progressive renal failure from renal hypoplasia [6]. All of these defects are controlled by a single recessive allele (hgn) located on chromosome 10 [7]. Sertoli cells in hgn/hgn testes have low proliferative activity during the early postnatal period, and there are too few Sertoli cells to normally distribute along the basement membrane of the seminiferous tubules. Furthermore, a considerable number of apoptotic cells have been detected in postnatal hgn/hgn testes, and, based on histological criteria, these apoptotic cells are likely Sertoli cells [8].

Physiological apoptotic cell death is observed during both testicular development and spermatogenesis, and it has been reported that these apoptotic cells are germ cells but not somatic cells [9, 10]. Germ cell apoptosis, which occurs directly or is secondarily induced by the dysfunction of somatic cells, has been well documented in testicular pathology [11]. To our knowledge, however, in both physiological and pathological conditions, there have been no other reports describing Sertoli cell apoptosis during the early postnatal period. To characterize the testicular phenotype of hgn/hgn rats, it is important to evaluate apoptotic cell death during postnatal testicular deve-lopment. Therefore, we assayed the distribution of Sertoli cells and the number of apoptotic cells in the postnatal testes of hgn/hgn and normal rats by immunostaining for the markers of testicular somatic and germ cells and by the TUNEL method for in situ detection of apoptotic cells. The markers used in the immunostaining were vimentin (VM) [12] for somatic cells, Mullerian inhibiting substance (MIS) [13] for Sertoli cells, and Vasa homologue (VSH) [14] for germ cells.

2 Materials and methods

2.1 Preparation of paraffin sections

The experimental procedure and care of animals were in accordance with the guidelines of the Animal Care and Use Committee of Nippon Veterinary and Life Science University [6, 8]. We used male rats from the hgn inbred strain maintained in our department [4, 5, 7, 8]. Sister-brother mating of the strain has continued for more than 50 generations. They were killed at postnatal days (PD) 0, 1, 3, 7 and 12 by overdose of ether, and their testes were removed. Male hgn/hgn rats were distinguished from phenotypically normal (+/hgn or +/+) littermates by weighing their testes using an electric balance [4, 7, 8]. Their testes were fixed in Bouin’s solution for 2_5 h. The fixative was replaced with alcohol, and the testes were embedded in paraffin and sectioned into 3 µm thick slices [6, 8].

2.2 Double staining

TUNEL staining was performed as previously reported using an in situ apoptosis detection kit (TaKaRa Bio, Shiga, Japan) [8]. After deparaffinization, the tissue sections were processed in a microwave oven (3 × 1 min) in apoptosis-preparation solution (Nara Pathological Institute, Nara, Japan) and then immersed in 0.01 mol/L Tris-buffered saline (TBS; pH 7.5). Fluorescein isothiocyanate (FITC)-conjugated dCTPs were incorporated into nick DNA for 90 min at 37ºC in the presence of terminal deoxynucleotidyl transferase (TdT). The sections were rinsed in TBS and incubated with Horseradish peroxides (HRP)-conjugated anti-FITC antibody for 30 min at 37ºC. The slides were incubated with 3,3´-diaminobenzidine tetrahydrochloride (DAB) for 10 min, and the reactions were stopped by immersion in tap water. The sections were subsequently processed for anti-VM or anti-VSH immunostaining. After immersion in 0.1 mol/L glycine-HCl buffer (pH 2.0) for 15 min, the slides were washed in PBS (0.1 mol/L, 1× 5 min [anti-VM]; 0.01 mol/L, 1 × 5 min [anti-VSH]) and incubated for 10 min in PBS containing 10% normal rabbit or goat serum to block non-specific antigen-antibody reactions. The sections were incubated with anti-VM antibody (clone V9, 1:40 dilution, LabVision Co., Fremont, CA, USA) [11] for 48 h at 4ºC or anti-mouse VSH antibody (rabbit polyclonal, 1:1000 dilution; the kind gift of Dr Toshiaki Noce, Mitsubishi Kagaku Institute of Life Sciences, Minami-Ooya, Machida, Tokyo, Japan) [14] for 24 h at 4ºC, and washed in PBS (3 × 5 min). The primary antibodies were detected with streptavidin and biotin complex methods (Histofine SAB-AP kit; Nichirei Co., Tokyo, Japan) [8]. A Fast Blue substrate kit (Nichirei Co., Tokyo, Japan) was used to detect the activity of alkaline phosphatase conjugated with streptavidin. After stopping the reaction by immersion in tap water, the stained sections were mounted in soluble mounting medium and examined under a light microscope (BX50, Olympus Co., Tokyo, Japan), with images obtained by a Penguin 600CL digital camera system (Pixcera Co., Oosaka, Japan) attached to the microscope [6, 8].

