Presence of leptin and its receptor in the ram reproductive system and in vitro effect of leptin on sperm quality

Chemicals

In this study all chemicals were purchased from Sigma-Aldrich Co. (USA) unless stated otherwise.

Animals

Suffolk White rams (Ovis aries) from Aoxin Animal Husbandry (Beijing, China), at the age of 1–2 years old with normal reproductive cycle, healthy and generally the similar body weight were selected for the experiments. All experimental protocols concerning the handling of animals were performed in accordance with the requirements of the Institutional Animal Care and Use Committee at the Xinjiang Agricultural University. The experiments were approved by the Animal Welfare and Animal Experimental Ethical Committee of Xinjiang Agricultural University (approval number 2020032).

Experiment design

Experiment 1: The serum samples were collected at 08:00, 14:00, 18:00, and 24:00 h from jugular vein of six mature Suffolk White rams (1–2 years old), the seminal plasma was collected at 14:00 and 24:00 h of three mature Suffolk White rams (1–2 years old). The blood and seminal plasma were used to estimate the circadian changes of serum and seminal plasma levels of leptin in the Suffolk White rams.

Experiment 2: Testis and epididymal tissues were collected from three adult (1–2 years old) healthy Suffolk White rams, these tissues were used for immunohistochemical analysis to detect expression of leptin/leptin receptor genes. The artificial vagina method was used to collect the semen from eight mature robust Suffolk White rams (1–2 years old). For collection of sperm from the epididymis, the whole epididymis of three adult Suffolk White rams were collected. The ejaculated and epididymal sperm of the Suffolk White rams were used for RT-PCR, RT-qPCR, WB to determine the presence of OB and OBR in ram testis, epididymis and sperm including mRNA expression, protein expression, and immunolocalization.

Experiment 3: The semen was collected from eight matured robust Suffolk White rams (1–2 years old). Evaluation of spermatozoa quality included motility parameters (progressive motility, VSL, VAP), mitochondrial membrane potential (MMP), viability, DNA fragmentation index (DFI), reactive oxygen species (ROS), acrosome integrity and acrosome reaction rate. Based on pilot study, we confirmed that supplementation of Leptin with 5 ng/mL for 2 h in vitro is the best concentration and treatment duration.

Testis and epididymal tissue collection

Testis and epididymal tissues were collected at Aoxin Animal Husbandry (Beijing, China) from three adult (1–2 years old) healthy Suffolk White rams.

The samples from castrated rams were immediately washed in phosphate-buffered saline (PBS) supplemented with antibiotics (penicillin, 100 IU/mL and streptomycin, 100 μg/mL). The tissues for histological analyses were incubated in fixative solution (4% paraformaldehyde) and other specimens were stored in liquid nitrogen for RT-PCR, Immunohistochemistry and Western blot (WB) analyses.

Sperm selection and preparation

The artificial vagina method was used to collect the semen from eight matured robust Suffolk White rams (1–2 years old). Semen analysis was performed at 37 °C: the observed sperm concentration and motility were >2 × 10⁹/mL and >80%, respectively. The semen was transferred to tubes and centrifuged at 400g for 4 min. The upper layer of semen was used for leptin measurement and the lower layer of semen was purified by the swim-up method. Briefly, 0.2 mL of semen was deposited at the bottom of the tubes and then IVF medium (G-IVF plus; Vitrolife, Gothenburg, Sweden) was added up to 1 mL. The tubes were placed at 37 °C for 40 min under 5.0% CO₂. The upper layer containing the mobile sperm was used for RT-PCR, RT-qPCR, WB, immunofluorescence and sperm parameters analyses.

For collection of sperm from the epididymis, the whole epididymis of three adult Suffolk White rams were sampled at Aoxin Animal Husbandry (Beijing, China) and transported to the lab with ice box. After making an incision in the epididymal, the content was mixed with IVF medium and centrifuged at 400g for 4 min. The analysis of sperm quality and the swim-up procedure were the same as above for the ejaculated sperm.

Estimation of serum and seminal leptin

The serum samples were collected at 08:00, 14:00, 18:00, and 24:00 h from jugular vein and the seminal plasma were collected at 14:00 and 24:00 h of six mature Suffolk White rams (1–2 years old). The concentration of leptin was measured by radioimmunoassay using Leptin Direct RIA TM Kit (RE54021; Immuno-Biological Laboratories, Hamburg, Germany) followed the manufcture’s instructions.

