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. 2020 Jul 24;238(1):86–95. doi: 10.1111/joa.13287

Three‐dimensional structure of seminiferous tubules in the Syrian hamster

Hiroki Nakata 1,✉, Miki Yoshiike 2, Shiari Nozawa 2, Yoko Sato 3, Shoichi Iseki 4, Teruaki Iwamoto 5, Atsushi Mizokami 6
PMCID: PMC7754951  PMID: 33189084

Abstract

The hamster is useful for the study of male reproductive biology. However, unlike in the mouse and rat, the gross structure of seminiferous tubules in the hamster is largely unknown. The aim of the present study was to clarify the precise 3‐dimensional (3D) structure of seminiferous tubules in hamsters. We reconstructed all seminiferous tubules in 3 and 1 testes from 0‐day (P0) and 10‐week (adult) Syrian hamsters, respectively, using serial paraffin sections and high‐performance 3D reconstruction software. In P0 hamsters, the average numbers of seminiferous tubules, terminating points, branching points, and blind ends per testis were 9.0, 89.7, 93.0, and 0.7, respectively. There were two types of tubules: shorter and dominant ones. The dominant tubules, 2–4 in number per testis and accounting for 86% of the total tubule length, had many terminating and branching points and appeared to be derived from the anastomosis of many shorter tubules. In an adult hamster, there were 11 seminiferous tubules with a total length of 22 m, 98 terminating points, 88 branching points, and 2 blind ends per testis. Three of the 11 tubules were dominant ones, accounting for 83% of the total length, and occupied the testis from the surface over the circumference to the center, while the others were short and occupied only one side of the testis. The amplitude and direction of the curves of tubules were random, and there were no funnel‐shaped networks of tubules present, in contrast to the mouse testis. The present study revealed the 3D structure of seminiferous tubules in developing and adult Syrian hamsters, which is different from that in mice and rats.

Keywords: 3D, hamster, seminiferous tubules, testis, three‐dimensional reconstruction

The present study revealed the 3D structure of seminiferous tubules in developing and adult Syrian hamsters, which is different from that in mice and rats.

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1. INTRODUCTION

The hamster is one of the most widely used experimental animals. It belongs to the Cricetidae family of rodents, whereas the mouse and rat belong to the Muridae family. Hamsters, Golden (Syrian) hamsters, in particular, are useful for the study of reproductive biology, because of their advantages including a constant estrous cycle, a short gestation period, and changes in testicular function dependent on the photoperiod (reviewed by Hirose and Ogura, 2018).

In order to investigate the functions of reproductive organs, the testis for example, precise knowledge of their structures is essential. The histology of seminiferous tubules in the testis (e.g., the stage, cycle, wave, and cell identification) has been extensively studied to date mainly in rats and mice (Clermont and Leblond, 1953; Oakberg, 1956; Perey et al., 1961; Russell et al., 1990; Hess and Franca, 2008; Ahmed and Rooij, 2009; Drumond et al., 2011; Meistrich and Hess, 2013; Nakata et al., 2015a; Nakata, 2019, 2019). The gross characteristics of seminiferous tubules (e.g., the number, length, path, and presence of branching points and blind ends) have also been studied in rats and mice using the 3‐dimensional (3D) reconstruction of serial histological sections (Curtis, 1918; Clermont and Huckins, 1961; Nakata et al., 2015b, 2017; Nakata, 2019, 2019). In three recent papers from our group, we reported high‐resolution 3D structures of whole seminiferous tubules in adult and postnatal developing mice using serial paraffin sections and high‐performance 3D reconstruction software. More recently, we also applied this technique to the analysis of efferent and epididymal ducts in the mouse (Nakata and Iseki, 2019).