2.3 Apoptotic cell counts

This experiment utilized testes from three normal and three hgn/hgn male rats at each time point. More than ten areas, each of which was defined by a square field (SF, 76738.16 µm²) in the finder of CCD camera of the digital camera system (Pixcera Co., Oosaka, Japan), were randamoly selected from the histological sections of each testis, and the numbers of TUNEL-positive cells were counted. The values were averaged and compared between the normal and hgn/hgn male rats using Mann-Whitney U-test.

2.4 Immunostaining of MIS

Tissue sections were processed in a microwave (5 × 3 min) in 0.01 mol/L citric acid buffer (pH 6.0) to reactivate their antigenicity, soaked in water, immersed in 2 mol/L HCl for 20 min, and soaked in PBS. The sections were immersed in methanol containing 3% periodic acid to inactivate internal peroxidases and incubated in PBS containing 10% normal rabbit serum for 60 min to block non-specific antigen-antibody reactions. Sections were incubated overnight at 4ºC with antibodies against MIS (goat polyclonal, 1:500 dilution; Santa Cruz Biotechnology, CA, USA). The sections were rinsed in PBS and incubated with Histofine SAB-PO (G) kit (Nichirei, Tokyo, Japan) for goat polyclonal primary antibodies. The slides were incubated with 3,3´-DAB and counterstained with hematoxylin.

3  Results

TUNEL and VM double staining revealed temporal and special distributions of VM-positive Sertoli cells and TUNEL-positive apoptotic cells in the seminiferous tubules during the early postnatal period. Figure 1 shows photographs of doubly stained testes on PD 1_12. Although VM was detected in several types of testicular somatic cells, in seminiferous tubules, VM was detected in the cytoplasm under the nucleus of each Sertoli cell. In normal testes, therefore, intense immunostaining associated with Sertoli cells formed an alignment along the basement membrane on PD 0_3 (Figure 1A, C, E), and, on PD 7_12, the VM filament of each Sertoli cell extended towards the tubular lumen (Figure 1G, I and Figure 2C). In hgn/hgn testes, there were fewer VM-positive Sertoli cells than in normal testes, and we often found the spot-like distribution of the VM associated with Sertoli cells in the tubular lumen (Figure 1B, D, F and Figure 2B). On PD 7_12, the Sertoli cells in hgn/hgn testes failed to form an axial extension of VM filaments (Figure 1H, J and Figure 2D). In hgn/hgn testes on PD 3_7, interstitial fibrous tissue, which was positive for VM, increased (Figure 1F, H).

There were few TUNEL-positive cells in the semini-ferous tubules of normal testes on PD 0_7, whereas many TUNEL-positive cells were observed in the tubules on PD 12 (Figure 1A, C, E, G, I and Figure 3). In hgn/hgn testes, however, a considerable number of TUNEL-positive cells were observed on all postnatal days examined, with the highest number of apoptotic cells detected on PD 1 (Figure 1B, D, F, H, J and Figure 3). In normal testes, TUNEL-positive cells were located in the central portion of the tubular lumen, and these cells did not stained with anti-VM antibody (Figure 1I and Figure 2A, C). In hgn/hgn testes on PD 1, most of apoptotic cells containing TUNEL-positive nuclei were observed near the basement membrane of the seminiferous tubules. TUNEL-positive cells were also found in the central portion of the tubules in hgn/hgn testes on PD 1, and immunostain-ing with antibody to VM was observed around the TUNEL-positive nuclei (Figure 2B). In hgn/hgn testes on PD 12, TUNEL-positive cells were located in the center of the tubules, and these cells were positive or negative for VM (Figure 2D).

