Cathepsin B plays a role in spermatogenesis and sperm maturation through regulating autophagy and apoptosis in mice

Animals

All animal experimental procedures were approved by the Ethics Committee of Shandong First Medical University (W202111230331, Jinan, China). Animal experiments were strictly carried out in accordance with the national and local animal protection laws. All mice were maintained under SPF conditions (22–24 °C, 50–55% humidity,12 h light/dark cycle) with free access to water and food in Laboratory Animal Center of Shandong First Medical University. All mice were treated humanely and with efforts to minimize suffering. To induce loss of consciousness and death with a minimum of pain and distress, all mice were euthanized by cervical dislocation to collected tissue samples for further analyses. There were no surviving animals at the end of study.

Quantitative RT-PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First strand cDNA was synthesized from total RNA using Primescript Reverse transcriptase (Takara, Tokyo, Japan). Then, a 10 µl mixture was made up containing 5 µl SYBR Premix Ex Taq reagent system (TakaRa, Tokyo, Japan), 0.2 µl Ctsb forward primer: 5′-ACCTTTGATGCACGGGAACA-3′, 0.2 µl Ctsb reverse primer: 5′-ACTCGGCCATTGGTGTGAAT-3′, 1 µl cDNA template, and 3.6 µl ddH₂O. RT-qPCR was performed on a BioRad Sequence Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with usual amplification condition (95 °C for 10min, (95 °C 15 s, 65 °C 30 s, 72 °C 30 s) × 40). Triplicates were performed for each reaction. The mRNA levels were calculated using the 2^−ΔΔCT method with glyceraldehyde phosphate dehydrogenase (GAPDH) as internal control, and the Gapdh primers were: forward, 5′-GATGCCCCCATGTTTGTGAT-3′, reverse, 5′-GGCATGGACTGTGGTCATGAG-3′.

Generation of Ctsb^-/- mice

Ctsb^-/- mice were generated using CRISPR/Cas9 technology. The single-guide RNA (sgRNA) target sequences were 5′-CCCTTGAGCGACAGGAAAAACCA-3′ and 5′-TTTCCAAAATTTAGCGGCCCTGG-3′. The sgRNAs were produced through in vitro transcription using a MEGAshortscript T7 kit (Ambion, Austin, TX, USA). The hCas9 mRNA was derived from pST1374-N-NLS-flag-linker-cas9, which was synthesized using the mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX, USA), and polyadenylated with a Poly(A) Polymerase Tailing kit (Life Technologies, Carlsbad, CA, USA). Wild-type C57BL/6 superovulated females at 5-6weeks of age were mated with adult C57BL/6 males to obtain zygotes. The Cas9 mRNA and sgRNA were injected into zygotes by microinjection. The injected zygotes were then transferred into the oviducts of pseudopregnant CD1 female mice. Genomic DNA was extracted from the tails of newborn founders. The genomic DNA fragments surrounding the sgRNA target sites were amplified by PCR (primers: Ctsb forward, 5′-ATGTAGCACATTCACTCTGTAAGC-3′; Ctsb reverse 1, 5′-CTTTTGGAAGTCCTGCAGTCAAA-3′; and Ctsb reverse 2, 5′-AAAGGGCCATGTTAAATTCCTTCTG- 3′).

Western blotting

Protein was extracted using Pierce™ RIPA Buffer (Thermo Fisher, NY, USA) with a protease inhibitor cocktail (Bimake, Houston, TX, USA). The denatured proteins were subjected to 10% sodium dodecyl polyacrylamide gel electrophoresis (SDS/PAGE) followed by electrotransfer onto a PVDF membrane. After being blocked with 5% skim milk in TBST for 2 h at room temperature, the membranes were incubated overnight with primary antibodies at 4 °C. The antibodies used were as follows: anti-CTSB (1:1,000; Abcam, Cambridge, UK), anti-LC3 (1:1,000; Proteintech, Chicago, USA), anti-ATG5 (1:1,000; Abcam, Cambridge, UK), anti-Caspase3 (1:1,000; Abcam, Cambridge, UK), anti-Cleaved Caspase3 (1:1,000; CST, Boston, USA), anti-β-actin (1:5,000; Abcam, Cambridge, UK), and anti-GAPDH (1:5,000; GeneTex, San Antonio, USA). After being washed with TBST, the membranes were incubated with secondary antibodies for 1 h at room temperature. The bands on Western blotting were quantified using Image J software and normalized to β-actin or GAPDH.

