Sirt6 is required for spermatogenesis in mice

Sirt6 is required for spermatogenesis

To study the potential role of Sirt6 in the testes, we first carried out immunostaining to investigate the location of SIRT6 in the seminiferous tubules of mice. We found that SIRT6 was specifically expressed in round spermatids and elongated spermatids (Figure 1A). Next, we generated Sirt6 global-knockout (hereafter called Sirt6^-/-) mice on a C57BL6/ICR mixed background. Unlike previous reports [31], we found that most of the Sirt6^-/- mice died within 4 weeks of age, while some Sirt6^-/- mice survived beyond 8 weeks. With the surviving Sirt6^-/- mice, we quantified pup numbers from 146 pregnant mice and identified the genotype of pups in each cage within a week of birth. Mating of Sirt6^+/- and Sirt6^+/- mice resulted in a mean litter size of about 9 pups, and gender distribution (699:751) was roughly consistent with Mendelian inheritance. The survival rate was about 4.13% from 218 Sirt6-knockout pups. We then performed western blot analysis to determine the expression levels of SIRT6 in the testes, and found that SIRT6 was significantly decreased in Sirt6^-/- testes compared to Sirt6^+/+ testes (Figure 1C). We characterized the 8 week-old knockout mice and found that they had lower body weight and were smaller than that of the Sirt6^+/+ littermates (Figure 1G). The testis size and weight were also significantly decreased compared with that of Sirt6^+/+ mice (Figure 1B, 1F and 1H). Further histological examination showed that Sirt6^-/- testes contained few elongated spermatids (Figure 1D), and in the cross-section of Sirt6^+/+ and Sirt6^-/- testes, we choose 300 seminiferous tubules of different sizes and determined by microscopy that the mean diameter of seminiferous tubules of Sirt6^-/- mice was significantly decreased compared with that in the control group (Figure 1E). Together, the loss of elongated spermatids and smaller testes in Sirt6^-/- mice suggest that Sirt6 deficiency severely impairs spermatogenesis.

Figure 1. SIRT6 protein expression and localization in mice testes. (A) Testicular sections of Sirt6^+/+ stained for SIRT6 (red) and DAPI (blue). SIRT6 is localized in the spermatids. 8-week mice, n=3. (B) The testes of Sirt6^-/- mice were smaller than those of the Sirt6^+/+ mice. 8-week mice, n=6, scale bar=3 mm. (C) SIRT6 protein levels were dramatically reduced in the testes of the Sirt6^-/- mice. ACTIN was used as the loading control. 8-week mice, n=3. (D) Histological analysis of Sirt6^+/+ and Sirt6^-/- mice seminiferous tubules by PAS-hematoxylin staining. 8-week mice, n=4. (E) The diameter of the seminiferous tubules in Sirt6^-/- mice was smaller than that in control mice. Sirt6^+/+, 177.43±2.36μm; Sirt6^-/-, 123.94±1.92μm. 8-week mice, n=6; 300 seminiferous tubules were used for each group. Data are presented as mean ± SEM. ***P < 0.001. (F) Quantification of testis weight of the Sirt6^+/+ and Sirt6^-/- mice. The testis weight of Sirt6^-/- mice was significantly reduced. Sirt6^+/+, 8.60±1.30% g; Sirt6^-/-, 4.49±1.40% g. 8-week mice, n=6. Data are presented as mean ± SEM. ***P < 0.001. (G) Quantification of body weight of the Sirt6^+/+ and Sirt6^-/- mice. The body weight of Sirt6^-/- mice was reduced. Sirt6^+/+, 27.00±0.55 g; Sirt6^-/-, 18.67±0.30g. 8-week mice, n=6. Data are presented as mean ± SEM. ***P < 0.001. (H) The testis weight/ body weight of Sirt6^-/- mice was also reduced. Sirt6^+/+, 0.32±0.07%; Sirt6^-/-, 0.04±0.07%. 8-week mice, n=6. Data are presented as mean ± SEM. ***P < 0.001.