Because VSH is located in the cytoplasm of germ cells [14], the immunostaining with antibody to VSH formed circles around the large nuclei of gonocytes. In TUNEL and VSH double staining on PD 1, the TUNEL-positive nucleus was surrounded by the immunostaining of VSH in normal testes (Figure 4A). In hgn/hgn testes, however, TUNEL-positive small nuclei were present outside the circles of VSH immunostaining in the cytoplasm of gonocytes (Figure 4B). MIS has been detected in the cytoplasm of Sertoli cells [13]. Because the number of Sertoli cells was apparently smaller in hgn/hgn testes than that in normal testes, the total area containing MIS in the seminiferous tubules was smaller in hgn/hgn testes, compared to that in normal testes (Figure 4C, D). In normal testis on PD 1, the nuclei of MIS-positive cells were uniform in shape and formed palisade-like distributions along the basement membrane of the tubules (Figure 4C). In hgn/hgn testis, the nuclei of MIS-positive Sertoli cells were irregular and small, and Sertoli cells often showed nuclear condensations (Figure 4D).

4 Discussion

We previously reported that the postnatal pathogenesis of hgn/hgn testes included apoptotic cell death in the seminiferous tubules. Because we also observed a decreased number of cytokeratin-positive cells and a disappeartween ance of follicle-stimulating hormone (FSH) mRNA expression during early postnatal development, we predicted that the apoptotic cells might be Sertoli cells [8]. Although Sertoli cell dysfunction is a major cause of male reproductive problems [1], there have been no other animal models with defective development of functional Sertoli cells during postnatal testicular development. In addition, to our knowledge, there have been no other reports describing apoptotic Sertili cell death during early postnatal development, the period during which Sertoli cells actively proliferate in normal testes.

The hpg mice have a decreased number of Leydig, Sertoli and germ cells [15]. By contrast to hgn/hgn rats, during the postnatal developmental stage, no apoptotic cell death has been reported in the Sertoli cells of hpg mice. The pathological difference between hpg mice and hgn/hgn rats might be a result of the difference be their primary causes. The hpg mice, which lack GnRH, show hypogonadotropic hypogonadism [15]. The primary cause of hypogonadism in hgn/hgn rats is present in the testes, because they have high levels of gonadotropins and low levels of testosterone in plasma [16].

We could not find any reports describing in situ detection of apoptotic Sertoli cells with double staining of TUNEL and immunostaining. In the present study, using TUNEL and VM double staining, we showed postnatal alteration in the number of TUNEL-positive apoptotic cells and identified VM-positive apoptotic cells in the seminiferous tubules of hgn/hgn testes. VM is an intermediate filament protein, which is expressed in somatic cells derived from mesenchymal cells. In the seminiferous tubules, VM is expressed by Sertoli cells, not by germ cells. In contrast to cytokeratin and MIS [12, 13], whose expressions are reduced with Sertoli cell differentiation, VM is expressed in Sertoli cells through their differentiation and maturation. To confirm the apoptotic cells as being Sertoli cells in hgn/hgn testes on PD 1, TUNEL and VSH double staining, as well as immunostaining of MIS, were also performed.

