Spermatogenesis is the process of differentiation of diploid type A spermatogonia to haploid spermatozoa. Several subtypes of A spermatogonia have been characterized in the adult mouse testis. These include A-single (As), A-paired (Apr), A-aligned (Aal), and A1–A4. However, in the immature testis, very little information is available on subtypes and morphological features of type A spermatogonia. Six-day-old mouse testes, fixed either in Bouin solution or 5% glutaraldehyde, were embedded in paraffin and Epon, respectively. Thick sections (∼1 μm) of Epon-embedded tissue were stained with toluidine blue and revealed three subtypes of spermatogonia by light microscopy. The smallest spermatogonia (subtype I) appeared as single cells and exhibited a round or oval flattened nucleus with one or two prominent dense nucleoli and a characteristic unstained round and centrally located vacuole. These cells bound toluidine blue more avidly and appeared darker in comparison with the other cell types. Electron microscopy of thin sections (90 nm) revealed a finely granulated chromatin homogeneously distributed in the nucleus and sparse organelles in the cytoplasm. The second subtype of spermatogonia (subtype II) also displayed dark staining but was larger than subtype I; there was no central vacuole in the nucleus and heterochromatin clumps were observed. The largest subtype of spermatogonia (subtype III) showed large heterochromatin clumps and a pale staining nucleus. Intercellular bridges were noted between subtypes II and III. Based on the dye avidity, the three subtypes were classified as dark, transitional, and pale spermatogonia, respectively. Image analyses of 30 different cells of each subtype revealed a decline in gray-scale intensity from subtype I to III. Five-micrometer sections of paraffin-embedded tissue were immunoassayed with an antibody against the glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα-1) receptor, a putative marker for undifferentiated spermatogonia, showing positive reaction only in germ cells. The pattern of GFRα-1 expression, coupled to the overall morphology of the cells, indicates that at this stage of development, mouse seminiferous tubules contain essentially As, Apr, and possibly Aal spermatogonia. Thus, the present study indicates the presence of subtypes of type A spermatogonia in the immature mouse testis similar to that described previously in adult monkey and man.
During embryonic development, spermatogonia arise from primordial germ cells (PGCs). PGCs migrate from the embryonal ectoderm to reach the genital ridges by 11 days post coitum in mice. At the genital ridge, future Sertoli cells enclose the PGCs, and seminiferous cords are formed. PGCs then differentiate into gonocytes within the seminiferous cords. In the late part of gestation, gonocytes undergo proliferation and the new progeny of gonocytes remain arrested in the G0/G1 phase of the cell cycle . Shortly after birth, the gonocytes resume proliferation to give rise to spermatogonia . Two major types of spermatogonia have been described in the adult rat. Although type A spermatogonia consist of a nucleus with pale-stained fine chromatin granules, type B spermatogonia contain a nucleus with darkly stained coarse chromatin granules arranged along the nuclear membrane [3, 4]. Several subtypes within the A spermatogonia of adult rodents have been described . These include subtypes A-single (As), A-paired (Apr), A-aligned (Aal), and A1–A4 in the mouse [6, 7]. The description of these subtypes came from classical morphological studies using whole mounts of adult seminiferous tubules that consist of not only spermatogonia but also more differentiated germ cell types such as spermatocytes, spermatids, and spermatozoa.
Studies on undifferentiated spermatogonia have been hampered because of lack of a specific marker for these cells. Nevertheless, the As, and possibly the Apr and Aal subtypes, express the glial cell-derived neurotrophic factor family membrane receptor alpha-1 (GFRα-1) . Glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, binds to GFRα-1 [8–10]. When occupied by its ligand, GFRα-1 activates the Ret (Rearranged during transfection) receptor tyrosine kinase, which mediates the intracellular response [11–13]. Recently, GDNF has been linked to the proliferation of undifferentiated type A spermatogonia [9, 14], and GFRα-1 expression could thus be used as a marker for this group of spermatogonia.