Histological analysis of testis and epididymal tissue

Paraffin sections were dewaxed with xylene, dehydrated with gradient alcohol, and then placed in distilled water. The samples were stained with hematoxylin solution (Servicebio, Wuhan, China) for 5 min. After washing with distilled water for 3 times, using 1% hydrochloric acid alcohol for color separation for 20 s. followed by incubation in 0.1M PBS pH7.4 for 3 min, The slices were washed through a graded alcohol concentrations for 2 min sequentially and stained with eosin (Servicebio, Wuhan, China) for 30 s. Finally, the samples were detained in 95% alcohol for 1 min, dehydrated in anhydrous ethanol and xylene.

Immunohistochemical analysis for expression of leptin/leptin receptor

The testis and epididymal tissues were fixed by paraffin-embedded (FFPE) method as described in section above. The paraffin slides were dewaxed with xylene, dehydrated with gradient concentrations of ethanol process, washed with deionized water, and placed in 3% H₂O₂ for 10 min. Thereafter, the samples were blocked with 1% Goat serum in PBS for 20 min. subsequently, the samples were incubated with the primary antibody rabbit anti-sheep OB antibody (1:100, ab3583; Abcam, Cambridge, UK) or rabbit anti-human OBR antibody (1:100, 20966-1-AP; Proteintech, Rosemont, IL, USA) in a humidified chamber at 4 °C, overnight. Then, the samples were incubated with the secondary antibodies (goat anti-rabbit IgG) at a dilution of 1:1,000 (Abcam, Cambridge, UK) for 60 min and then reacted with 3,3′-diaminobenzidine (DAB) as chromogen substrate (Servicebio, Wuhan, China). The negative controls (NG) were processed identically as above, except for primary antibody being IgG. The expression of OB and OBR in immunohistochemical staining was evaluated using ImageJ software analysis color intensity with relative grey value. Immunohistochemical analysis was repeated at least three times for each experiment.

Immunofluorescence detection of leptin and leptin receptor

Fresh ejaculated sperm were selected by the swim-up procedure, centrifuged, and fixed for 10 min with 4% paraformaldehyde solution. After the removal fixative solution, sperms were washed in TBSTx (0.1% Triton X-100 in TBS) and placed in a blocking solution (5% BSA in TBSTx) for 1 hour. After blocking, the specimens were incubated with primary antibodies OB (1:100, ab3583; Abcam, Cambridge, UK) and OBR (1:100, 20966-1-AP, Proteintech, Rosemont, IL, USA) antibodies at 4 °C, overnight in a humidified box. For the control sperm slides, normal rabbit serum was used. Sperms were then washed (centrifuging at 300g for 2 min and resuspending in PBS) four times and incubated for 2 h with the secondary antibody (Alexa Fluor488, P0176; Beyotime Institute of Biotechnology, Shanghai, China). After washing two times, the slides were coated with 10 μL concentrated sperm suspension and covered with a coverslip. The slides were analyzed by a confocal microscope (SP8; Leica, Richmond, IL, USA). Negative controls (NG) were were processed identically as above, except for the lack of primary antibodies. PBS was used in its amount instead of the primary antibody. Immunofluorescence analysis was repeated at least three times for each experiment.

Analysis of mRNA expression of OB and OBR using reverse-transcription PCR and quantitative RT-PCR

The total RNA was extracted from testis, epididymal tissue, adipose tissue, ejaculated and epididymal sperms with Trizol LS extraction kit and Turbo DNA-free kit (Ambion, Austin, TX, USA) using a standard protocol. The applied PCR primers and the expected amplification product lengths were shown in Table 1. RT-PCR procedures were performed as per the manufacturers’ guidelines (Cells-to-cDNA TM Kit, AM1722; Ambion Company, Austin, TX, USA). The PCR conditions were as follows: initial denaturation at 95 °C for 10 min, 35 denaturation cycles at 94 °C for 30 s, annealing at Tm for 45 s, and then extension at 72 °C for 15 s, followed by final extension at 72 °C for 10 min. The levels of relevant mRNAs, including the products of OB and OBR in epididymal and ejaculated sperm were quantified by real-time qPCR using One-Step SYBR RT-PCR Kit (Takara, Japan) in a light cycler (Roche Applied Science, Penzberg, Germany). The qPCR program included a 10 min incubation at 95 °C to activate FastStart DNA polymerase, followed by 35 cycles of 95 °C for 10 s, 60 °C for 15 s and 72 °C for the appropriate extension time with single fluorescence acquisition. Each sample was run in triplicate and the mean cycle threshold (Ct) value was transformed to relative expression level by the 2^−ΔΔCt method (Livak & Schmittgen, 2001). GADPH was used as an internal control for normalization.

