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Regular Article |
a Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
b Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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meiosis, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Due to the restricted nature of the as mutation, which affects only spermatogenesis, homozygous rats provide an excellent model for studying spermatogenesis. The as locus was identified by genetic analysis of a spontaneously aspermatic rat, which appeared in an inbred Wistar strain. The mutant strain was established and named the TT line [2]. The as/as testis shows arrest of spermatogenesis, mainly at the pachytene spermatocyte stage, whereas the heterozygous males and all the females are free from any abnormalities [3]. Although the as locus is known to be localized in a region close to malate dehydrogenase 2 on rat chromosome 12 [4], the gene(s) responsible for the arrest of spermatogenesis are still unknown. In the spermatogenesis of as/as rats, a characteristic inclusion-like body appears in the cytoplasm of the pachytene spermatocytes. Although a previous study indicated that this organelle could be of ribosomal origin [5], the pathogenesis remains unclear. To understand the function of the gene(s) at the as locus, it is necessary to clarify whether the site that causes arrest of spermatogenesis is in the germ cell or in the somatic cell components that support spermatogenesis.
The spermatogonial transplantation technique developed by Brinster et al. [6, 7] is a powerful tool for evaluating the potency of germ cell differentiation into spermatozoa and the somatic cell environment in which spermatogenesis takes place. To date, several cases of hereditary spermatogenesis defect in mice have been studied to identify whether the phenotype is the result of defective germ cells or a testicular environment that is unfavorable for spermatogenesis (e.g., jsd/jsd mice [8]). We also applied this technique to the as/as rat to pinpoint the site responsible for arrest of spermatogenesis. The unsuccessful results of transplantation of normal germ cells into the as/as testis have led to morphological reevaluations. We examined the as/as testis by electron microscopy, including a functional analysis of the blood-testis barrier.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protocols for the use of animals in the present study were approved by the Animal Care Committee of Graduate School of Medicine, Chiba University. Rats used in this report were obtained from a breeding colony raised at the National Institute of Agrobiological Sciences. Immunodeficient mice were purchased from Charles River Japan (Atsugi, Japan).
Spermatogonial Transplantation
In experiment 1, as homozygous germ cells were transplanted into the testes of immunodeficient mice and normal rats. Donor cells were collected from adult as homozygous testes. Testicular single-cell suspensions were prepared by sequential enzyme digestion as described by Brinster et al. [6, 7] with some modifications. Under ether anesthesia, blood was collected and the testes were removed and placed in Dulbecco modified PBS (DPBS; Nissui Pharmaceutical Co., Tokyo, Japan) with penicillin G and streptomycin. After removing the tunica albuginea, the testes were placed in DPBS containing 0.25% (w/v) collagenase type IV, 0.05% (w/v) DNase type I, and antibiotics at 32°C. After 10–15 min of incubation with manual agitation every 5 min, the dispersed seminiferous tubules were digested with 0.25% (w/v) trypsin, 0.05% DNase type I, and antibiotics in DPBS at 32°C and manually agitated every 5 min. The cell suspension was centrifuged at 600 x g for 5 min at 18°C. After rinsing two or three times with the injection medium [6], the collected cells were filtered through a cell strainer of 40-µm pore size (Falcon 2342; Becton Dickinson Co., Franklin Lakes, NJ). The cells were collected by centrifugation at 600 x g for 5 min at 16°C and then resuspended in the injection medium to a final concentration of 107–108 cells/ml. A trypan blue exclusion test showed the viability of the cells to be >79%.
In the case of xenograft transplantation, immunodeficient mice (BALB/cAnNCrj-nu/nu) were used as the recipient animals. The recipient rats used were phenotypic normal males (+/+ or +/as) in the TT line to avoid immunoreaction after transplantation. Recipient animals were given busulfan in advance at doses of 40 mg/kg body weight for mice and 10 mg/kg body weight for rats. High doses of busulfan may cause edema in the rat testis, so the lower dose was used for rats (T. Ogawa, personal communication). All of the rats survived treatment without any changes to external appearance. The mice underwent bone marrow transplantation 4 days after busulfan administration, according to the methods of Dobrinski et al. [9]. The as homozygous germ cells were transplanted into the immunodeficient mice 1 mo after busulfan administration, when the recipients were 12 wk of age. Transplantation into the normal rat testis was performed at least 2 wk after busulfan treatment when the recipients were 4–5 wk of age.
