Ethics statement
All experiments in the current study were approved by the Institutional Animal Care and Use Committee of the Northwest A&F University, Shaanxi, China.
Chemicals and reagents
Unless otherwise stated, all chemicals and reagents used in the present study were purchased from Sigma-Aldrich China. The R848 (TLR7/8 ligand) was obtained from Novus Biologicals (Littleton, CO, USA).
Semen collection and Swim-up test
Semen was collected twice weekly from six Guanzhong dairy goat bucks (2–3 years old) using an artificial vagina at the Shaanxi Aonike Guanzhong Dairy Goat Breeding Farm, Fuping, Shaanxi, China. The ejaculate was transported to the lab, and sperm quality was assessed within 30 min. All semen samples with sperm motility >75% were mixed to avoid the influence of the individual bucks used in the study. The swim-up test is shown in Fig. 1, as described previously [13]. The sperm were incubated in a goat semen extender, as described previously [17], containing different concentrations of R848 for 0, 15, 30, 45, and 60 min. Next, the percentage of sperm in the upper and lower layers was evaluated (number of upper or lower layer sperm/total sperm). The separated sperm were used for further analysis.
Sperm motility parameters assessment using the computer-aided sperm analysis (CASA) system
Sperm were incubated in goat semen extender containing 1 μM R848 at 37℃ for 30 min. Then, 10 μL of semen was placed on a pre-warmed glass slide (CELL-VU® DRM-600, Millennium Sciences, New York, NY, USA) with a coverslip. Sperm motility parameters were evaluated using a computer-aided sperm analysis (CASA) system (HVIEW-SSAV8.0, FuZhouHongShiYe Software Technology Co., Ltd., China). The sperm motility parameters measured included sperm motility per se, average path velocity (VAP), average straight-line velocity (VSL), and average curvilinear velocity (VCL). All experiments were replicated five times.
Immunofluorescence (IF) of testis and epididymis
Testes and epididymis of adult dairy goat bucks were collected and fixed in 4% (w/v) paraformaldehyde overnight and embedded in paraffin. Sections were probed with an anti-TLR7 antibody (bs-6601; Bioss Antibodies, Woburn, MA, USA) and anti-TLR8 antibody (ab180610; Abcam, Cambridge, MA, USA) at a ratio of 1:200. After washing with PBS, the antigens were visualised using Cy3-conjugated goat anti-rabbit IgG (ab6939; Abcam), 30 μL fluorescein isothiocyanate-peanut agglutinin (FITC-PNA, 100 μg/mL in PBS), and 1 μg/mL DAPI. Section staining was monitored and photographed using an epifluorescence microscope (Nikon 80i; Nikon, Tokyo, Japan). Three replications were performed.
Immunofluorescence of sperm
The sperm of the dairy goat bucks was spread over slides, air-dried, and fixed using 100% methanol for 10 min. After air-drying, the sample slides were rinsed with PBS three times for 5 min each time. The sperm samples were then permeabilised with 0.5% (v/v) Triton X-100/PBS at room temperature for 30 min. The sample slide was rinsed with PBS three times for 5 min each time. The sperm were incubated with blocking solution (5% BSA) at 37°C for 30 min. Sperm were probed with anti-TLR7 or anti-TLR8 antibodies at a ratio of 1:100, and incubated overnight at 4°C. After washing with PBS, the antigens were visualised with Cy3-conjugated goat anti-rabbit IgG (1:2000; ab6939, Abcam) and 1 μg/mL DAPI. Sperm staining was monitored and photographed using an epifluorescence microscope (Nikon 80i; Nikon). Three replications were performed.
Analysis of sperm membrane integrity
Sperm membrane integrity was evaluated using SYBR-14/propidium iodide (PI) following the procedure described by [18]. Briefly, the sperm (100 μL) was stained using 0.1 μL of SYBR-14 working solution (100 μM in DMSO) for 10 min at 37°C, and then 0.5 μL propidium iodide (PI) working solution (2.4 M in water) was added for 10 min at 37°C. Sperm staining was monitored and photographed using an epifluorescence microscope (Nikon 80i; Nikon). More than 200 spermatozoa per slide were assessed. Three replicates were evaluated for each semen sample by the same observer.
