Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells

Journal Pre-proof Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells Jun Ito, Kanna Meguro, Kazuki Komatsu, Takuya Ohdaira, Rina Shoji, Takahisa Yamada, Satoshi Sugimura, Yohei Fujishima, Akifumi Nakata, Manabu Fukumoto, Tomisato Miura, Hideaki Yamashiro

PII:

S0378-4320(19)30250-7

DOI:

https://doi.org/10.1016/j.anireprosci.2020.106288

Reference:

ANIREP 106288

To appear in:

Animal Reproduction Science

Received Date:

14 March 2019

Revised Date:

27 December 2019

Accepted Date:

16 January 2020

Please cite this article as: Ito J, Meguro K, Komatsu K, Ohdaira T, Shoji R, Yamada T, Sugimura S, Fujishima Y, Nakata A, Fukumoto M, Miura T, Yamashiro H, Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells, Animal Reproduction Science (2020), doi: https://doi.org/10.1016/j.anireprosci.2020.106288

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Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells

Jun Itoa, Kanna Meguroa, Kazuki Komatsua, Takuya Ohdairaa, Rina Shojia, Takahisa

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Yamadaa, Satoshi Sugimurab, Yohei Fujishimac, Akifumi Nakatad, Manabu Fukumotoe, Tomisato Miurac, Hideaki Yamashiroa,* a

Graduate School of Science and Technology, Niigata University, Niigata, 959-2181,

Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo,

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b

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Japan;

Graduate School of Health Sciences, Hirosaki University, Aomori, 036-8560, Japan;

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Division of Life Science, Hokkaido University of Science, Hokkaido, 006-8590, Japan;

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c

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183-0054, Japan;

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Japan

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Department of Molecular Pathology, Tokyo Medical University, Tokyo, 160-8402,

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*Corresponding

Author:

E-mail

address:

(H.Yamashiro)

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[email protected]

Highlights  Spermatogenesis is controlled by proliferation and apoptosis in male germ cells.  Large Japanese field mice could be used as an animal model to study spermatogenesis.

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 Additionally, the effects of ecological and anthropogenic factors can be studied.

ABSTRACT

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The aim of this study was to investigate the proliferation and apoptosis of male germ

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cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan.

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Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells

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were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase

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(TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay.

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The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the 2

seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.

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Keywords: Apodemus speciosus; Seasonal reproductive cycle; Spermatogenesis; Testis 1. Introduction

Many mammalian seasonal breeders such as white-footed mice (Johnston and Zucker,

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1980), brown hare (Simeunvic et al., 2000), roe deer (Roelants et al., 2002), Iberian

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mole (Dadhich et al., 2010; Dadhich et al., 2013), and large Japanese field mice

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(Apodemus speciosus) (Meguro et al., 2019 in press) have responses to cyclic seasonal changes with physiological and behavioural changes being associated with the seasonal

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changes. Seasonal breeding animals have a particular type of spermatogenesis

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regulation, as male testis activation and involution are dependent on environmental conditions, including photoperiod, temperature, and food availability. Thus, researchers

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study the mechanisms controlling reproductive processes to understand the effects of ecological factors on animal reproduction. Testicular function is controlled both genetically and hormonally, but environmental conditions may also affect these functions. Spermatogenesis is thus an indicator of the effects of ecological and

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anthropogenic factors on animal reproduction (Dadhich et al., 2010). Proliferating cell nuclear antigen (PCNA) is a widely used marker for germ cell proliferation in seasonal breeding animals (Tokunaga et al., 1999; Morales et al., 2002; Strbenc et al., 2003). There is a specific pattern of PCNA staining in mice testes for the

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intermediate, A, and B types of spermatogonia and primary spermatocytes in the leptotene and zygotene phases when there are immunoreactive evaluations using the

PCNA antibody (Chapman and Wolgemuth, 1994). The number of PCNA-positive germ

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cells in the seminiferous tubules of brown hares decreases slightly from September to

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November and increases again in mid-November (Strbenc et al., 2003). In monkeys, the

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density of PCNA-positive germ cells in the seminiferous epithelium decreases by ¼ to ⅓ during the nonbreeding season compared with the density during the breeding season

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(Tokunaga et al., 1999). In brown hares and monkeys, the proliferative potential of male

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germ cells depends on the stage of the reproductive cycle. In contrast, in Syrian hamsters, the number of PCNA-positive germ cells decreases only during the regressing