Western blot analysis for OB and OBR protein expression

Western blot was used for analysis of OB and OBR proteins in testis, epididymal tissue, ejaculated and epididymal sperm of rams as described by the method of Zhang (Zhang et al., 2017). Briefly, tissues or cells were lysed with a lysis buffer (HX1862; Huaxingbo, Hong Kong, China). Equal amounts of protein were resolved using 12% SDS-PAGE gel and transferred to PVDF membranes (ISEQ00010; Millipore, Burlington, MA, USA). After they were blocked with 5% nonfat milk or BSA (A3311; Sigma-Aldrich, St. Louis, MI, USA) at 37 °C for 60 min, the membranes were incubated with primary antibodies against OB (1:1,000, ab3583, Abcam, Cambridge, UK), OBR (1:1,000, 20966-1-AP, Proteintech, Rosemont, IL, USA) and GAPDH ( 1:2,500, WL01114; Wanleibio, Hebei, China) at 4 °C, overnight. The membranes were then washed 3–5 times with TBST buffer (HX1893; Huaxingbo, Hong Kong, China) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, 7074; Cell Signaling Technology, Danvers, MA, USA). Western blotting detection reagents (GE Healthcare, Uppsala, Sweden), and then were visualized using a cooled CCD camera (Nikon, Tokyo, Japan) based chemiluminescence detector (GE Healthcare, Uppsala, Sweden). Protein bands were then analyzed by computer software (Image-Quant 350; GE Healthcare, Uppsala, Sweden).

Spermatozoa motility parameters

The upper layer containing the mobile sperm as described in section (Sperm selection and preparation) placed in Disposable Sperm Counting Plate (SAS, Beijing, China), The concentration of sperm and motility was measured by computer-assisted sperm analyzer (CASA, SAS, Beijing, China).

Spermatozoa viability (Plasma Membrane Integrity)

The Eosin–Nigrosin test (SAS, Beijing, China) was used to assess membrane integrity of sperm for only one-step staining. The nigrosine created a black background, making stained sperm easier to distinguish. The samples were observed with a 400× phase microscope (CX41; Olympus, Tokyo, Japan), the head of live sperm was white or light pink, the head of dead sperm was red or dark pink.

Mitochondrial membrane potential (MMP)

MMP were assessed with a JC-1 Assay Kit (SAS, Beijing, China) based on the manufacturer’s instruction. JC-1 emission fluorescence changed from red (~595 nm) to green (~535 nm) as the MMP changes from aggregates to monomers. sperm was diluted to 500 μL, 1–2 × 10⁶ /ml in PBS, added to 500 μL of JC-1 staining working solution, incubated at 37 °C for 15 min in the dark, centrifuged at 300g for 5 min, and the supernatant was discarded, Resuspend in 1.0 ml of JC-1 staining buffer (1×), centrifuged at 300g for 5 min, discard the supernatant, add 300 μL of JC-1 staining buffer (1×) to resuspend, Finally, the samples were detected by flow cytometer (LSR Fortessa, BD, USA).

Sperm chromatin dispersion test (SCD)

The SCD test is assessed with a SCD Assay Kit (SAS, Beijing, China) based on this principle that when the head of spermatozoon lysed, intact DNA loops of sperm expanded, but fragmented DNA of sperm does not expand or is minimally expanded. The semen was diluted in DPBS to a concentration of 5 × 10⁶/ml and 60 μL was added to Eppendorf tubes containing low-melting point aqueous agarose. The sperm suspension in the agarose was mixed well, Aliquots of 30 μL suspension were placed onto a agarose precoated glass slice, The slice was placed on a cold surface at 4 °C for 5 min, The coverslip was gently removed immersed in lysis solution I for 7 min, placed in lysis solution II for 25 min, washed in distilled water, the slide was dehydrated in gradient ethanol (70%, 90%, 100%) for 2 min each, let the slide to dry in an oven at 37 °C, a layer of dye solution was covered for 15 min and the slide was washed in tap water, then air dry, the sperm was assessed under a light microscope (Olympus CX41; Olympus, Tokyo, Japan) at 400-fold magnification.

Sperm reactive oxygen species (ROS)

Intracellular ROS of sperm was assessed with a ROS Assay Kit (SAS, Beijing, China) containing the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) according to the manufacturer’s instruction. In brief, 1 µL H2DCFDA and 5 µL PI were added into the sperm suspension and mixed well, incubated at 37 °C for 30 min in the dark. the samples were centrifuged, suspended with PBS. Then, the samples were detected by flow cytometer.