Transplantations were performed by efferent duct injection, according to the method of Ogawa et al. [10]. Animals were anesthetized with ketamine, and the testis was exposed through a midline incision. Under a dissecting microscope (Stemi2000; Zeiss, Jena, Germany), a micropipette with a tip approximately 150 µm in outer diameter was threaded into the efferent duct and gently inserted toward the testis. When the tip reached the rete, the donor cell suspension was injected with a microinjector (IM-6; Narishige, Tokyo, Japan) connected to the micropipette. Filling of the seminiferous tubules with the cell suspension was easily monitored because of the added blue dye. Between 50% and 95% of the surface tubules were filled with the donor cell suspension until the flow of the solution stopped. After the injection, the testis was returned to the body cavity, and the incision was sutured.
In experiment 2, the germ cells collected from the testis of normal rats 10–14 days of age were transplanted into the as homozygous testis. A donor cell suspension was prepared as above. The viability of the cells, confirmed by a trypan blue exclusion test, was >97%. The as homozygous adult rats were pretreated with 10 mg/kg busulfan, and the cell suspension was transplanted by efferent duct injection. The busulfan treatment and the transplantation caused no marked change in the external appearance of the rats.
The recipient animals were killed by deep ether anesthesia 100–130 days after transplantation. The testes were removed and fixed in Bouin solution. Serial 6-µm sections were cut from the paraffin-embedded testes and stained with hematoxylin and eosin. The sections were observed under light microscopy. When necessary, the cauda epididymides were removed and scratched in DPBS to collect spermatozoa. The DPBS was centrifuged at 800 x g for 5 min at room temperature, and the supernatant was discarded. The precipitate was resuspended in a small aliquot of DPBS and smeared on a glass slide. Epididiymal sperm was examined under light microscopy.
Electron Microscopy
The as/as male rats (12–13 wk of age) were deeply anesthetized with ether and perfused through the heart with 3% glutaraldehyde in 10 mM Hepes saline (pH 7.3). The perfusion-fixed testes were cut into small pieces (1 mm3) and fixed for another 2 h. After fixation with osmium tetroxide, the specimens were processed for conventional electron microscopy. Ultrathin sections (about 80 nm) stained with uranyl acetate and lead citrate were observed with an electron microscope (JEM 2000EXII; JEOL, Tokyo, Japan). The semithin sections (1 µm) were stained with toluidine blue and used for light microscopy.
Evaluation of the Blood-Testis Barrier
Permeability of the blood-testis barrier in the as/as testis was examined using cytochrome c as an intercellular tracer. As the control, normal littermates were examined. Dissolved cytochrome c (equine heart origin, MW 12 384; Sigma, St. Louis, MO) in DPBS at a concentration of 150–200 mg/ml was administered to the as/as and normal male rats (12–13 wk of age) by either efferent duct microinjection into the seminiferous tubules as described above or by interstitial injection beneath the tunica albuginea. Each procedure was performed under deep ketamine anesthesia. Approximately 5 min after administration of the cytochrome c solution, the animals were perfused with 2% paraformaldehyde, 3% glutaraldehyde in 100 mM cacodylate buffer (pH 7.2) through the heart and then processed with diaminobenzidine (DAB) as described by Toyama et al. [11]. Ultrathin sections were examined with an electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In experiment 1, between 70% and 95% of the surface tubules in 14 testes of seven immunodeficient mice were filled with the as/as germ cell suspension after injection (Table 1). On histological examination, most of the seminiferous tubules consisted of Sertoli cells and spermatogonia, because of administration of busulfan, but the endogenous spermatogenesis remained in >30% of tubules. The elongated spermatids in the tubules were identified as mouse sperm cells from their morphological appearance (Fig. 1a). In 6 of the 14 testes, pachytene spermatocytes with inclusion-like bodies in the cytoplasm were observed in the seminiferous epithelium of several tubules. The spermatocytes were located near the lumen and appeared to slough out of the epithelium. The seminiferous epithelium consisted of Sertoli cells, spermatogonia, and preleptotene to pachytene spermatocytes; however, no round or elongated spermatids were observed. In a testicular section, one to eight tubules (0.6%–5.1% of total tubules) contained these characteristic spermatocytes. By the analyses of the serial sections, the total length of the portions that contained these spermatocytes was between 200 and 850 µm.
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When the as/as germ cells were transplanted into the rat testis, >50% of the surface tubules in six testes of six animals were filled with the cell suspension at the time of injection. Normal rat spermatogenesis was observed in about 40% of the tubules in the six testes, and the rest of the tubules consisted of Sertoli cells and spermatogonia at 15 wk after transplantation. In four of the six testes, arrest of spermatogenesis at the pachytene spermatocyte stage was detected, accompanied by the appearance of inclusion-like bodies in the cytoplasm (Fig. 1b). The pachytene spermatocytes tended to move closer to the lumen. The other types of cells present in the epithelium were spermatogonia, preleptotene to leptotene spermatocytes, and Sertoli cells. The cellular association was similar to that observed in the normal testis, although spermatids were lacking. In each of the four testes, one tubule had these characteristic spermatocytes. The total length of the portions of the tubule that contained the spermatocytes with inclusion-like bodies was between 170 and 1300 µm, as determined by analyses of the serial sections.