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phase of spermatogenesis and increases until the breeding season (Seco-Rovira et al., 2015). The results of these studies indicate that there is variation in the proliferative potential of male germ cells in seasonal breeding animals. The PCNA protein also has important functions in many aspects of DNA replication and replication-associated

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processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly (Boehm et al., 2016). Spontaneous germ cell apoptosis is common in normal testes and is an important factor involved in regulating germ cell development and sperm output (Sinha Hikim and

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Swerdloff, 1999). Results from this previous study indicated that there was spontaneous apoptosis of a few differentiating spermatogonia, in particular type A spermatogonia, and spermatocytes during the meiotic divisions in rats and hamsters. In mice,

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spontaneous apoptosis was most commonly observed in spermatocytes, including

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dividing spermatocytes, less frequently in spermatogonia, and seldom in spermatids.

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Germ-cell apoptosis seems to be an important process for preserving the genomic integrity of male gametes by eliminating irreparably damaged cells (Gartner et al.,

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2008). Apoptosis is also observed during the transitional phase in the seminiferous

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epithelial germ cells of several seasonal breeding animals such as hamsters (Furuta et al., 1994; Morales et al., 2002), white-footed mice (Young et al., 2001), European brown

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hares (Strbenc et al., 2003), and large hairy armadillos (Luaces et al., 2014), and is regulated by hormonally-controlled processes. Apoptosis increases when there are lesser androgen concentrations in the hormonal milieu of the testes (Lee et al., 1999; Woolveridge et al., 1999) and there is evidence that the photoperiod regulates germ cell

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apoptosis in these species (Blottner et al, 1995; Blottner et al., 1999; Young et al., 1999). With different processes, such as entrance of male germ cells into the epididymis during the inactive phase, there is regulation of spermatogenesis in Iberian moles (Dadhich et al., 2010). The detailed changes in proliferation and apoptosis of male germ cells in the

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testis during the seasonal reproductive cycle, and the resulting regulation of spermatogenesis in the testes of large Japanese field mice have not been studied.

Based on our knowledge of the reproductive biology of large Japanese field

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mice, we consider this species to be an excellent animal model for studying

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spermatogenesis and the effects of environmental changes. In the present study, there is

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evaluation of the physiological dynamics of male germ cell proliferation and apoptosis

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during the seasonal reproductive cycle of the large Japanese field mice.

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2. Materials and methods

2.1. Collection of large Japanese field mice

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The study protocol was designed in accordance with the laboratory animal care and

use guidelines, and all procedures were conducted in accordance with the guidelines of the Ethics Committee for Care and Use of Laboratory Animals for Research of Niigata University, Japan (Protocol No. 26-80-2). The capture of undomesticated rodents in the

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sampling area was permitted by the Niigata City Office. Large Japanese field mice were captured from March 2015 to December 2015 at Kakuta Mountain in Niigata (Table 1). Individual large Japanese field mice were captured using Sherman-type live traps baited with peanuts, as described previously (Akiyama et al., 2015). Large Japanese field mice

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(n = 21) were used in this experiment. The mice were killed by cervical dislocation on the day of capture. It was difficult to determine the age of the mice, and therefore, large Japanese field mice with a body weight of more than 30 g were included in the present

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study. The testes were isolated from each mouse, and one testis from each mouse was

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fixed in Bouin’s solution. The other testis was frozen at -80 °C for further studies.

2.2. Histological analysis of testis for classification of stages of spermatogenesis

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Individual testes (n = 21) were fixed in Bouin’s solution. Fixed testes were embedded

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in paraffin, and then stained using hematoxylin and eosin (HE), according to a previously described method (Akiyama et al., 2015). The testes were then briefly

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dehydrated in a series of different concentrations of alcohol, made transparent by treatment with toluene, embedded in paraffin, and cut into 5 μm-thick sections before staining. To reduce the effects of the cross-sectional position of the seminiferous tubules, there

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was counting of the number of male germ cells in seminiferous tubules with intact basal lamina in each section. There was also examination of the diameters of the seminiferous tubules and the total number of elongated spermatids and male germ cells in random groups of ten seminiferous tubules in a blinded fashion.