Histological analysis and fertility test

The testes and epididymides were isolated from the adult male mice and fixed with Bouin’s solution for 12 h at room temperature. Next, the tissues were dehydrated via an ethanol series from 30% to 100%. Afterwards, the tissue samples underwent xylene dehydration, clearing, and embedded into paraffin. Tissue sections (4 µm) were spread onto slides and dried overnight at 55 °C. After deparaffination and rehydration by immersing in serial concentrations of ethanol, tissue sections were stained with hematoxylin and eosin (H&E) for histological analysis. Sperm samples from the cauda epididymis were spread onto slides and air dried overnight. Finally, after being fixed with 4% paraformaldehyde in PBS for 30 min, sperm samples were stained with H&E according to standard protocols. At least 200 sperm cells were used to assess the sperm morphology in each mouse, 4–6 mice were evaluated in each group.

To test the reproductive ability, 2-month-old WT or Ctsb^-/- males were caged with adult WT females in a ratio of 1:2 for 3 months. The number of pups per litter was recorded. Four mating cages were set for each group.

Computer-assisted sperm analysis (CASA)

The cauda epididymis was detached in each adult male mouse, washed with PBS, and placed in an M2 medium (Sigma-Aldrich, St. Louis, MO, USA). Sperm cells were allowed to exude from incisions of the cauda epididymis for 30 min at 37 °C under 5% CO₂, after which any debris was removed and volume was increased to one mL with M2 medium. 10 µL of the exudate was used to assess sperm motility and sperm count by using the CASA system (Tsinghua Tongfang, Beijing, China).

Immunohistochemistry

The tissues were fixed with 4% PFA for 12 h at room temperature, then dehydrated and embedded into paraffin. Immunohistochemistry was performed on paraffin-embedded sections. After deparaffination and rehydration, tissue sections were subjected to antigen retrieval. Endogenous peroxidase was inhibited through incubation with 3% H₂O₂ for 10 min at room temperature, and tissue sections were blocked in 10% normal goat serum for 30 min at room temperature. Then, the tissue sections were incubated in primary antibodies at 4 °C overnight. The primary antibodies used in this study included a rabbit monoclonal anti-CTSB antibody (1:200, Abcam, Cambridge, UK) and a rabbit monoclonal anti-SOX9 antibody (1:200, ABclonal, Wuhan, China). The staining process was performed using a Streptavidin-Peroxidase-Biotin kit (SP-9000, ZSGB-BIO, Beijing, China), and chromogenic reaction was conducted with a DAB kit (ZLI-9017; ZSGB-BIO, Beijing, China) according to the manufacturer’s instructions.

Immunofluorescence staining

Immunofluorescence staining was performed on paraffin-embedded sections as previously described (Wen et al., 2020). Specifically, after deparaffination and rehydration, tissue sections were subjected to antigen retrieval and permeabilization. Then, the sections were blocked with 5% normal goat serum and incubated with primary antibody (γ-H2AX; Abcam, Cambridge, UK) at 4 °C overnight and secondary antibody at 37 °C for 1 h. The nuclear was stained with DAPI, and the images were captured using ZEISS LSM 880 confocal laser scanning microscope (ZEISS, Oberkochen, Germany).