In experiment 2, the injection of normal germ cells was successful in five testes of the five as/as animals. Between 50% and 70% of the surface tubules were filled with the suspension. The five testes were thoroughly examined histologically. In these testes 30%–50% of the tubules contained Sertoli cells exclusively or Sertoli cells and several spermatogonia in the epithelium, but in the rest, spermatogenesis was arrested at the pachytene spermatocyte stage. These pachytene spermatocytes were identified as endogenous because of the appearance of the inclusion-like bodies in the cytoplasm. In two of the five testes, a portion of the tubules contained elongated spermatids found randomly in the seminiferous epithelium. A few spermatozoa with abnormally shaped heads and tails were found in the ipsilateral epididymis. This incomplete spermatogenesis is occasionally observed in the intact as/as testis (data not shown). No tubules in the five testes showed complete spermatogenesis, and no morphologically normal spermatozoa were collected from the epididymides.
Morphology of the as/as Testis
In the seminiferous epithelium of the as/as testis, pachytene spermatocytes with the inclusion-like body were located near the lumen (Fig. 2). The bottom of the seminiferous epithelium was occupied by spermatogonia and preleptotene to leptotene spermatocytes. Multinucleated giant cells and other degenerating cells were often observed near the lumen. The cytoplasm of these multinucleated giant cells often contained inclusion-like bodies, indicating that these giant cells were derived from the pachytene spermatocytes with the inclusion-like bodies. The projections of the Sertoli cells were conspicuous.
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When the pachytene spermatocytes in the as/as testis were examined by electron microscopy, the synaptonemal complex was often observed in the nuclei. This observation was consistent with those by Báyes et al. [12]. Three kinds of dense material clusters were observed in the cytoplasm (Fig. 3, a–c). The first type was associated with small round vesicles (60–90 nm across) and contained denser materials. This type of cluster was irregularly shaped, and several patches of clusters were seen in any section through a pachytene cell. The second type was observed among mitochondria (Fig. 3, a and c). The dense material completely filled the space between three or more mitochondria, stretching from the outer membrane of one mitochondrion to that of another. The material particles were the finest of the three types of clusters. The third type was the largest of the three types of clusters (Fig. 3, a and b). One or more clusters were observed in the cytoplasm close to the nucleus. These cluster was round to ovoid, without a limiting membrane. The particles they comprised were the largest of the three types of clusters, which resembled ribosomes as described by Atagi et al. [5]. No meshwork of fibrils overlaying the particles was observed.
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The Sertoli cells did not show any morphological abnormalities. Complete ectoplasmic specializations between adjoining Sertoli cells were observed (Fig. 4). A subsurface cistern of the endoplasmic reticulum ran parallel to the Sertoli cell plasma membrane, and a layer of actin filaments lay between the membrane and subsurface cistern. Tight junctions were observed in the specializations; thus, the ultrastructures of the ectoplasmic specializations between adjoining Sertoli cells in the as/as testis were comparable to those seen in the normal testis.
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Permeability of the Blood-Testis Barrier
The function of the blood-testis barrier was evaluated using cytochrome c as an intercellular tracer. When cytochrome c was injected into the interstitium, penetration of the tracer from the basal lamina into intercellular spaces in the seminiferous epithelium was visualized with the reaction product of DAB. In the normal testis at 13 wk of age, the tracer stopped at the basal margin of the ectoplasmic specialization between adjoining Sertoli cells (Fig. 5, a and b). In contrast, when the tracer was injected into the interstitium of the as/as testis, the reaction product was observed in the intercellular spaces of the adluminal compartment and in those of the basal compartment (Fig. 5c). These results provide evidence of penetration by the tracer through the tight junction, indicating that the blood-testis barrier was defective.