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The diameter of seminiferous tubules was used as a criterion for classification, because there was a correlation between the presence of sperm in the end part of the

cauda epididymis and seminiferous tubule diameter. The phases of spermatogenesis

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during the seasonal reproductive cycle were identified as the active, transitional, and

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inactive phases when the diameters of the seminiferous tubules were more than 170 μm,

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between 110 to 170 μm, and less than 110 μm, respectively (Table 2). This classification

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is a modified version of a method used in a previous study (Akiyama et al., 2015).

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2.3. Immunofluorescence staining

Immunostaining was performed utilising a modified version of a previously described

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method (Takino et al., 2017). Individual testes (n = 21) were fixed in Bouin’s solution, embedded in paraffin, cut into 5 µm sections, and placed onto slides. The paraffin from sections was removed with use of xylene and then re-hydrated using graded ethanol solutions. The slides were prepared in 0.01 M sodium citrate buffer in an oven at 121 °C

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for 10 min. The slides were subsequently washed twice in phosphate buffered saline (PBS) and incubated with blocking buffer (5% bovine serum albumin in PBS) for 40 min at room temperature. Rabbit anti-PCNA antibody (ab2426, Abcam, Cambridge, UK) at a dilution of 1:500 was used as the primary antibody. Alexa Fluor

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488-conjugated donkey anti-rabbit IgG H&L (ab150073, Abcam) at a dilution of 1:200 was used as the secondary antibody. The number of PCNA-positive germ cells was determined using a confocal laser scanning microscope (Olympus, BX61, Tokyo,

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2.4. In situ detection of apoptotic cells

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Japan).

To detect apoptotic cells in the seminiferous tubules, a terminal deoxynucleotidyl

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transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling

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(TUNEL) assay was performed on 21 individual paraffin-embedded sections using an Apoptosis in situ Detection Kit (Wako, Osaka, Japan) by modifying the manufacturer’s

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protocol. The number of seminiferous tubules surrounded by an intact basal lamina that enclosed TUNEL-positive germ cells was determined for each sample. After the number of TUNEL-positive germ cells for each seminiferous tubule and the total number of seminiferous tubules were determined, the number of apoptotic cells per seminiferous

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tubule was calculated. Based on the number of male germ cells observed using H&E staining, the percentage of apoptotic cells per seminiferous tubule was calculated. To determine the frequency of apoptotic cells present during spermatogenesis, the numbers of distinct TUNEL-positive germ cells along the basal lamina and in the central lumen

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were determined.

2.5. Statistical analyses

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Statistical analyses were performed using STATVIEW Version 5.0 (Abacus Concepts

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Inc., Berkeley, CA, USA). Normality assumptions for continuous variables were tested

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using the Bartlett test. For approximately normally distributed data, descriptive statistics, such as mean, standard deviation, and range values, were calculated. Differences

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between treatment groups were analysed using an one-way analysis of variance

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(ANOVA) with Fisher’s protected least-significant difference post-hoc test. Data are

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expressed as the mean ± SD. P value of less than 0.05 indicated statistical significance.

3. Results

3.1. Testis size in large Japanese field mice There were time-of-year correlations with active (n = 11), transitional (n = 6) and

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inactive (n = 4) phases of spermatogenic activity of large Japanese field mice (Table 1). Annual seasonal changes in the testicular morphology in the mice were similar to those previously described (Akiyama et al., 2015). The average body weights during the active, transitional and inactive phases were 42.5 ± 6.1 g, 41.8 ± 8.0 g and 38.8 ± 5.4 g,

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respectively. The testis length during the active phase (14.9 ± 1.6 mm) was greater than that during the transitional phase (10.5 ± 2.4 mm) and there were further decreases in

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3.2. Histological analysis using H&E staining

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length during the inactive phase (7.8 ± 2.4 mm).

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Representative photomicrographs illustrating the histology of seminiferous tubules are shown in Figure 1. I. The numbers of male germ cells, elongated spermatids per

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seminiferous tubule, and the diameters of seminiferous tubules in the 21 individual

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testis sections were determined (Fig. 1. Ⅱ). The average diameter of seminiferous tubules during the active, transition, and inactive phases was 188.0 ± 10.1 µm, 125.3 ±

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6.2 µm, and 82.3 ± 10.1 µm, respectively (Fig. 1. Ⅱ A). The diameter of the seminiferous tubules was less during the transitional and inactive phases than during the active phase (P < 0.05). In addition, the numbers of male germ cells and elongated spermatids were less during the transitional and inactive phases than during the active

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phase (P < 0.05; Fig. 1. Ⅱ B and C). There was a difference in the number of male germ cells per Sertoli cell in tissues during the active and inactive phases (P < 0.05; Fig. 1. Ⅱ D).