The FITC conjugate Arachis hypogaea (peanut) agglutinin (PNA) was used to assess the sperm acrosome. The sperms collected from cauda epididymis were carefully washed with PBS, then coated on slides and air-dried. The samples were fixed with 4% PFA, washed with PBS, and stained with PNA at 37 °C for 30 min. For the acrosome staining in testis, testicular paraffin sections were dewaxed and rehydrated, then PNA was stained at 37 °C for 30 min.

Transmission electron microscopy (TEM)

The caput epididymides were rapidly isolated from WT and Ctsb^-/- mice and fixed with 2.5% glutaraldehyde overnight at 4 °C. After post-fixation with 1% osmic acid, the caput epididymides were infused in 10% gelatin, dehydrated in sucrose, and then frozen in liquid nitrogen. Cryosections (50 nm) were prepared using a cryo-ultramicrotome (Leica, Wetzlar, Germany) and observed with a JEM-1200EX microscope (JEOL, Tokyo, Japan) following the manufacturer’s protocol.

TUNEL assay

Cell apoptosis was measured using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay with an in situ cell death detection kit (Roche, Basel, Switzerland) following the manufacturer’s protocols.

Statistical analysis

All statistical data were analyzed by using GraphPad Prism, RT-qPCR data and Western blot data were normalized to GAPDH or β-actin. Data were expressed as mean ± standard deviation (SD). The Student’s t test was used for data comparison, and P < 0.05 was considered statistically significant.

Expression analysis of Ctsb in mice

We adopted an RT-qPCR assay to elucidate the relative expression levels of Ctsb in different mouse tissues. As the results show, Ctsb demonstrated ubiquitous expression in mouse tissues (Fig. 1A). In the male reproductive system, a small amount of Ctsb was expressed in the testis, but Ctsb was abundantly expressed in the caput epididymis and cauda epididymis. Next, we used immunohistochemistry to further confirm the expression levels of CTSB in the male reproductive system. Consistent with the results of RT-qPCR, CTSB was highly expressed in both the caput epididymis and cauda epididymis, and a small amount of CTSB was present in the testes mainly in Leydig cells (Fig. 1C). Furthermore, we found that CTSB was primarily expressed in the tail of spermatozoa (Fig. 1B). The abundant expression of CTSB in spermatozoa and the male reproductive system implied that CTSB might participate in spermatogenesis or sperm maturation.

Generation of Ctsb^-/- mice using CRISPR/Cas9

To study the function of CTSB in male reproduction, CRISPR/Cas9 was used to knockout the Ctsb gene. We designed two sgRNAs that were used to delete a large portion of the coding sequence of Ctsb (Fig. 2A). The resultant deletion was confirmed by sequencing and PCR (Figs. 2B and 2C). Western blotting showed no band at the expected size for the CTSB protein in Ctsb^-/- mice (Fig. 2D), and CTSB was undetectable by immunohistochemistry analyses in the homozygote testis and epididymides of Ctsb^-/- mice (Fig. 1C). These results consistently indicated that Ctsb^-/- mice were successfully generated. To test the fertility of the Ctsb^-/- male mice, adult WT and Ctsb^-/- male mice were mated with adult WT female mice at a ratio of 1:2 for three months. The number of pups from each litter was recorded, and these results showed a smaller average number of pups for Ctsb^-/- male mice (Fig. 2E).

Impaired sperm quality in Ctsb^-/- mice

To investigate the function of CTSB in the male reproductive system, we paid close attention to the development of the testis and epididymis in Ctsb^-/- mice. No significant differences in weight and morphology of the testis, epididymis, or seminal vesicle were found between adult WT and Ctsb^-/- mice (Figs. 3A and 3B). Histology analysis showed slight degeneration of the seminiferous tubules characterized by vacuolization of the epithelium in Ctsb^-/- mice (Fig. 3C). Additionally, Ctsb^-/- mice had a decreased number and sparse arrangement of epithelial cells in the caput epididymis (Fig. 3C). Next, we analyzed whether the processes of meiosis and spermiogenesis were normal in Ctsb^-/- mice. The expression of γH2AX was used to identify the meiotic process, whereas PNA was used to identify acrosome development. Immunofluorescence analysis showed a normal presence of γH2AX-positive spermatocytes (Fig. 3D), PNA staining showed normal development of acrosome in the round spermatids at stage VII and VIII (Fig. 3E) of Ctsb^-/- mice. Ctsb^-/- mice also had normal levels of SOX9-positive Sertoli cells (Figs. 3F and 3G).