Analysis of sperm acrosome integrity
Based on the method described by Ren et al. (2019) [17], acrosome integrity was measured using fluorescein isothiocyanate-peanut agglutinin (PNA-FITC) staining. Briefly, sperm (30 μL) was spread over slides, air-dried, and fixed using 100% methanol for 10 min. The fixed sperm were then stained using 30 μL PNA-FITC (100 μg/mL in PBS), incubated at 37°C for 30 min, and rinsed thrice with PBS, and nuclear staining was performed using 1 μmol/L DAPI. The surface dye was washed three times using PBS and air-dried in the dark. Spermatozoa staining was monitored and photographed using an epifluorescence microscope (Nikon 80i; Nikon). More than 100 spermatozoa per slide were assessed and requested five more fields per sample. Three replicates were evaluated for each semen sample by the same observer.
Analysis of sperm mitochondrial activity and ATP levels
The sperm mitochondrial activity changes were evaluated using a JC-1 (5,5',6, 6'-tetrachloro-1,1',3,3'-tetraethyl-imidazolcarbocyanine iodide) Mitochondrial Membrane Potential Detection Kit (Beyotime Institute of Biotechnology, Haimen, China), following the manufacturer's instructions. Briefly, sperm samples (2 × 106/mL) were stained with 28 μL of JC-1 (stock solution) in PBS (final volume, 100 μL). After incubation at 37°C for 30 min in the dark, the samples were centrifuged at 600 × g for 5 min, resuspended in JC-1 buffer, and placed on ice. Sperm samples were immediately analysed in a flow cytometer (FAC-SCalibur; BD Biosciences, San Jose, CA, USA) with excitation at 490 nm and emission at 530 nm (JC-1 monomer) and excitation at 525 nm and emission at 590 nm (JC-1 polymer). Sperm mitochondrial activity was calculated using the following equation:
Sperm mitochondrial activity (%) = the value of JC-1 polymer/(JC-1 monomer + JC-1 polymer)*100%.
At least three technical replicates were evaluated for each sperm sample.
ATP levels of dairy goat sperm were assessed using an ATP Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, 500 μL semen (8 × 107 sperm/mL) was centrifuged and resuspended in ATP assay lysate (200 μL) to release intracellular ATP at 4°C. The sperm was then centrifuged at 12,000 × g at 4°C for 5 min, and the supernatant was aspirated. The ATP standard solution (0.5 mmol/L) was diluted to concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µmol/L in succession by ATP assay lysate. The supernatants (50 μL) and standards (50 μL) were added to a 100-μL ATP detection working solution in opaque 96-well plates. The samples' fluorescence intensity was detected using a multi-detection microplate reader (Bio Tek Synergy H1; Bio Tek, Winooski, VT, USA). At least three technical replicates were evaluated for each sperm sample.
In vitro fertilisation (IVF) using the lower layer TLR7/8-activated sperm
Goat ovaries were obtained from a local abattoir and transported to the laboratory within 4 h in 0.9% sodium chloride solution at 38°C. Thirty to forty COCs were placed in a 200-µL drop of BO-IVM medium (IVF Bioscience, Falmouth, United Kingdom), and were incubated for 24 h (38.5°C, 5% CO2). TLR7/8-activated sperm (50 µL, 2 × 107 spermatozoa/mL) was added to the 30–40 cumulus-oocyte complexes in a 500-μL six-well dish containing BO-IVF (IVF Bioscience) medium and mineral oil and incubated for 18 h of at 38.5°C and 5% CO2. Then, the unfertilised sperm and cumulus cells were removed from the zygotes using 0.1% hyaluronidase PBS. Next, the zygotes were transferred to BO-IVC medium (IVF Bioscience) for culture. Embryos were collected for sex determination.