Localisation

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semi-quantitative

analysis

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proliferation

using

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3.3.

immunofluorescence

To confirm the localisation of germ cells with PCNA-positive nuclei in seminiferous

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tubules, there was immunofluorescence staining of the testis sections. Germ cells with

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PCNA-positive nuclei were observed in the first cell layer from the basal membrane in

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the seminiferous tubules during the active and inactive phases (Fig. 2. I). Furthermore, germ cells with PCNA-positive nuclei were also observed in one or two more layers of

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the seminiferous tubules during the transitional phases. Seminiferous tubules during the

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transitional phase were present on the central lumenal side, and some seminiferous tubules were completely filled with germ cells with PCNA-positive nuclei.

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The number of germ cells with PCNA-positive nuclei per seminiferous tubule during

the active, transitional, and inactive phases was 33.5 ± 5.3, 33.8 ± 2.3 and 17.8 ± 6.6, respectively (Fig. 2. Ⅱ). The number of germ cells with PCNA-positive nuclei during the inactive phase was less than that during the active and transitional phases (P < 0.05).

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3.4. Detection of apoptotic cells using the TUNEL and DNA ladder formation assays To identify apoptotic cells in situ, testis sections were analysed using the TUNEL assay. The number of apoptotic cells and the rate of apoptosis was calculated. In

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seminiferous tubules, spermatogonia, spermatocytes, and spermatids were stained (Fig. 3. I). There was no difference in the number of apoptotic cells per seminiferous tubule

among the different phases of spermatogenesis (Fig. 3. Ⅱ A). The percentages of

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TUNEL-positive germ cells during the active, transitional, and inactive phases were 3.2

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± 1.9%, 3.9 ± 5.4% and 10.7 ± 5.3%, respectively, with the percentage for the inactive

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phase being greater than those during the other phases (P < 0.05; Fig. 3. Ⅱ B). In addition, the percentages of TUNEL-positive germ cells in and along the basal

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lamina during the active, transitional, and inactive phases were 43.0 ± 8.5%, 16.0 ±

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8.3%, and 39.2 ± 6.7%, respectively (Fig. 3. Ⅱ C). The proportions of cells in and along the central lumen during the active, transitional, and inactive phases were 57.0 ±

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8.5%, 84.0 ± 8.3%, and 60.8% ± 6.7%, respectively (Fig. 3. Ⅱ D).

4. Discussion The present study was undertaken to investigate the physiological dynamics of

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proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice. Phases of spermatogenesis during the seasonal reproductive cycle were classified into active, transitional, and inactive phases based on seminiferous tubule diameter of mice in each phase. In seasonal breeding animals, there

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are seasonal-associated changes in testicular weight, testicular mass, seminiferous tubule diameter, number of male germ cells, and number of elongated spermatids. The results from use of H&E and immunofluorescence staining with a PCNA antibody,

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which allows for identification of spermatogonia types A and B, and primary

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spermatocytes in the leptotene, zygotene, and pachytene phases (Chapman and

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Wolgemuth, 1994) indicated the number of male germ cells in the seminiferous tubules, including secondary spermatocytes and elongated spermatids, decreased during the

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transitional compared with active phase. Furthermore, the numbers of spermatogonia

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and primary spermatocytes also decreased until there was onset of the inactive phase. Collectively, these findings indicate the germ cell proliferation capacity changed

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markedly during the phases of spermatogenesis, because the regulation of the number of spermatogonia and primary spermatocytes is important for controlling the production of male germ cells. Further investigation using well-established protein markers is needed to define the full spectrum of germ cells that are present in the active, transitional, and

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inactive phases in large Japanese field mice testes. In addition, there were male germ cells with PCNA-positive nuclei in the first cell layer from the basal membrane in the seminiferous tubules during the active and inactive phases, whereas male germ cells with PCNA-positive nuclei were only detected

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in one or two layers of the seminiferous tubules during the transitional phase. During the transitional phase, some seminiferous tubules were present at the side of the central

lumen, and some of these were completely filled with germ cells with PCNA-positive

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nuclei. In other seasonal breeding animals such as the armadillo, spermatocytes and