Sirt6 is dispensable to meiosis during spermatogenesis

Chromosomal synapsis depends on the formation of the synaptonemal complex, which is comprised of three main proteins: SYCP1, SYCP2, and SYCP3 [33]. SYCP1 is the central element that links two homologous chromosomes for pairing and helps in the formation of the chiasmata in the pachytene stage. SYCP3 and SYCP2 are the lateral elements of the synaptonemal complex and are distributed along the chromosome axis [33]. The phosphorylation of histone H2AX at serine 139 (termed γH2AX) is essential for the production of programmed DNA double-strand breaks (DSBs) which are required for the initiation of meiotic recombination [34]. Concomitant with DSB formation and repair, γH2AX accumulates in the nucleus during the leptotene (distributed globally) and zygotene stages, decreases in the autosomes and concentrates only in the sex chromosomes during the pachytene stage. To investigate the mechanism underlying the infertility of Sirt6^-/- mice, we first examined meiotic progression by immunostaining the axial components of the synaptonemal complex and γH2Ax. Immunostaining of SYCP3 and γH2Ax in spermatocytes revealed that the initiation of DSB formation appeared starting in the leptotene stage and remained until the pachytene stage, and DNA damage was repaired in all autosomes and concentrated only in the sex chromosomes in both Sirt6^+/+ and Sirt6^-/- mice (Figure 2A and 2B), suggesting that Sirt6 is not essential for DSB repair in meiosis. We then examined homolog synapses via immunostaining of the synaptonemal complex protein SYCP1 that loads only to the synapsed chromosomes [35]. SYCP1 was found to be colocalized with SYCP3 (except on XY chromosomes) in most of the pachytene spermatocytes (Figure 2C and 2D), indicating that full synapsis was acquired in the spermatocytes of both Sirt6^+/+ and Sirt6^-/- mice. On the basis of these findings, we concluded that Sirt6 is not essential to the meiotic progression of germ cells during spermatogenesis. Thus, it might be the subsequent spermiogenesis stages that were affected in Sirt6^-/- mice.

Figure 2. Sirt6 is not essential to meiosis during spermatogenesis. (A, B) Spermatocytes of Sirt6^+/+ and Sirt6^-/- stained with SYCP3 (green) and γH2AX (red) antibodies, 8-week mice, n=4; 200 cells were used for each group. (C, D) Spermatocytes of Sirt6^+/+ and Sirt6^-/- stained for SYCP3 (green) and SYCP1 (red), 8-week mice, n=4; 200 cells were used for each group.

Loss of Sirt6 results in spermatogenic arrest at the elongated spermatid stage

The spermatogenesis process in mice is usually divided into 12 stages to describe germ cell development, which was characterized in tissue stained with Periodic Acid Schiff (PAS) and hematoxylin [36]. To determine exactly which spermatogenic stage was affected by SIRT6 deficiency, we performed PAS and hematoxylin staining and observed that spermatogonia, spermatocytes and round spermatids had a significantly reduced number of elongated spermatids in Sirt6^-/- testes (Figure 3A), suggesting elongated spermatids development was disrupted in Sirt6^-/- mice.

Figure 3. Sirt6 deficiency leads to spermatogenic arrest at the elongated spermatid stage. (A) Paraffin-embedded sections of seminiferous tubules and testes from Sirt6^+/+ and Sirt6^-/- were stained with PAS-hematoxylin. B, B type spermatogonial stem cells; L, leptotene spermatocytes; D, diplotene spermatocytes; P, pachytene spermatocytes; rST, round spermatids; eST, elongated spermatids; M, meiotic divisions. 8-week mice, n=4. (B) Different developmental steps of single round spermatid and elongated spermatids from Sirt6^+/+ and Sirt6^-/- testes. 8-week mice, n=4. (C) Quantification of the proportion of round spermatids in the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 27.91±1.03%; Sirt6^-/-, 25.27±1.96%. 8-week mice, n=4; 96 seminiferous tubules were used for each group. (D) Quantification of the proportion of elongated spermatids in the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 26.67±0.48%; Sirt6^-/-, 2.64±0.12%. 8-week mice, n=4; 96 seminiferous tubules were used for each group. Data are presented as mean ± SEM. ***P < 0.001.