The testis of the hamster has several anatomical and functional differences from that of the mouse; for example, while the body weight of the hamster is only 4 times greater than that of the mouse, the testicular volume is more than 15 times greater; the rete testis is located on the cranial pole of the testis in the hamster but on the lateral side of the testis in the mouse; and the size of the testis changes depending on seasons in the hamster but not in the mouse, because the former is a seasonal breeder but the latter is not. Experimental exposure of hamsters to a short daily photoperiod can induce male reproductive quiescence via testicular regression (Sinha‐Hikim et al., 1988a; 1988b). Changes in the morphology and function of hamster testis dependent on the photoperiod have been investigated to date (Morales et al., 2007; de Nascimento et al., 2009; Beltrán‐Frutos et al., 2018; Matzkin et al., 2019). In regard to the histology of seminiferous epithelium in hamsters, the process of spermatogenesis from spermatogonial stem cells to spermatozoa with regular temporal and spatial intervals called the cycle and stage, respectively, is known to be basically similar to that of mice and rats (Clermont and Trott, 1969). On the other hand, to our knowledge, there has been no report on the gross 3D structure of seminiferous tubules in hamsters. Therefore, in the present study, we reconstructed and analyzed all seminiferous tubules in postnatal 0‐day and 10‐week Syrian hamster testes using serial paraffin sections and high‐performance 3D reconstruction software.

2. MATERIALS AND METHODS

2.1. Animals

The present animal study was approved by the Animal Research Committee of St. Marianna University School of Medicine (1712012). Pregnant Syrian hamsters (Slc:Syrian) at 10 dpc (days post‐conception) and adult male hamsters (10 weeks of age at the time of experiments), reared under 14‐hr light/10‐hr dark conditions to keep them in the reproductive phase, were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Adult males were reared no longer than 1 week before experiments under standard 12‐hr light/12‐hr dark laboratory conditions with free access to standard food and water, which was confirmed not to influence their reproductive activity. Newborns were used within 24 hr (P0) after delivery from pregnant hamsters at 14–15 dpc.

2.2. Tissue preparation

Hamsters were sacrificed by CO2 asphyxiation. The testes were dissected out, immersed in 10% formalin neutral buffer solution (for newborns) or Bouin's solution (for adults) overnight, dehydrated in a graded ethanol series, and embedded in paraffin. Serial 5‐µm‐thick sections were made using a microtome with intervals of 10 and 50 µm for the P0 and adult testes, respectively, and then mounted on glass slides.

2.3. Immunohistochemistry (IHC) and periodic acid‐Schiff–hematoxylin (PAS‐H) staining

Using the formalin‐fixed, paraffin‐embedded tissue sections of P0 testes, IHC with the enzyme detection method was performed. The sections, after deparaffinization and rehydration, were treated with 1% skim milk in PBS for 1 hr at room temperature to prevent non‐specific antibody binding, and then incubated with a mouse monoclonal anti‐alpha smooth muscle actin (αSMA) antibody (ab21027; Abcam) at a 1:200 dilution for 1 hr at room temperature to immunostain myoid cells surrounding the basement membrane of seminiferous tubules. After washing in PBS, the sites of immunoreaction were visualized by incubating the sections successively with a horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG antibody (DAKO) at a 1:200 dilution for 1 hr and a peroxidase substrate (ImmPACT DAB EqV Substrate, SK‐4103; Vector Laboratories) for 10 min, and then counterstained with hematoxylin. The sections were digitized using a BX51 microscope (Olympus) equipped with a DP74 digital camera (Olympus) attached to a standard PC. The Bouin‐fixed, paraffin‐embedded tissue sections of an adult testis were treated with PAS‐H to stain the basement membrane of seminiferous tubules, as previously described (Nakata et al., 2017). The sections were digitized using a whole‐slide scanner (Nanozoomer 2.0‐HT; Hamamatsu Photonics) with a 20‐fold objective lens, and the resulting digital images of the sections were visualized with viewer software (NDP.view2; Hamamatsu Photonics).