Artificial insemination (AI) using the lower layer TLR7/8-activated sperm
A CIDR (CIDR®, EaziBreed; Zoetis, Parsippany-Troy Hills, NJ, USA) was intravaginally placed in the donor Guanzhong dairy goat does (n = 6) on day 0. On day 14, the goats were injected with FSH (Folltropin-V®; Bioniche Animal Health, Belleville, Canada) from the morning. In total, 180 mg of FSH was injected for three days (50, 50, 30, 30, 10, and 10 mg) at 12 h intervals. The CIDR was removed from the does on day 17. After 24 h, the donor goats were intra-cervically inseminated with TLR7/8-activated sperm as described in our previous report [17]. Briefly, two inseminations after oestrus (12 h apart) were performed, and embryos were collected for sex determination after five days.
Sex determination of goat embryos by PCR
Each of the blastocysts in vitro was transferred to PBS in 0.01% BSA for washing, and then individually placed in a 0.2-mL enzyme-free centrifuge tube and dispensed with 2.5 μL of genome extract (1 mg/mL Proteinase K, 0.5% Triton X-100, 50 mM Tris-HCl, pH = 8.0). The single blastocyst was then put in a Polymerase Chain Reaction (PCR) machine and incubated at 65°C for 3 h and at 95°C for 10 min. PCR analyses of single blastocysts were performed with KOD FX Neo (KFX-201, Toyobo Co., Ltd., Life Science Department, Osaka, Japan) according to the manufacturer's instructions. Primer sets recognising the Y- (SRY) and X-chromosome (B-ACTIN) were generated and used in PCR reactions (Table 1). The following PCR amplification conditions were used: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 68°C for 40 s, with a final extension at 68°C for 5 min. Subsequently, the PCR products were visualised by 2% agarose gel electrophoresis.
Determination of X/Y sperm ratio by Flow cytometry
After incubating the sperm with 1 μM R848 for 30 min at 37°C, the upper or lower sperm layer was centrifuged at 400 x g at 37°C for 5 min. The sperm samples were then diluted to a concentration of 2 x 108 sperm/mL with 500 μL goat extender containing 8 μL Hoechst 33342 (2 mg/mL) and were tailed off by ultrasound for 1 s. The sperm was incubated at 37°C for 50 min in the dark, mixing every 10 min. A high-speed sorting flow cytometer (BD FACSAria™ III Cell Sorter; BD, Franklin Lakes, NJ, USA) was used to analyse the proportion of X/Y sperm.
Western blot analyses
As described in our previous study [19], to obtain total protein lysates, the sperm were lysed on ice for 30 min using a cell lysis buffer containing 250 μL RIPA and 2.5 μL PMSF. A BCA assay kit was used to determine the total protein concentrations, and then the protein samples with loading buffer were boiled at 100°C for 10 min. Protein samples (20 μg protein each lane) were electrophoresed on 10% SDS-polyacrylamide gels. The proteins were separated by PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with the indicated primary antibodies. Blots were incubated with primary antibodies overnight at 4°C. The primary antibodies used were: phospho-AKT (10176-2-AP; Proteintech Group, Inc., China), phospho-NFκB (3031s; Cell Signaling Technology, Boston, MA, USA), phospho-GSK3α/β (9331s; Cell Signaling Technology), total AKT (ab81283; Abcam), total NFκB (8242s; Cell Signaling Technology), and total GSK3α/β (5676s; Cell Signaling Technology). Finally, after washing with TBST, the blots were incubated with an alkaline phosphatase-conjugated secondary antibody (1:10000 dilutions in TBST) for 1 h at 37°C. The reactive proteins were visualised with chemiluminescence (ECL) western blot reagents and quantified using ImageJ software. Protein levels were normalised to anti-alpha tubulin (ab7291; Abcam).
Statistical analyses
All experiments were replicated at least three times independently. All results are expressed as the mean ± standard error of the mean (SEM). Sperm motility, VAP, VCL, and VSL were compared using the independent-samples t-test. Other data were analysed by one-way ANOVA, and multiple comparisons with Duncan's multiple range tests were performed using SPSS version 20 for Windows (SPSS Inc., Chicago, IL, USA) (*, P<0.05; **, P<0.01).