Moreover, the differentiation of haploid spermatids can be divided into 16 steps, and step-by-step spermatid differentiation can also be characterized with PAS and hematoxylin staining. The morphology of round spermatids in both Sirt6^+/+ and Sirt6^-/- testes was fully developed and consisted of a well-assembled acrosome and nucleus (Figure 3B). During spermiogenesis, the round-shaped nucleus undergoes elongation and becomes falciform. These sperm-specific nuclear structures and shapes are considered to contribute to the generation of a more hydrodynamic sperm head that can quickly transit through the female reproductive tract and protects the genetic material from physical and chemical damages [37]. In the elongated spermatids of Sirt6^+/+ mouse, the acrosome was found to stop growing and was observed to change its morphology during steps 9-16. After a series of highly complex morphological changes, such as spermatid elongation and cytoplasm removal, spermiogenesis eventually results in producing mature sperm. The spermiogenesis process was considered fully normal in Sirt6^+/+ mice as indicated by mature sperm found in the testes. However, in Sirt6^-/- mice, many of the spermatids were observed to have irregularly shaped heads (Figure 3B). Next, we quantified the proportion of round spermatids in both Sirt6^+/+ and Sirt6^-/- testes, and found that the round spermatid developmental status of Sirt6^-/- mice is consistent with that of Sirt6^+/+ mice (Figure 3C). However, the proportion of elongated spermatids was decreased significantly in Sirt6^-/- testes (Figure 3D). Together, these findings indicate that SIRT6 deficiency blocks the spermiogenesis process, and loss of Sirt6 results in spermatogenic arrest at the elongated spermatid stage.

SIRT6-deficiency leads to malformed spermatids with impaired acrosomes

The acrosome is a unique membranous organelle located at the anterior portion of the sperm nucleus. The organelle plays an important role in the dispersion of cumulus cells and/or sperm penetration of the zona pellucida of the oocyte during fertilization [38]. Acrosome biogenesis consists of the following phases: the Golgi phase with round spermatids from step 1 to step 3 in the seminiferous tubules; the Cap phase with round spermatids from step 4 to step 8 in the seminiferous tubules; and the acrosome and maturation phases with elongated spermatids from stage step 9 to step 16 in the seminiferous tubules [39]. To test whether Sirt6 knockout has any impact on acrosomes biogenesis, single-sperm immunofluorescence was performed. The acrosomes of round spermatids from both Sirt6^+/+ and Sirt6^-/- mice were found to be well-developed while acrosomes of elongate spermatids from Sirt6^-/- mice showed severe defects compared to Sirt6^+/+ mice (Figure 4A). Moreover, the acrosome signal of elongated spermatids of Sirt6^-/- mice showed weaker fluorescence intensity than that of Sirt6^+/+ mice (Figure 4B), indicating acrosome biogenesis is affected in Sirt6^-/- mice. Altogether, more than 90% of elongated spermatids were abnormal in the Sirt6^-/- mice compared with 4.22% in the Sirt6^+/+ mice (Figure 4C), suggesting elongated spermatids are disrupted severely in Sirt6^-/- mice. These results indicate that SIRT6-deficiency leads to malformed elongated spermatids with impaired acrosomes.

Figure 4. SIRT6-deficiency leads to malformed spermatozoa with impaired acrosomes. (A) Immunofluorescence staining of PNA (green) and DAPI (blue) in Sirt6^+/+ and Sirt6^-/- spermatids. The acrosome is disrupted in Sirt6^-/- spermatids. (B) Quantification of intensity of elongated spermatids in (A). Sirt6^+/+, 33.84±0.90; Sirt6^-/-, 27.79±0.84, 8-week mice, n=4; 150 cells were used for each group. Data are presented as mean ± SEM. *P < 0.05. (C) Quantification of disrupted elongated spermatids of the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 4.22±0.57%; Sirt6^-/-, 91.16±1.08%, 8-week mice, n=4; 200 cells were used for each group. Data are presented as mean ± SEM. ***P < 0.001.