2.4. Reconstruction processing

The 3D reconstruction was performed as previously described (Nakata et al., 2015b, 2017) with a slight modification. Briefly, segmentation of seminiferous tubules for the 3D reconstruction was performed by extracting the brown areas of DAB‐stained myoid cells in digital images of P0 sections with ImageJ software (NIH; http://imagej.nih.gov/ij/) or manually marking the PAS‐H‐stained basement membrane in digital images of adult sections with Photoshop CS6 software (Adobe Systems, Inc.). After segmentation, the images of P0 and adult sections were converted into grayscale in JPEG format with Photoshop CS6 software at a resolution of 1465 and 7264 nm·pixel−1, respectively. Using Amira 6.3.0 software (Visage Imaging GmbH), the serial images were aligned automatically followed by manual adjustment, and the inside of the segmented outlines of a selected tubule was filled with a particular color using threshold processing and traced from section to section. This procedure was repeatedly applied to all seminiferous tubules with different colors, and they were then 3D‐reconstructed. To draw the core lines of seminiferous tubules, individual traced tubules in cross sections were shrunk concentrically by 8 (11.72 µm) and 5 (36.3 µm) pixels in all directions, their resolution was changed to 5860 and 29,056 nm·pixel−1, respectively, and then, they were reconstructed into thin tubules, in which the core lines were drawn using the same software. The rete testis and whole testis were also 3D‐reconstructed by filling the inside of their outlines with threshold processing. In the case of the P0 testis, only the position of the center of the rete testis was marked with a black sphere.

3. RESULTS

3.1. 3D structure of all seminiferous tubules in P0 hamsters

Figure 1 presents the process of reconstruction of seminiferous tubules from a representative P0 testis (#3). The outlines of seminiferous tubules were labeled with IHC for αSMA that stains myoid cells (Figure 1a), the DAB color was extracted with ImageJ software and converted to a grayscale (Figure 1b), and the inside of individual seminiferous tubules was filled with different colors using threshold processing and traced from section to section along the entire length of the tubules (Figure 1c). Thereafter, individual seminiferous tubules and their core lines labeled with respective colors were 3D‐reconstructed and superimposed on the testis together with the rete testis (Figure 1d,e, respectively). A single seminiferous tubule was defined as a structure connected to the rete testis at least with one extremity and having a continuous lumen, regardless of the presence of branching points and/or blind ends. All seminiferous tubules in 3 testes from 3 different P0 hamsters (#1, #2, and #3) were reconstructed, and their core lines were drawn. Table 1 summarizes the morphometric parameters of the core lines in P0 testes. The average number and total length of seminiferous tubules per testis were 9.0 ± 0.8 and 62.0 ± 6.1 mm, respectively, and the average numbers of terminating points (connections with the rete testis), branching points, blind ends, and “rings” per testis were 89.7 ± 5.6, 93.0 ± 13.1, 0.7 ± 0.5, and 10.3 ± 4.2, respectively. A ring was defined as a closed circle formed by bifurcated tubule portions between 2 branching points. The presence of a ring added 2 branching points while not adding terminating points to each tubule. Within all 27 seminiferous tubules from the 3 testes, only two blind ends were found and located near the cranial poles of the testes (Figure 1e). There were two types of seminiferous tubules: shorter tubules, which had no more than 5 branching points, and dominant tubules, which had more than 5 branching points. The average lengths of the shorter and dominant tubules were 1.8 (n = 18) and 16.1 (n = 9) mm, respectively. The shortest and longest tubules were 0.2 and 27.6 mm, respectively. The dominant tubules, 2 to 4 in number per testis, accounted for 86% of the total length of tubules, and the number of branching points in these dominant tubules accounted for 94% of that in all tubules.

FIGURE 1.

FIGURE 1

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Photographs of a transverse section of the testis from #3 P0 hamster treated with immunohistochemistry for αSMA with DAB substrate (a), after extraction of DAB color for segmentation of seminiferous tubules (b), and after tracing all seminiferous tubules in different colors (c). All reconstructed seminiferous tubules (d) and their core lines (e) in different colors are superimposed on the reconstructed figure of the testis and viewed from 3 directions. The positions of the rete testis and blind end are shown as black and gray spheres, respectively (d and e). All scales, 200 µm

TABLE 1.