In the immunostaining of VM, we observed defective distributions of VM filaments in the Sertoli cells of hgn/hgn testes. Abnormal spot-like distributions of VM filaments in the tubular lumen of hgn/hgn testes indicate the detachment of apoptotic Sertoli cells from the tubular basement membrane [8]. During the postnatal deve-lopment of normal testes, VM filaments extend towards the tubular lumen in coordination with the cytological differentiation of Sertoli cells [12]. Because abnormal distribution of VM filaments is related to defective differentiation of Sertoli cells, the absence of axially oriented VM filaments in early postnatal hgn/hgn testes strongly suggest that the normal allele of the hgn locus directly or indirectly controls the differentiation of Sertoli cells.

In normal testes development during the early postnatal period, Sertoli cells proliferate rapidly, and spermatogonia differentiate from gonocytes and resume mitosis [1_3]. During this time, the cells undergoing physiological apoptosis are germ cells, increasing gradually up to 3 weeks after birth. This is regarded as the establishment of the proper ratio of germ cells to functional Sertoli cells [9, 10]. We found few TUNEL-positive apoptotic cells in normal testes on PD 0_7, whereas a considerable number of apoptotic cells, which were positive for TUNEL but negative for VM, were detected on PD 12. Therefore, it is likely that most of the TUNEL-positive cells in normal testes are germ cells.

In hgn/hgn testes on PD 1, most of the TUNEL-positive cells were located near the basement membrane. Some of the TUNEL-positive cells on PD 1 and most on PD 12 were located in the center of the tubules, and these cells were often stained with anti-VM antibody. However, the cellular distribution of VM is apparently abnormal in the TUNEL-positive apoptotic cells. It has been reported that the progression of apoptosis involves the disruption of cytoskeletal network [17]. This process might be facilitated by the cleavage of VM filaments [17]. Although, at present, the biochemical condition of VM filaments in apoptotic Sertoli cells is unclear, the presence of VM immunostaining associated with TUNEL-positive neculei indicates that hgn/hgn testes include apoptotic Sertoli cells during their postnatal development.

This observation is also supported by the result of TUNEL and VSH double staining. Genes homologous to Vasa, which encodes Asp-Glu-Ala-Asp (DEAD)-family protein of ATP-dependent RNA helicase in Drosophila, have been cloned in many animal species, and the expression of VSH is highly specific for gem cells [14]. The immunostaining with antibody to VSH formed circles around the large nuclei of gonocytes. In hgn/hgn testis on PD 1, TUNEL-positive nuclei were certainly present outside of the circular staining of VSH associated gonocytes.

MIS, a specific secretory product of Sertoli cells, causes regression of the mullerian ducts in embryonic development, and it has been reported that MIS is expressed in postnatal Sertoli cells [13]. Although we could not establish TUNEL and MIS double staining for technical reasons, immunostaining of MIS revealed that MIS-positive Sertoli cells had condensed small nuclei characteristic of apoptotic cells. These results indicate that premature Sertoli cells are the major cell type showing TUNEL-positive apoptosis in hgn/hgn testes during the early postnatal period. In addition, the absence of functional Sertoli cells from hgn/hgn testes indicates that the hgn/hgn rat is a unique model for studying Sertoli cell dysfunction.

Recently, we localized the hgn locus to an 840 kb region on rat chromosome 10 [18]. In this region, we detected the insertion of a 25 bp duplication into an exon of the Spag5/astrin/MAP126 gene, which codes for a protein associated with the spindle microtubule (H. Suzuki, personal communication). Although Spag5 knockout mice have normal fertility [19], the depletion of Spag5 by RNA interference in HeLa cells causes mitotic arrest, with dispersed distribution of chromosomes and consequent apoptotic cell death [20]. Abnormal mitotic cells with dispersed chromosomes have been observed in hgn/hgn testes during the early postnatal period [8]. Immunological staining with antibodies to GATA-4, which is a marker for Sertoli cells, indicates that the cells showing abnormal mitosis in hgn/hgn testes are Sertoli cells (Dr H. Suzuki, personal observation). Therefore, apoptotic cell death of Sertoli cells would result from the disorganization of the spindle apparatus caused by defective Spag5. Confirmation of this hypothesis, however, requires further investigation.