In a 6-day-old male mouse, the seminiferous tubules consist of only one type of undifferentiated germ cell (i.e., type A spermatogonia). These cells are being extensively used for studies on spermatogonial biology and transplantation and the development of spermatogonial cell lines [15–18]. However, very little is known about the composition of different cell subtypes and their surface markers in a 6-day-old mouse testis. The present study was undertaken to define the subtypes of spermatogonia found in the 6-day-old neonatal mouse testis.
Materials and Methods
Preparation of Testis Samples for Morphological Examination
Male 6-day-old Balb/c mice pups (Charles River Breeding Laboratories, Wilmington, MA) were killed by carbon dioxide inhalation in accordance with the Animal Welfare Act and institutional guidelines. Testes were immediately excised and sliced in half. The halves were fixed with either Bouins solution or 5% glutaraldehyde in collidine buffer (0.05 M, pH 7.4). Tissues fixed in Bouin solution were processed and embedded in paraffin. Tissues fixed in glutaraldehyde were cut into ∼1-mm cubes and placed in the same fixative for an additional 1 h before being washed three times with collidine buffer and postfixed in a 1:1 mixture of 4% osmium tetroxide and 2% potassium ferrocyanide for 2 h. Following postfixation, the cubes were dehydrated in ascending grades of ethanol and absolute ethanol for 2 h, infiltrated with propylene oxide and propylene oxide-epoxy resin (Epon, Electron Microscopy Science, Fort Washington, PA), and finally embedded in Epon.
For light microscopy, sections of ∼1 μm thickness were cut from tissue blocks with an ultramicrotome. The sections were mounted on glass slides and stained with toluidine blue (1% in 0.1% borax) and examined under an Axioplan-2 microscope (Zeiss, Göttingen, Germany). Digital images were obtained at 1200 dpi resolution and stored for further analysis.
For electron microscopy, sections of 90 nm (gold interference color) were collected on copper grids (200 mesh) and counterstained with lead citrate and uranyl acetate. The sections were examined and photographed with a JEOL 110 (Jeol, Ltd., Tokyo, Japan) transmission electron microscope.
Using Adobe Photoshop software (version 5.0 Adobe System Inc., Mountain View, CA), the recorded images under the light microscope were adjusted to a 600 dpi and 180% sharpness with a threshold set to 0 and radius set to 2.0 pixels. The contrast/gray level was arranged to represent a sigmoid curve. Analysis of the individual digital image was performed by calculating the main gray value for areas of equal size on the nucleus. Care was taken to avoid areas of the nucleoli, clumps of heterochromatin, or both, following the procedure outlined in a recent publication .
Immunocytochemistry on Paraffin Sections
Sections (5 μm thick) were prepared on microscope slides and rehydrated before high-temperature antigen unmasking in 0.01 sodium citrate buffer (pH 6). After incubation in the blocking solution (0.2% donkey serum in PBS), the sections were incubated overnight at 4°C with a goat anti-mouse GFRα-1 antibody at dilutions of 1:50 to 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA. cat. no. sc-6157). After washing with PBS, the sections were incubated with a donkey anti-goat secondary antibody conjugated to biotin (Santa Cruz Biotechnology, cat. no. sc-2042). Finally, the sections were incubated with streptavidin-peroxidase, and the enzyme revealed with a VIP kit (Vector Laboratories, Burlingame, CA), giving a purple precipitate on the reaction sites. Appropriate controls for immunostaining with absence of primary antibody were included.
Morphological Characterization of Spermatogonia in 6-Day-Old Mouse Testis by Light Microscopy
The morphological features of all the cellular elements found in the testis were preserved very well. The cross-sections of the seminiferous cords exhibited a compact mass of cells and the absence of a lumen at this age. The predominant cell type observed within the seminiferous cords was the Sertoli cell. At the base of the seminiferous epithelium, the Sertoli cells appeared to be triangular in shape with an oval-shaped nucleus located perpendicular to the base. In cross-sections, the nuclei were circular in shape and smaller in size, compared with the nucleus of the germ cell type. The only type of germ cell found was the type A spermatogonia. Neither gonocytes nor type B spermatogonia were observed in the 6-day-old mouse testis (Fig. 1).