Sirt6 deficiency results in increased apoptotic spermatids

A large number of degenerated cells with highly condensed nuclei were found in the seminiferous epithelium of Sirt6^-/- mice (Figure 3B, from step 9 to step 16). It is likely that these elongated spermatids underwent cell death. To confirm this possibility, we performed immunofluorescence staining via the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and found that TUNEL positive cells per tubule and TUNEL positive tubules were increased in Sirt6^-/- spermatids (Figure 5A–5C). To confirm this result, we performed cleaved caspase-3 staining to label apoptotic cells, which showed that Sirt6 deletion significantly elevated the number of apoptotic spermatids (Figure 5D–5F). These results suggest that elongated spermatids may be lost due to cell death in Sirt6^-/- mice.

Figure 5. Sirt6 deficiency results in increased apoptotic spermatids. (A) Paraffin embedded sections from Sirt6^+/+ and Sirt6^-/- testes were stained with TUNEL kit (green) and SYCP3 (red) to determine the presence of apoptotic cells. 8-week mice, n=6. (B) Quantification of TUNEL positive cells per tubule of the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 0.45±0.1; Sirt6^-/-, 3.03±0.25. 8-week mice, n=6; 533 cells were used for each group. Data are presented as mean ± SEM. ***P < 0.001. (C) Quantification of TUNEL positive tubules of the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 6.00±0.49; Sirt6^-/-, 34.67±0.53. 8-week mice, n=6; 228 seminiferous tubules were used for each group. Data are presented as mean ± SEM. ***P < 0.001. (D) Paraffin embedded sections from Sirt6^+/+ and Sirt6^-/- testes were stained with Cl-caspase3 (green), SYCP3 (red) and DAPI (blue) to determine the presence of apoptotic cells. 8-week mice, n=6. (E) Quantification of Cl-caspase3 positive cells per tubule of the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 0.40±0.13; Sirt6^-/-, 4.88±0.34. 8-week mice, n=6; 497cells were used for each group. Data are presented as mean ± SEM. ***P < 0.001. (F) Quantification of Cl-caspase3 positive tubules of the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 7.83±0.50; Sirt6^-/-, 39.00±0.59. 8-week mice, n=6; 201 seminiferous tubules were used for each group. Data are presented as mean ± SEM. ***P < 0.001.

Histone deacetylation activities of SIRT6 might not be required for spermatogenesis

In vitro screening of acetylated histone tail peptides revealed that the deacetylation activity of SIRT6 could increase acetylation of H3K9, and Sirt6-deficent cells have been shown to contain hyperacetylated H3K9 at telomeres [40]. In addition to H3K9, other lysines such as H3K18 and H3K56 are also reported to be substrates of SIRT6 [19, 41], so we tested whether Sirt6 regulates histone acetylation of these sites during spermatogenesis. H3K9ac was localized in round spermatids and elongated spermatids (Figure 6B), but no differences in H3K9ac levels were found between Sirt6^+/+ and Sirt6^-/- testes (Figure 6C). Western blotting also confirmed that H3K9ac levels showed no significant differences between Sirt6^+/+ and Sirt6^-/- testes (Figure 6A). As for other SIRT6 deacetylation targets, H3K18ac was localized in elongated spermatids in testes (Supplementary Figure 1), but the levels of H3K18ac in Sirt6^-/- testes were similar to that of the Sirt6^+/+ mice (Figure 6D). Similar to H3K9ac and H3K18ac levels, no difference was found between the H3K56ac levels of Sirt6^+/+ and Sirt6^-/- testes (Figure 6E). These results suggest that deacetylation activities of SIRT6 on H3K9ac, H3K18ac and H3K56c are not required for spermatogenesis.