Parameters in reconstructed seminiferous tubules in P0 testes.

Testis name Tubules Total length (mm) Terminating points a Branching points Blind ends Rings b
#1 8 63.2 92 95 1 9
#2 9 54.0 82 76 0 6
#3 10 68.8 95 108 1 16
Average 9.0 ± 0.8 c 62.0 ± 6.1 89.7 ± 5.6 93.0 ± 13.1 0.7 ± 0.5 10.3 ± 4.2
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a

Connections with the rete testis.

b

Closed circles formed by bifurcated tubule portions between 2 branching points.

c

Mean ± SD.

Based on a representative P0 testis (#3), the morphometric parameters of the core lines of individual seminiferous tubules are summarized in Table 2, and the 3D‐reconstructed figures of the core lines of all seminiferous tubules (T1–T10 in the order of their connection with the rete testis) are shown in the horizontal (Figure 2a) and vertical (Figure 2b) planes. T5, T7, T8, and T10, with greater lengths and many terminating and branching points, were regarded as dominant tubules, whereas T1, T2, T3, T4, T6, and T9 were regarded as shorter tubules. In T2, only one extremity was connected to the rete testis whereas the other extremity terminated as a blind end without reaching the rete testis or showing further branching. The overall pathways of tubules in the horizontal plane viewed from the top were either simple thin loops connected to the rete testis (shorter tubules) or complex networks of anastomosed thin loops (dominant tubules). In both cases, the tubules locally ran straight without forming zigzag convolutions and, as a whole, ran inside the testis without forming large circles along the surface of the testis. In the vertical plane viewed laterally, the tubules were distributed in a regular stacked pattern in the order of their connections with the rete testis. The shorter tubules ran with a very small vertical amplitude, whereas the dominant tubules occupied large vertical areas. These characteristics of the pathways of seminiferous tubules in hamsters differ from those in mice (Nakata et al., 2017).

TABLE 2.

Parameters in individual reconstructed seminiferous tubules in #3 P0 testis.

Tubule name Length (mm) Terminating points a Branching points Blind ends Rings b
T1 0.2 3 1 0 0
T2 0.5 1 0 1 0
T3 1.5 2 0 0 0
T4 2.4 4 2 0 0
T5 19.3 25 29 0 3
T6 3.0 4 2 0 0
T7 13.8 19 21 0 2
T8 12.0 15 19 0 3
T9 2.0 3 1 0 0
T10 14.2 19 33 0 8
Total 68.8 95 108 1 16
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a

Connections with the rete testis.

b

Closed circles formed by bifurcated tubule portions between 2 branching points.

FIGURE 2.

FIGURE 2

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Core lines of reconstructed seminiferous tubules in the testis from #3 P0 hamster. The core lines of individual seminiferous tubules named T1–T10 in the order of their positions in the testis from the cranial side toward the bottom are shown in the horizontal plane viewed from the top (a) and in the vertical plane viewed from the lateral (b). The positions of the rete testis and blind end are shown as black and gray spheres, respectively

3.2. 3D structure of all seminiferous tubules in the adult hamster

The basement membranes of all seminiferous tubules in an adult testis, stained with PAS‐H, were segmented manually in all 189 images of serial sections with intervals of 50 µm (Figure 3a,b), and the tracing of individual seminiferous tubules labeled with different colors from section to section was performed manually (Figure 3c). The rete testis lay down on the cranial pole of the testis with its longer axis extending from ventral to dorsal directions of the testis. There were 11 seminiferous tubules in the testis, which were named T1–T11 in the order of their initial connections with the rete testis from the ventral toward dorsal side. On the surface of the testis observed from the outside (Figure 3d), T1 and T3 were restricted to narrow cranial–ventral areas close to the rete testis, whereas T4, T6, and T11 were distributed over broad areas surrounding the testis and covered 90% of the whole surface. T8 was observed only on the right‐side surface, whereas T5, T7, and T10 were observed on the left‐side surface. T2 and T9 were hardly observed from the outside. In transverse sections of the testis (Figure 4), the tubules starting from ventral portions of the rete testis (e.g., T1–T5) tended to occupy broader areas in the upper cross sections (e.g., b–d) and those starting from dorsal portions (e.g., T8–T11) in the lower cross sections (e.g., f–h). In each cross section, individual tubules distributed irregularly without forming concentric circles, as observed in the mouse testis.