A careful examination of several spermatogonia revealed minor structural differences and differential toluidine blue uptake among them (Fig. 2). Based on these structural and staining differences, type A spermatogonia were classified into three subtypes. In subtype I, the cells were smaller in size, compared with the other two subtypes. The shape of these cells in tangential and cross-section of the tubules showed a flattened to round profile. No intercellular bridges were apparent between cells of subtype I and the other spermatogonial subtypes. They exhibited a round or oval-shaped flattened nucleus. A round central vacuole within the nucleus was the characteristic feature of subtype I. The vacuole was visible when the nucleus was sectioned in the central plane of the cell. Vacuoles were no bigger than 3 microns and the average size of the spermatogonial nucleus is approximately 10 microns. If sections are cut at the periphery of the nucleus, the vacuoles are likely to be missed. Nevertheless, we classified the spermatogonial subtypes only when the nucleus was centrally sectioned. One or two dense nucleoli were also observed inside the nucleus. Chromatin appeared to be homogeneously distributed in the nucleoplasm (Fig. 2). These cells avidly bound toluidine blue dye. Both nucleoplasm and cytoplasm were well stained. Thus, these cells were identified as “dark spermatogonia.” The density of these cells appeared to be low. Very few cells among the dark spermatogonia were in the dividing phase of the cell cycle.
The spermatogonia belonging to the second subtype (subtype II) were larger in size than the cells in subtype I (Fig. 2). The nucleus was round in shape and showed a large number of heterochromatin clumps. Unlike subtype I, a nucleolar vacuole was absent in subtype II cells (Fig. 2). Both the nucleoplasm and cytoplasm stained darkly with the dye. These cells appeared to be in an intermediary stage between subtypes I and III and were termed as “transitional spermatogonia.” Several subtype II cells were undergoing division as evidenced by the mitotic figures (Fig. 3A). Intercellular bridges were apparent between the cells of subtype II (Fig. 4A).
The third spermatogonial subtype was also larger than subtype I (Fig. 2). The nucleus exhibited a large number of heterochromatin clumps. The most characteristic feature of this subtype was the reduced avidity for toluidine blue dye. Both the nucleus and cytoplasm were stained poorly by the dye and appeared pale in comparison with subtypes I and II. Based on their appearance, these cells were termed “pale spermatogonia.” Many of the subtype III cells exhibited mitotic figures, indicating that they were undergoing replication (Fig. 3B). Intercellular bridges were observed between the cells of subtype III and between the cells of subtypes II and III (Fig. 4B and Fig. 4C).
Morphological Characterization of Spermatogonia in 6-Day-Old Mouse Testis by Electron Microscopy
Morphological analyses of the different subtypes of spermatogonia under the electron microscope confirmed the observations made at the level of the light microscope. The major criterion used to distinguish the subtypes under the electron microscope was the presence of the central vacuole in subtype I and the degree of chromatin condensation for the subtypes II and III.
The cells of subtype I (Fig. 5A) revealed a scarce number of organelles within the cytoplasm. The predominant type of organelle observed were mitochondria. Endoplasmic reticulum was less well developed. In most of the cells, a well developed Golgi apparatus was observed. Occasionally, the cytoplasm exhibited round vacuoles lined by a double membrane. The vacuoles were either empty or contained small round vesicles. The nucleus was normally round in shape with a homogeneously distributed darkly stained chromatin. A pale stained round central area was apparent within the nucleus. Two reticulate nucleoli normally associated with small amounts of heterochromatin were often observed. They were generally positioned near the nuclear membrane opposite to each other separated by the central clear space. Occasionally small amounts of stained heterochromatin were observed.