Next, sperm quality, including morphology and motility, was examined in Ctsb^-/- mice. H&E staining showed that morphologically abnormal sperm was increased in the cauda epididymis of Ctsb^-/- mice (Fig. 4A). The morphologically abnormal sperm were compared in testis, caput epididymis, and cauda epididymis between WT and Ctsb^-/- mice (Fig. 4B). The ratio of morphologically abnormal sperm in testis and caput epididymis was significantly increased in Ctsb^-/- mice. However, the ratio of morphologically abnormal sperm in cauda epididymis was more pronounced in Ctsb^-/- mice. Specifically, the proportions of sperm cells with abnormal heads, coiled tails, and that were decapitated were all markedly increased in the cauda epididymis of Ctsb^-/- mice (Fig. 4C). Meanwhile, PNA-staining showed that Ctsb^-/- mice had a higher percent of sperm cells with abnormal acrosome morphology (Figs. 4D and 4E). Compared to WT mice, the sperm count was decreased in Ctsb^-/- mice (Fig. 4F). CASA analysis was used to evaluate sperm motility. The proportion of progressive motility sperm was decreased, and the proportion of immobilized sperm was elevated in Ctsb^-/- mice (Fig. 4G). The increased level of morphologically abnormal sperm and attenuated sperm motility suggested that sperm quality was impaired in Ctsb^-/- mice.

Dysregulated autophagic and apoptotic activity in Ctsb^-/- mice

To study the mechanism of sperm quality damage in Ctsb^-/- mice, we investigated autophagic activity in the male reproductive system. The expression levels of ATG5, an autophagy protein with a core role in germ cell development, and autophagy-associated gene LC3, a widely used marker for mammalian autophagy, were analyzed by Western blotting. Our results suggested that autophagy was suppressed in the testis of Ctsb^-/- mice, as indicated by the decreased protein levels of ATG5 and LC3II/LC3I (Figs. 5A and 5B). As consistent with the observations in testis, ATG5 expression and the ratio of LC3II/LC3I were markedly decreased in the epididymis of Ctsb^-/- mice (Figs. 5C and 5D). Immunohistochemistry staining of LC3 and ATG5 also showed decreased autophagic activity in the testis and caput epididymis of Ctsb^-/- mice (Fig. 5E). Next, we used TEM to examine the ultrastructural autophagic processes in the epididymis of WT and Ctsb^-/- mice. We found that the number of autophagic vacuoles surrounded by double-membrane structures was significantly decreased in Ctsb^-/- mice (Fig. 5F). Ctsb ^-/- mice also had a significant number of epididymis cells with apoptosis changes, which mainly involved chromatin condensation and distributes along the nuclear membrane (Fig. 5F). The TUNEL assay was used to assess the apoptotic activity in the male reproductive system of WT and Ctsb^-/- mice. Our data revealed that, compared to WT mice, the number of TUNEL-positive cells was significantly increased in both testis and epididymis of Ctsb^-/- mice (Fig. 5G). Also, Western blotting results showed that the level of cleaved-caspase3 was elevated in both testis (Fig. 5H) and epididymis (Fig. 5I) of Ctsb^-/- mice, further indicating increased apoptotic activity. Together, these observations would suggest that Ctsb knockout inhibits autophagic activity and promotes apoptotic activity in the male reproductive system of mice.