Figure 6. Deacetylation activity of SIRT6 on H3K9ac, H3K18ac and H3K56c is not required for spermatogenesis. (A) The levels of H3K9ac in Sirt6^+/+ and Sirt6^-/- testes were detected by western blot. 8-week mice, n=3. (B) Localization of H3K9ac in Sirt6^+/+ and Sirt6^-/- seminiferous tubules. Testes sections of Sirt6^+/+ and Sirt6^-/- stained with SYCP3 (green) and H3K9ac (red) antibodies. 8-week mice, n=4. (C) Quantification of H3K9ac intensity of round spermatids and elongated spermatids in the Sirt6^+/+ and Sirt6^-/- mice. Sirt6^+/+, 15.82±0.48; Sirt6^-/-, 14.78±0.58. 8-week mice, n=4; 200 cells were used for each group. Data are presented as mean ± SEM. (D) The levels of H3K18ac in Sirt6^+/+ and Sirt6^-/- testes were detected by western blot. 8-week mice, n=3. (E) The levels of H3K56ac in Sirt6^+/+ and Sirt6^-/- testes were detected by western blot. 8-week mice, n=3. (F) A functional diagram of Sirt6 in spermatogenesis.

Animals

Mice were maintained in a 12:12 light/ dark cycle with food and water available ad libitum in cages held at 23±2°C. Sirt6 global knockout (Sirt6^-/-) mice were generated using Sirt6-floxed (Sirt6^flox/flox) mice on a C57BL6/ICR mixed background. Mice harboring a floxed conditional knockout cassette of C57BL6 Sirt6 were purchased from EUCOMM. Mice carrying the floxed Sirt6 allele (Sirt6^+/flox) were mated to mice expressing Cre recombinase under control of the ZP3 promoter (Zp3-Cre) [56]. To generate heterozygous Sirt6-knockout (Sirt6^+/−) mice, these Sirt6^+/flox; Zp3-Cre or Sirt6^{flox /flox}; Zp3-Cre mice were further mated with WT female mice. Zp3-Cre mice were purchased from Jackson Laboratory. WT ICR mice were purchased from SPF (Beijing) Biotechnology, LLC.

All the 8-week male mice were sacrificed by cervical dislocation before testes collection. All the animal experiments were approved by the Animal Research Panel of the Committee on Research Practice of the University of the Chinese Academy of Sciences (Approval number: IOZ20150041).

Tissue collection and histological analysis

For histological examination, at least three adult mice for each genotype were analyzed. The testes were dissected and fixed with Bouin’s fixative for up to 24 h at 4 °C. Next, the testes were dehydrated using graded ethanol and embedded in paraffin. 5μm sections were obtained and transferred to glass slides. After deparaffinization, sections were stained with H&E or stained with PAS-hematoxylin to determine the seminiferous epithelia cycle stages.

Chromosome spreads of spermatocyte

Chromosome spreads of spermatocytes were performed using a drying-down technique as previously described [57]. Briefly, the testes were dissected, and the tubules were washed with phosphate-buffered saline (PBS), pH 7.4 at room temperature. Next, the tubules were placed in a hypotonic extraction buffer containing 30 mM Tris, 50 mM sucrose, 17 mM trisodium citrate dihydrate, 5 mM EDTA, 0.5 mM DTT and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.2, for 30 min at room temperature. Subsequently, the tubules were homogenized in 100 mM sucrose, pH 8.2, on a clean glass slide and were pipetted repeatedly to make a suspension. The cell suspensions were placed on slides pre-coated with 1% paraformaldehyde (PFA) (Solarbio), pH 9.2, and 0.15% Triton X-100. The slides were dried for at least 2 h in a closed box with high humidity in room temperature. Finally, the slides were washed twice with PBS for 10 min and stained with antibodies according to standard protocols.