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spermatids detach from the side of the basal lamina and translocate to the central lumen

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due to a decreased abundance of cell adhesion factors during testis regression, and these cells may be eliminated as a result of apoptosis (Luaces et al., 2014). There is a large,

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dynamic network of linked PCNA molecules at and around the replication fork area of

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DNA (Boehm et al., 2016). Such a protein network would serve to increase the local concentration of all the proteins necessary for normal replication, translesion synthesis,

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error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly and regulate the various processes during DNA replication. The limitations of PCNA as a proliferation marker in spermatogenesis studies, therefore, need to be considered. Furthermore, the functions of PCNA in DNA replication and in

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replication-coupled DNA damage tolerance and repair processes should be examined. Spontaneous apoptosis affects germ cell development. Thus, there is a probability that apoptosis leads to modulation of spermatogenesis in seasonal breeding animals (Sinha Hikim and Swerdloff, 1999). In the present study, there was examination of the number,

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percentage, and localisation of apoptotic germ cells in seminiferous tubules in testis sections during each phase of spermatogenesis using the TUNEL assay. The number of

TUNEL-positive germ cells per seminiferous tubule during spermatogenesis was not

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different. The rate of TUNEL-positive germ cells to total male germ cells as determined

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using H&E staining procedures, however, was greater during the inactive compared

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with other phases of spermatogenesis. Approximately 40% of the total TUNEL-positive germ cells, identified as spermatogonia, in the seminiferous tubules were localised

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along the basal lamina during the active and inactive phases. During the transitional

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phases, however, only approximately 20% of these cells were localised to the basal lamina. To the best of our knowledge, this is the first study in which results indicate

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there is an increased apoptosis in the central lumen side during the regressing phase with there being a decreased number of germ cells, including spermatocytes or spermatids. In addition, apoptosis along the basal lamina was induced to a greater extent during the inactive phase than during the regressing phase. The process of apoptosis

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may result in elimination of germ cells with meiotic or mitotic errors during the periods when there is a greater rate of germ cell proliferation. In addition, apoptosis may function to remove cells with a PCNA-positive nucleus, such as spermatogonia and primary spermatocytes, and thus control the number of male germ cells in the testes of

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large Japanese field mice in response to seasonal changes. In contrast, Leydig cells, which are located within the interstitial compartment of the

testis, are steroidogenic cells that have primary functions in the synthesis and secretion

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of testicular androgens, of which testosterone is the most important during

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spermatogenesis (Martin, 2016). In moles, the depletion of most of the adluminal

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portion of the germinative epithelium during testis regression implies there is a dramatic remodeling of the somatic cells of the gonad (Dadhich et al., 2013). The marked

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reduction in the seminiferous-tubule diameter leads to changes in Leydig cells with the

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result being the occupying of the lesser available inter-tubular space, forming a continuous matrix of interstitial tissue in which the much smaller tubules are finally

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embedded. There was no apoptosis and cell proliferation in the Leydig cells of large Japanese field mice during the seasonal breeding cycle, as reported for the brown hare (Strbenc et al., 2003). Testosterone synthesis by Leydig cells probably contributed to the transition to active phases of spermatogenesis in large Japanese field mice testes.

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5. Conclusions Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells, including spermatogonial and primary spermatocytes.

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Notably, this species of wild mice can be used as an animal model to study spermatogenesis and the effects of ecological and anthropogenic factors on animal

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reproduction.

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Author statements JI and HY were responsible for the design, carried out the experiment, and drafted the manuscript. KM, KK, TO, RS, TY, SS and YF carried out experiment. AN, MF and TM were collaborated and responsible for coordination of the study.

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Conflicts of interest

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The authors declare no competing financial interests.

Acknowledgements This research was supported by the Japan Society for the Promotion of Science, and entrusted to the Japan Atomic Energy Agency (JAEA) by the Ministry of Education, 18

Culture, Sports, Science, and Technology of Japan (MEXT). This work was also supported by the Japan Society for the Promotion of Science (JSPS)

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Takino, S., Yamashiro, H., Sugano, Y., Fujishima, Y., Nakata, A., Kasai, K., Hayashi, G., Urushihara, Y., Suzuki, M., Shinoda, H., Miura, T., Fukumoto, M., 2017. Analysis of the effect of chronic and low-dose radiation exposure on spermatogenic cells of male large Japanese field mice (Apodemus speciosus) after the Fukushima Daiichi

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Nuclear Power Plant accident. Radiat. Res. 187, 161–168. Tokunaga, Y., Imai, S., Toril, R., Maeda, T., 1999. Cytoplasmic liberation of protein gene product 9.5 during the seasonal regulation of spermatogenesis in the monkey (Macaca fuscata). Endocrinology 140, 1875–1883.