FIGURE 3.

FIGURE 3

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Photograph of a longitudinal section of the testis from an adult hamster treated with PAS‐H stain (a), after manual segmentation of seminiferous tubules (b), and after tracing of all seminiferous tubules in different colors (c). All reconstructed seminiferous tubules in different colors are superimposed on the reconstructed testis and viewed from 6 directions (d). Individual seminiferous tubules are named T1 (white), T2 (green), T3 (magenta), T4 (light blue), T5 (orange), T6 (light green), T7 (yellow), T8 (navy blue), T9 (cobalt blue), T10 (violet), and T11 (red) in the order of their connections with the rete testis (black) from the ventral toward dorsal side. Scales: 1 mm (a and b) and 5 mm (d)

FIGURE 4.

FIGURE 4

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Transverse sections of all reconstructed seminiferous tubules in the testis from an adult hamster. The testis was cut in 10 horizontal planes from the cranial to caudal poles (a), and transverse sections appearing in the 2nd to 8th planes are shown (b–h). The same colors as in Figure 3 are assigned to T1–T11

We also visualized core lines of the tubules in order to examine the detailed paths of individual tubules, including branching points and blind ends. The software sometimes drew erroneous lines, partly because sections with 50‐µm intervals were used. These mistakes were corrected manually by tracing the core lines in all serial sections. Figure 5 shows the core lines of all 11 seminiferous tubules with respective colors superimposed on the testis together with the rete testis, and Table 3 shows the morphometric parameters for individual core lines. T1 and T2 had blind ends without connection to the rete testis. There were a total of 88 branching points in the seminiferous tubules. T3 and T9 had no branching point, T5, T8, and T10 had one, T7 had two, and T4, T6, and T11 had 29, 12, and 42 points, respectively. Some of the tubule portions arising from a branching point terminated in the rete testis, whereas most of them connected to another branching point, giving rise to a large anastomosing tubule. In T4 and T11, there were 3 and 2 “rings,” respectively (Table 3). Looking at the paths of the core lines of individual tubules (Figure 5), T1 and T2 occupied very small areas near the rete testis, and T3, T5, T7, T8, T9, and T10 occupied only the right or left side of the testis. In contrast, T4, T6, and T11 occupied broad areas of the testis from the surface over circumference to the center. All seminiferous tubules followed winding paths, but the amplitude and direction of the curves were random without the regularity seen in the mouse tubules. Furthermore, the funnel‐like tapering of the caudal portions of tubule networks seen in the mouse testis was not recognized. There were 98 connection points (terminating points) of seminiferous tubules in total with the rete testis. The total length of all seminiferous tubules was 22 m. T1 and T2, which had blind ends, were extremely short. The average length of T3, T5, T7, T8, T9, and T10, which had 0‐2 branching points and no blind end, was 602 mm. In contrast, T4, T6, and T11 were markedly long and had many branching points. The length of T4, T6, and T11 accounted for 83% of that of all tubules, and the number of branching points in them accounted for 94% of that in all tubules. The average numbers of branching points per tubule and per tubule length were 8.0/tubule and 4.0/m, respectively. From these results, T4, T6, and T11 were regarded as the dominant tubules, whereas the others were the shorter tubules. The characteristics of the pathway of seminiferous tubules in this single adult testis, including the total numbers of terminating points, branching points, blind ends, and rings, and the presence of shorter and dominant tubules, were basically similar to those in the 3 P0 testes (Tables 1, 2, 3). Therefore, we consider that the results obtained in the present study are applicable to postnatal hamsters in general.

FIGURE 5.