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When cytochrome c was injected into the lumen of seminiferous tubules of a normal testis, the tracer was observed in the intercellular space of the adluminal compartment, but not beyond the adluminal margin of the ectoplasmic specialization (Fig. 6a). In the case of the as/as testis, however, cytochrome c injected into the lumen penetrated through the specializations from the lumen to the basal compartment (Fig. 6, b and c). Unexpectedly, no reaction product was observed in the intercellular space of the basal compartment and the interstitial tissue. Thus, only a small quantity of cytochrome c passed through the narrow intercellular space in the ectoplasmic specialization, reached the basal compartment, and diffused into the interstitial tissue. A total of 100 specializations were observed and all showed dysfunction of the blood-testis barrier.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Electron microscopy of the as/as testis revealed three types of dense material clusters in the cytoplasm of pachytene spermatocytes. The morphological characteristics of two types of clusters were consistent with those of the intermitochondrial substance and the chromatoid body, as reported by Russell and Frank [15]. However, the characteristics of the third type were consistent with previous observations by Atagi et al. [5], which was at variance with all six types of nuage in the spermatocytes [15]. The pathological mechanisms that lead to aggregation of ribosomes at this stage of spermatogenesis will become clear when the function of the as mutant gene(s) is fully known. The fusion of inclusion-like body-containing pachytene spermatocytes, resulting in the formation of multinucleated giant cells, might indicate that the as mutant gene(s) causes aggregation of ribosomes, which in turn triggers cell degeneration.
The restoration of spermatogenesis in the as/as testis was not observed despite thorough histological examination of the five recipient testes. In experiment 1, colonization of transplanted as/as germ cells was successful, and the cells differentiated up to the pachytene spermatocytes in both recipient rat and mouse testis. Therefore, technical problems could not account for the unsuccessful restoration of spermatogenesis. The results suggested that the failure of the somatic cell components could result in an environment that cannot support spermatogenesis. The ectoplasmic specialization between adjoining Sertoli cells is a main component of the blood-testis barrier [16–18]. Electron microscopy of the as/as testis revealed that the ectoplasmic specialization between adjoining Sertoli cells was morphologically normal; however, the results of the tracer examination with cytochrome c revealed a dysfunction of the barrier. Atagi et al. [5] examined the barrier function in the as/as testis using lanthanum as a tracer and concluded that the barrier was fuctioning normally. The discrepancy between studies can be attributed mainly to the different methods employed. We applied cytochrome c in two ways, from basal to adluminal and from adluminal to basal. Both of the results indicated the dysfunction of the barrier in the as/as testis. The barrier function becomes complete after 18–20 days of age in the normal rat [19–21]. Because the function of the as/as rats was immature at 4 wk of age (data not shown), the mutant rat seems unable to establish the barrier system at any point in their life span. This permanent dysfunction of the barrier system in the as/as rat could provide a useful model for studying the role of the blood-testis barrier in sustaining spermatogenesis.
Dysfunction of the blood-testis barrier has been demonstrated in the cryptorchid testes of Tfm and Sxr male mice [22]. In these animals, high abdominal temperature appears to be the factor affecting the function of the Sertoli cells. Several chemical agents delay the establishment of the blood-testis barrier [11, 19]. For example, neonatal administration of diethylstilbestrol (DES) delayed formation of the specializations between Sertoli cells for 5 wk, resulting in retardation of the development of the barrier in rats [11]. The DES-injected animals also showed meiotic arrest of spermatogenesis. Also, Claudin-11 null mice lack junction strands in the testis and lack elongated spermatids [23]. The DES treatment and the Claudin-11 null mice provide evidence that the barrier function is required for the process of meiosis [24]. An unfavorable environment for spermatogenesis caused by the dysfunctional blood-testis barrier might prevent restoration of spermatogenesis after transplantation of normal germ cells, in the as/as testis. However, unknown defects on somatic cell components by the as mutant gene(s) are possibly related to the results. Further investigation is necessary to examine this possibility.
Several molecules may be involved in the ectoplasmic specializations between adjoining Sertoli cells. Actin, which is the main component of the specializations [25], fimbrin and vinculin [26, 27], espin [28], cadherin-associated molecules [29], and 
6ß1 integrin [30] have so far been reported. In addition, several tight junction proteins such as occludin [31], ZO-1 [32], and claudin 11 [33] are expressed in the rat and/or mouse testis. The mechanisms by which the as mutant gene(s) affects the formation of the blood-testis barrier and how the meiotic arrest of spermatogenesis accompanying the generation of inclusion-like body occurs remain to be determined. The results so far suggest that this mutation may be caused by the deletion or translocation in a region of chromosomes. The as locus is localized on a region of rat chromosome 12 [4] that corresponds to a region on human chromosome 7 and on mouse chromosome 5 where several genes coding cell adhesion molecules are localized (Genome Database: http://gdbwww.gdb.org/). The responsible gene(s) will be cloned as part of our continuing genetic analysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence. FAX: 81 298 38 7408; jgucci{at}nias.affrc.go.jp 
Accepted: April 12, 2002.
Received: December 29, 2001.
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REFERENCES |
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6ß1 integrins, and their formation is controlled by follicle-stimulating hormone. Biol Reprod 1998 58:371-378
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