Antibodies

Rabbit anti-SIRT6 polyclonal antibody was purchased from sigma (S4197) for western blotting and Abcam(ab62739) for immunofluorescence staining. Mouse anti-SYCP3 (150292) and rabbit anti-SYCP3 (ab15093) were purchased from Abcam (Cambridge, MA). Rabbit anti-SYCP1 (NB300-228c) was purchased from Novus Biologicals (Littleton, CO). Mouse anti-γH2AX (05-636) was purchased from Merck Millipore (Darmstadt, Germany). Rabbit anti-H2A (ab18975) was purchased from Abcam (Cambridge, MA). Rabbit anti-H3K9ac(P30050M) and mouse anti-ACTIN antibody (M20011L) were purchased from Abmart, H3K18ac and H3K56ac antibody kits (9927T) were purchased from Cell Signaling Technology. Alexa Fluor 488 -lectin PNA was purchased from Molecular Probes. Goat FITC-conjugated secondary antibodies for rabbit, goat FITC-conjugated secondary antibodies for mouse, goat TRITC conjugated secondary antibodies for rabbit, and goat TRITC conjugated secondary antibodies for mouse were purchased from Zhong Shan Jin Qiao (Beijing, China). Alexa Fluor 680-conjugated goat anti-mouse and Alexa Fluor 800-conjugated goat anti-rabbit secondary antibodies, used in western blot analysis, were purchased from Invitrogen.

Immunoblotting

Tissue extracts were prepared in cold RIPA buffer (25 mM Tris–HCl, pH 7.6, 350 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with 1 mM PMSF and a protein inhibitor cocktail (Roche Diagnostics, 04693116001, Rotkreuz, Switzer-land) using a Dounce homogenizer. After a brief sonication, the cell lysates were incubated on ice for 30 min. The samples were centrifuged at ~14 000 ×g for 15 min at 4 °C to pellet the cell debris, and the supernatant was transferred to a new tube for further analysis. Protein lysates were separated via SDS-PAGE and electro-transferred to nitrocellulose membranes. After incubation with respective primary at 4 °C overnight and secondary antibodies in room temperature one hour, the membrane was scanned using an ODYSSEY Sa Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Immunofluorescence and TUNEL assay

Testes were collected and immediately embedded in OCT compound (Tissue-Tek) and cut into 7μm sections using a microtome-cryostat (CM1950, Leica). Sections were fixed in 4% PFA for 10 min and then rinsed three times with PBS at room temperature (pH7.4), treated with 0.1% Triton X-100 for 10 min and rinsed three times with PBS. The samples were then blocked in 5% bovine serum albumin (BSA) in PBS for 30 min, and incubated with the respective primary antibody at 4°C overnight. After rinsing three times with PBS, the sections were incubated with a FITC-conjugated secondary antibody at 1:200 for 1 h at 37°C. DAPI (D3571, Life Technologies) was used to label nuclei. Images were captured immediately using a TCS SP8 microscope (Leica, Wetzlar, Germany). To examine chromosome immunofluorescence, the chromosome spreads of spermatocytes were rinsed with PBS three times and blocked with 5% BSA, followed by staining as detailed above. To detect apoptotic cells in the testis, we used the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay kit (In Situ Cell Death Detection Kit; Roche, 11684795910) [58]. Briefly, sections of the testes were deparaffinized and boiled for 15 min in sodium citrate buffer for antigen retrieval. Next, the slides were treated with H₂O₂ for 10 min at room temperature and sodium citrate for 2 min on ice. The slides were rinsed twice with PBS, and the TUNEL reaction mixture was added and incubated in a humidified atmosphere for 60 min at 37 °C in the dark, followed by staining as described above.

Hormone measurement

Serum levels of mouse testosterone were measured using a radioimmunoassay kit (Beijing Sinouk Institute of Biological Technology) as previously described [59].

Statistical analysis

All experiments were repeated at least three times and the data are presented as the mean ±SEM. In each group, we used Sirt6^+/+ littermates as negative controls. Statistical analyses were conducted using GraphPad PRISM version 8.02. The statistical significance of the differences between the mean values was measured by a Student’s t-test with a paired two-tailed distribution. The data were considered significant when the P-value was less than 0.05 (*), 0.01 (**) or 0.001(***).

Ethical approval

All of the animal experiments were performed according to approved institutional animal care and use committee (IACUC) protocols (#08-133) of the Institute of Zoology, Chinese Academy of Sciences. All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.