FIGURE 5

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Core lines of all reconstructed seminiferous tubules in the testis from an adult hamster. The core lines of T1–T11, represented by the same colors as in Figure 2, are superimposed together (All) or separately (T1–T11) on the reconstructed figures of the rete testis (black) and testis (gray) and shown in vertical planes viewed from 2 directions. b is a rotation of a by 90°

TABLE 3.

Parameters in individual reconstructed seminiferous tubules in an adult testis.

Tubule name Length (mm) Terminating points a Branching points Blind ends Rings b
T1 56.6 1 0 1 0
T2 9.5 1 0 1 0
T3 487.3 2 0 0 0
T4 5177.8 25 29 0 3
T5 478.8 3 1 0 0
T6 4589.7 14 12 0 0
T7 778.1 4 2 0 0
T8 790.3 3 1 0 0
T9 288.6 2 0 0 0
T10 788.3 3 1 0 0
T11 8523.7 40 42 0 2
Total 21,968.7 98 88 2 5
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a

Connections with the rete testis.

b

Closed circles formed by bifurcated tubule portions between 2 branching points.

4. DISCUSSION

In the present study, we reconstructed all seminiferous tubules in 3 and 1 testes from P0 and adult Syrian hamsters, respectively, using serial paraffin sections and high‐performance 3D reconstruction software. To the best of our knowledge, this is the first study on the 3D reconstruction of hamster seminiferous tubules. The total length of adult seminiferous tubules obtained in the present study, approximately 22 m, is comparable to the results in past studies without using 3D reconstruction (Sinha‐Hikim et al., 1988b; Seco‐Rovira et al., 2015).

The gross structure of seminiferous tubules in the hamster, as revealed in the present study, differs in several aspects from that in the mouse and rat reported previously (Clermont and Huckins, 1961; Nakata et al., 2015b, 2017). First, the lengths of individual seminiferous tubules in mouse and rat testes are relatively even, whereas the seminiferous tubules in the hamster testis have two distinct types: shorter and dominant tubules. This difference is attributable to the difference in the number of branching points. In our previous study on 3 P0 mice, 72% of the tubules had only 0 or 1 branching point and the other tubules had a maximum of 4, with the average number of branching points per tubule being 1.1 (Nakata et al., 2017). In contrast, in the present study on 3 P0 hamsters, the average number of branching points per tubule was 10.3, and 94% were concentrated in dominant tubules numbering 2–4 per testis and accounting for 86% of the total length of tubules.

Second, the pathways of seminiferous tubules are different between the mouse/rat and hamster both locally and as a whole. In the P0 mouse testis, the tubules locally run in zigzag convolutions and, as a whole, form large circles along the surface of the testis (Nakata et al., 2017). In contrast, in the P0 hamster testis in the present study, the tubules locally run straight and, as a whole, form thin loops (in shorter tubules) or complex networks of anastomosed thin loops (in dominant tubules). In adult mouse and rat testes, the zigzag convolutions of the seminiferous tubules further increase their amplitude and form cranial and caudal hairpin turns, with the cranial turns in contact with the surface of the testis, whereas the caudal turns are located closer to the center of the testis, resulting in funnel‐shaped networks of individual tubules with tapered caudal portions. Furthermore, the caudally located networks surround the preceding cranially located networks from the bottom and outside, making a “stacked‐cup” pattern of networks (Clermont and Huckins, 1961; Nakata et al., 2015b, 2017). In contrast, in the present adult hamster testis, such regular paths of seminiferous tubules are not seen: The curves in each tubule have random amplitudes and directions, and there is no caudal tapering nor stacked‐cup pattern in the tubule networks.