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Woolveridge, I., de Boer-Brouwer, M., Taylor, M.F., Teerds, K.J., Wu, F.C.W., Morris, I.D., 1999. Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes. Biol. Reprod. 60, 461–470.

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Young, K.A., Zirkin, B.R., Nelson, R.J., 1999. Short photoperiods evoke testicular

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apoptosis in white-footed mice (Peromyscus leucopus). Endocrinology 140, 3133–

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3139.

Young, K.A., Nelson, R.J., 2001. Mediation of seasonal testicular regression by

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ur

na

apoptosis. Reproduction 122, 677–685.

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Fig. 1. Histological analysis of the testis and classification of the stages of spermatogenesis; Ⅰ: Photomicrographs of haematoxylin-eosin stained sections of large Japanese field mice testes. A, active phase; B, transitional phase; C, inactive period. Spg,

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spermatogonia; Spc, spermatocytes; Spm; spermatid; Espm, elongated spermatids; Bars:

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50 µm

25

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Fig. 2. Localisation of germ cells with PCNA-positive nuclei in seminiferous tubules by

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immunofluorescence staining;Ⅰ: active phase; transitional phase; inactive phase and

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negative control for active phase; PCNA staining spermatogonia, and/or primary spermatocytes; Ⅱ: Mean number of PCNA-positive cells per seminiferous tubule;

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Values are presented as mean ± SD;

a–b

Different superscripts denote differences (P <

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0.05); Bars: 100 µm

26

27

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Fig. 3. Apoptosis determined using TUNEL staining of seminiferous tubules of large

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Japanese field mice; Ⅰ: Brown stained nuclei of apoptotic cells, A, active phase; B,

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high magnification of A; C, transitional phase; D, high magnification of C; E, inactive period; F, high magnification of E; Black arrows indicate TUNEL-positive germ cells

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(spermatogonia or spermatocytes); Bars: 50 µm (A, C, and E), 10 µm (B, D, and F); Ⅱ:

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A, average number of TUNEL-positive male germ cells per seminiferous tubule; B, average rate of TUNEL-positive male germ cells; C, the rate of TUNEL-positive male germ cells along the basal lamina; D, the rate of TUNEL-positive male germ cells in the central lumen; Values are presented as mean ± SD; differences (P < 0.05) 28

a–b

Different superscripts denote

Table 1 Body weight and testis size of large Japanese field mice Spermatogenesis stage Sampling Date

Mice

Body weight (g)

Testis length (mm)

Testis diameter (mm)

2015/4/10

1

32.7

14

8

2015/4/10 2015/4/10

2 3

32.3

16

8

38.6

15

9

2015/5/8 2015/5/12 2015/7/22 2015/8/26 2015/8/26 2015/10/27 2015/11/11 2015/11/13

4 5 6 7 8 9 10 11

45.3

16

9

15

7

13

7

16

8

17

9

15

6

13

6

42.2

12

7

42.5 ± 6.1

14.9 ± 1.6

7.6 ± 1.1

41.5

12

5

38.7

10

5

31.8

7

4

48.0

11

5

37.1

9

5

53.9

14

7

41.8 ± 8.0

10.5 ± 2.4

5.2 ± 0.9

1 2 3 4 5 6

ur

na

2015/5/14 2015/6/5 2015/6/5 2015/6/16 2015/6/26 2015/7/10

45.3

53.6 44.1 42.7

n=6

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44.9

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Transition

45.4

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n = 11

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Active

1

36.8

6

4

2015/6/5

2

37.5

6

3

2015/11/20 2015/12/1

3 4

34.2

8

4

46.6

11

4

38.8 ± 5.4

7.8 ± 2.4

3.8 ± 0.5

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Inactive 2015/5/21

n=4

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Table 2 Classification criteria for reproductive stage based on the diameter of seminiferous tubules Spermatogenesis stage Diameter of seminiferous

Transitional (n =

11)

6)

>170 µm

110–170 µm

Inactive (n = 4)

<110 µm

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tubule (µm)

Active (n =

30