References

References

1 Sharpe RM, McKinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction 2003; 125: 769_84.

2 Orth JM, Gunsalus GL, Lamperti AA. Evidence from Sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of Sertoli cells produced during perinatal development. Endocrinology 1988; 122: 787_94.

3 Clermont Y, Perey B. Quantitative study of the cell population of the seminiferous tubules in immature rats. Am J Anat 1957; 100: 241_67.

4 Suzuki H, Inaba M, Suzuki K. Temporal and spatial distribution of alkaline phosphatase activity in male hypogonadic rat (hgn/hgn) testis during postnatal development. J Vet Med Sci 1998; 60: 671_9.

5 Suzuki H, Hakamata Y, Kamei T, Kikukawa K, Suzuki K. Reduced fertility in female homozygotes for hgn (male hypogonadism) selected by hgn-associated hypoplastic kidney. Cong Anom 1992; 32: 167_78.

6 Suzuki H, Tokuriki T, Saito K, Hishida A, Suzuki K. Glomerular hyperfiltration and hypertrophy in the rat hypoplastic kidney as a model of oligomeganephronic disease. Nephrol Dial Transplant 2005; 20: 1362_9.

7 Suzuki H, Kokado M, Saito K, Kunieda T, Suzuki K. A locus responsible for hypogonadism (hgn) is located on rat Chromosome 10. Mamm Genome 1999; 10: 1106_7.

8 Suzuki H, Yagi M, Saito K, Suzuki K. Dysplastic development of seminiferous tubules and interstitial tissue in rat hypogonadic (hgn/hgn) testes. Biol Reprod 2004; 71: 104_16.

9 Rodriguez I, Ody C, Araki K, Garcia I, Vassalli P. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J 1997; 16: 2262_70.

10 Boulogne B, Olaso R, Levacher C, Durand P, Habert R. Apoptosis and mitosis in gonocytes of the rat testis during foetal and neonatal development. Int J Androl 1999; 22: 356_65.

11 Boekelheide K. Mechanisms of toxic damage to spermato-genesis. J Natl Cancer Inst Monogr 2005; 34: 6_8.

12 Paranko J, Kallajoki M, Pelliniemi LJ, Lehto VP, Virtanen I. Transient coexpression of cytokeratin and vimentin in differentiating rat Sertoli cells. Dev Biol 1986; 117: 35_44.

13 Kuroda T, Lee MM, Haqq CM, Powell DM, Manganaro TF, Donahoe PK. Mullerian inhibiting substance ontogeny and its modulation by follicle-stimulating hormone in the rat testes. Endocrinology 1990; 127: 1825_32.

14 Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, Noce T. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech Dev 2000; 93: 139_49.

15 Baker PJ, O'Shaughnessy PJ. Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice. Reproduction 2001; 122: 227_34.

16 Hakamata Y, Kikukawa K, Kamei T, Suzuki K, Taya K, Sasamoto S. A new male hypogonadism mutant rat (hgn/hgn): concentrations of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum and the responsiveness of accessory sex organs to exogenous T, FSH, human chorionic gonadotropin, and luteinizing hormone-releasing hormone. Biol Reprod 1988; 38: 1145_53.

17 Morishima N. Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action. Genes Cells 1999; 4: 401_14.

18 Suzuki H, Nakamiya E, Daigo K, Saito K, Suzuki K. Fine mapping of the region including hypogonadism (hgn) locus on rat chromosome 10. J Vet Med Sci 2004; 66: 1151_4.

19 Fitzgerald CJ, Oko RJ, van der Hoorn FA. Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers. Mol Reprod Dev 2006; 73: 92_100.

20 Gruber J, Harborth J, Schnabel J, Weber K, Hatzfeld M. The mitotic-spindle-associated protein astrin is essential for progression through mitosis. J Cell Sci 2002; 115: 4053_9.