The testes of the mouse and rat develop from the testis cords of embryonic gonads that form a series of large C‐shaped arches placed side by side (Jost et al., 1981; Combes et al., 2009). These testis cords, numbering 10–30, may develop into the postnatal seminiferous tubules of the mouse and rat basically in the original arrangements, with minor modifications by branching and anastomosis. In contrast, the human testis cords are composed of a network of numerous thin loops anastomosed to each other (Bremer, 1911). On the other hand, the structure of the testis cords in the hamster is not well understood. If the testis cords in the hamster are similar to those in humans and composed of an assembly of many anastomosed thin loops instead of large C‐shaped arches as in the mouse and rat, it may account for the postnatal patterns of hamster seminiferous tubules as observed in the present study. We hypothesize that such thin loops of embryonic hamster testis cords undergo extensive anastomosis to give rise to the dominant seminiferous tubules by the time of birth. The reason why the frequency of anastomosis is larger in the hamster than in the mouse and rat may be that the testis cords are more densely packed and so the distance between their basement membranes is closer in the former. In order to support this possibility, we calculated the ratio of the volume of seminiferous tubules to that of the testis in P0 hamsters and P0 mice obtained in our present and past (Nakata et al., 2017) studies, respectively. The value in hamsters, 34.4 ± 1.7%, was twofold of that in mice, 16.1 ± 3.0% (n = 3, p < 0.05 by unpaired Student's t test). Further studies will be necessary to clarify how seminiferous tubules develop from testis cords in hamster embryos.

For the automatic segmentation of seminiferous tubules in P0 hamsters in the present study, myoid cells surrounding the basement membrane were immunostained with anti‐αSMA antibody, followed by splitting the DAB color using ImageJ software. Compared with the technique used in our previous study in P0 mice (Nakata et al., 2017), that is, immunofluorescence staining of the basement membrane with anti‐laminin antibody, the present technique is easier for the segmentation of seminiferous tubules, because the myoid cell layer is more conspicuous than the basement membrane, and the DAB color can be observed in a bright field and is more stable than fluorescence. However, we did not adopt the present or previous technique in the adult hamster testis, which was too large to uniformly immunostain all portions. Also, in the adult hamster testis, PAS‐H stained the basement membrane only weakly and it also stained cells in the seminiferous epithelium, making automatic segmentation of the basement membrane difficult, as was performed in the adult mouse testis (Nakata et al., 2017). Therefore, we had to perform manual segmentation of the basement membrane. These are the reasons why only a single adult hamster testis was analyzed in the present study. The hamster testis is 15 times larger than the mouse testis, and the human testis is 10 times larger than the former. Therefore, in a future study, 3D reconstruction of whole seminiferous tubules in the adult human testis may be possible but would require considerable time and labor using the present procedure. The application of artificial intelligence based on deep learning of pattern recognition may automate both the segmentation and tracing steps, thereby making it easier to perform 3D reconstruction from numerous serial paraffin sections.

The present study revealed the 3D structure of seminiferous tubules in developing and adult Syrian hamsters, which is different from that in mice and rats. The changes associated with aging or short and long photoperiods will be the subject of future studies. Although the procedure used in the present study is simple, it is a powerful tool to analyze organs with complex tubular structures, and will be useful for studying the male reproductive tract not only in rodents but also in humans under normal and pathological states, such as infertility.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

HN, TI, MY, and YS designed the project. HN, MY, and SN performed the research. HN, SI, and AM analyzed the data and wrote the paper. All authors discussed the data and commented on the paper.

ACKNOWLEDGEMENTS

We thank Mr. Itsuro Kamimura (Maxnet Co., Ltd.) for advice on using Amira software, and Dr. Toshihisa Hatta (Kanazawa Medical University) and Dr. Satoru Honma (Kanazawa Medical University) for the use of Nanozoomer 2.0‐HT. We also thank Mr. Shuichi Yamazaki for technical assistance in making paraffin sections, and Ms. Ryoko Yamagishi and Ms. Sachiko Okabe for assistance in segmentation.

Raoof M, Yoshiike M, Nozawa S, et al. Three‐dimensional structure of seminiferous tubules in the Syrian hamster. J. Anat. 2020;238:86–95. 10.1111/joa.13287

Funding information

This work was supported by JSPS KAKENHI Grant Numbers JP16K18976, JP18K09153, and JP19K16743, and the Takeda Science Foundation.

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