The spermatogonia belonging to subtype II exhibited a large round nucleus and a cytoplasm with a significant increase in the number of organelles (Fig. 5B). The nucleus consisted of a reticulate nucleolus and several heterochromatin clumps. A relatively larger amount of chromatin was bound to the nuclear membrane, compared with subtype I cells. The cytoplasm exhibited a well-developed Golgi apparatus and a significant amount of smooth endoplasmic reticulum unlike subtype I cells.
The differences in nuclear morphology and features between subtypes II and III (Fig. 5C) were less apparent at the level of the electron microscope. Similar to subtype II, subtype III cells exhibited a round nucleus containing a reticulate nucleolus and heterchromatin clumps attached to the nuclear membrane. The major change was the increase in the amount of heterochromatin clumps bound to the nuclear membrane in subtype III. A well-developed Golgi apparatus and smooth endoplasmic reticulum were also apparent in these cells.
Quantitative Analysis of Different Types of Spermatogonia in the Seminiferous Cords of 6-Day-Old Mouse
All three subtypes were randomly distributed in the seminiferous cord (Fig. 6). The dark spermatogonia (subtype I) formed 11% of the total number of spermatogonia counted in 60 cross-sections of seminiferous cords. The transitional subtype of spermatogonia (subtype II) that were the most predominant represent 47% of the total spermatogonia, and the remaining 42% of spermatogonia belonged to the class of pale spermatogonia (subtype III).
Image Analysis of Different Types of Spermatogonia
Image analyses were performed on 30 cells of each kind. Cells analyzed were selected from cross-sections of different tissue blocks to ensure that observed differences are particular to the cell type and not caused by differences in the dye binding ability between tissue samples. A gray scale histogram showed no big dispersion indicating the homogeneity on the selected areas. On a scale of values ranging from 0 (black) to 255 (white), a gray scale histogram for each subtype of type A spermatogonia was drawn. Subtypes I and II did not show significant differences in the staining pattern; nevertheless, a lightening was clearly evident in subtype III (Fig. 7).
Expression of GFRα-1 in the 6-Day-Old Seminiferous Cord
Paraffin-embedded sections of 6-day-old mouse testis showed positive immunoreaction to GFRα-1. The positive cells were germ cells because they exhibited a round shape with a high nuclear:cytoplasmic ratio (Fig. 8A). Moreover, intercellular bridges (Fig.8A, arrows) connecting some of these positive cells indicated that they were Apr, possibly Aal spermatogonia (Fig. 8, arrowheads). Other cells found within the seminiferous epithelium might also be As (Fig. 8A, asterisk). Sertoli cells did not show immunoreactivity. Sections immunoassayed in the absence of primary antibody did not show any staining (Fig. 8B).
Since the first description of the germ cell by von Ebner in 1871 , spermatogenesis and the cells involved in that process have now been studied for more than a century. The process has been described from the migrating primordial germ cell in the embryo to the development of mature spermatozoa in the postpubertal male. In the latter half of fetal development prior to parturition, the primordial germ cells differentiate into gonocytes in the male. These gonocytes are located centrally within the seminiferous cords. In rodents, 3 days after birth, the gonocytes proliferate and migrate towards the base of the seminiferous epithelium. This process of migration is generally complete by day 6 in the mouse, and the germinal cells located at the base of the seminiferous epithelium are termed type A spermatogonia . Type A spermatogonia undergo several mitotic divisions to yield intermediate type and type B spermatogonia during their differentiation pathway into spermatozoa. Interestingly, in the adult testis, several subtypes of type A spermatogonia have been identified. Based on morphological features, two types of spermatogonia were initially described in the rodent testis in the early part of the 20th century . Later, studies conducted using whole mounts of seminiferous tubules led to further classification of the spermatogonia. The whole-mount approach assesses a cell type on its topographical localization rather than on morphological features. Furthermore, the classification of spermatogonia by this approach is also dependent upon the stage of the seminiferous epithelium cycle and the number of aligned cells rather than on nuclear features [7, 21].
In the present study, we identified three type A spermatogonia subtypes (I, II, and III) in 6-day-old mouse testis that we classified as dark, transitional, and pale subtypes. In a recent article, Chiarini-Garcia and Russell  observed that approximately 1% of the undifferentiated cells in the adult mouse (As to Aal) were darker than the others and classified them as type Aal based on the stage of the seminiferous epithelial cycle in which they were found. These cells exhibited a central vacuole and a fine chromatin granulation in similarity with the dark type A spermatogonia (subtype I) that we found in the 6-day-old mouse testis. Because the type A dark cells were never seen connected by bridges, we tentatively classified them as type As spermatogonia. The presence of intercellular bridges between “transitional-transitional,” “transitional-pale,” and “pale-pale” subtypes suggests that the transitional and the pale subtypes were possibly more differentiated than the dark subtype and might be Apr and Aal spermatogonia. Furthermore, the A transitional and A pale showed a substantial increase in the amount of endoplasmic reticulum and the number of mitotic figures, compared with the A dark. In the transitional subtype, chromatin condensation was relatively less, compared with the pale subtype, and thus, the pale subtype could be considered to be at a later stage of differentiation.
By using an antibody against GFRα-1, we obtained positive immunoreaction exclusively in germ cells. In recent publications, GDNF, secreted by Sertoli cells, has been directly linked with undifferentiated spermatogonial proliferation [9, 14]. GDNF binds to its receptor, GFRα-1, located on the spermatogonia , to trigger the intracellular response [11–13]. We now confirm the presence of GFRα-1 in neonatal spermatogenesis, located exclusively on the undifferentiated type A spermatogonia. Thus, although GFRα-1 seems to be expressed at a higher level in As spermatogonia, the protein is not specific for one particular spermatogonial subtype.
Models of mammalian spermatogonial differentiation have been established, emphasizing the differences between rodents and primates. A model widely accepted for rodents was originally proposed by Huckins  and Oakberg  in which single isolated spermatogonia (As) were considered the stem cells. These cells can renew themselves to maintain the stem cell population or upon mitotic division give rise to two daughter cells with incomplete cytokinesis that remain connected by an intercellular bridge  and become Apr. The Apr spermatogonia is considered a differentiated cell. From the Apr spermatogonia, cells continue dividing by mitosis, and then germ cells in clones of interconnected cells of increasing size are called Aal (Fig. 9A). Nevertheless, it is not known whether the intercellular bridges between Apr and Aal spermatogonia constitute an irreversible differentiation step . Finally, the Aal cells differentiate into A1 spermatogonia, which after a series of synchronous divisions will give rise to primary spermatocytes through differentiation into A2, A3, A4, intermediate, and type B spermatogonia. Unlike rodents, the model for spermatogonial differentiation in primates consists of a reserve stem cell (“dark” spermatogonia) undergoing differentiation into a renewing stem cell (“pale” spermatogonia; Fig. 9B). These “pale” spermatogonia undergo several mitotic divisions to form chains that will finally differentiate into type B spermatogonia .
The types of spermatogonia observed in the 6-day-old mouse testis closely resemble the spermatogonia found and described in monkey by Clermont and Leblond  and later in humans by Clermont [26–28]. It is likely that the order in which these spermatogonia arise from one another is the same in rodents and primates (Fig. 9C). Although dark and pale type of classification has not been applied to the rodent model previously, darker cells with nuclear vacuoles have been described in mice and were considered as possible stem cells (type A0) [29, 30].
In conclusion, we present morphological evidence for the presence of three subtypes of spermatogonia in the immature 6-day-old mouse testis. These subtypes resemble closely the morphology of the subtypes described for adult primates.