Sperm acrosome integrity (AI) and acrosome reaction (AR)

AI and AR of sperm are measured using a AR Assay Kit (SAS, Beijing, China) containing Pisum sativum agglutinin (PSA) labelled with fluorescein isothiocyanate (FITC) (PSA-FITC). Briefly, the control tube contains 0 ng/mL leptin and the test tube contains 5 ng/mL leptin incubated in Biggers-Whitten-Whittingham (BWW) medium supplemented with bovine serum albumin (Solarbio, Beijing, China). The samples were incubated at 37 °C, 5.0% CO₂, for 3 h in air incubator. The samples were stained with 1.5 µL PI and 2.5 µL PSA-FITC for 10 min in the dark, centrifuged, suspended with PBS and analyzed by flow cytometer. Acrosome Integrity (AI%) was defined as the percentage (%) of FITC-PSA-positive sperms, the inducibility of acrosome reaction (AR%) was defined as the percentage (%) of FITC-PSA-negative sperms having undergone acrosome reaction.

Statistical analysis

Data are expressed as mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by student t test using SPSS 21.0 statistical software. *P < 0.05 was considered statistically significance. **P < 0.01 was considered highly significant difference. At least three independent experiments were repeated for each finding.

The circadian profile of serum and seminal plasma levels of leptin in the Suffolk White rams

The concentrations of serum leptin in the Suffolk White rams were not show an obvious circadian rhythm even the highest level was detected at the 24:00 h (Fig. 1A). There were no significant differences in the leptin levels in the seminal plasma of the Suffolk White rams between two time points of 14:00 and 24:00 (P = 0.748) (Fig. 1B).

Expression of OB and OBR in testis, epididymis, and sperm of the Suffolk White rams

In the testis, the OB expression was observed in spermatogonium, primary spermatocytes, secondary spermatocytes, spermatids, and Sertoli cells (Fig. 2B). Surprisingly, the primary and secondary spermatocytes showed the highest expression OB (P < 0.01) (Fig. 2Bb). Also, OBR expression has been identified in spermatogonium, primary and secondary spermatocytes, spermatids, and Sertoli cells (Fig. 2C). The strongest expression of OBR was observed in secondary spermatocytes (P < 0.05) (Fig. 2Cb).

In the epididymis, it was observed that OB was expressed in the stereocilia of the epididymis (Fig. 3Bd), while OBR was expressed in the stereocilia of the epididymal corpus and epididymal cauda (Fig. 3Cf). OB expression in columnar cells of epididymal caput was stronger than that in columnar cells of epididymal cauda (P = 0.007), while no signals has been detected in columnar cells of epididymal corpus tissues (Fig. 3B). OBR expression was detected in columnar cells of epididymis (Fig. 3C). The soluble OBR expression was also found in the epididymal lumen (Fig. 3Cc).

Immunolocalization of OB and OBR in sperm of the Suffolk White rams

The immunocytochemical assay revealed that both OB and OBR were expressed in the whole sperm including the head and tail (the middle piece, principal piece, and end piece) (Fig. 4A).

Detection of OB and OBR in testis, epididymal tissue, ejaculated and epididymal sperm with RT-PCR

The RT-PCR analysis identified mRNA expression of both OB and OBR in testis, epididymal tissue, ejaculated and epididymal sperm (Fig. 4B). This finding was supported by the WB analysis which confirmed the presence of that the proteins of both OB and OBR in testis, epididymal tissue, ejaculated and epididymal sperm. The leptin protein of the Suffolk White rams was recorded as 16 kDa and that of leptin receptor was observed to be 96 kDa (Fig. 4C). The RT-qPCR results indicated OB and OBR gene expression levels in epididymal sperm were significantly higher (P < 0.01) than that in the ejaculated sperm (Fig. 4D).

Effect of leptin supplementation on Spermatozoon motility, MMP and viability

Incubation of sperms with leptin 5 ng/mL for 2 h, compared with the control group (0 ng/mL), significantly improved the sperm progressive motility (P = 0.005), VSL (P = 0.014), VAP (P = 0.015) compared to the control group (Table 2). JC-1 staining showed that, 5 ng/mL Leptin- treated group significantly increased MMP of ram sperm compared to the control (P = 0.003) (Figs. 5A, 5B). Eosin-nigrosine staining showed that 5 ng/mL Leptin-treatment was significantly increased the survival rate of the sperms compared to the control. (P = 0.015) (Figs. 5C, 5D).

Effect of leptin on Spermatozoon DNA fragmentation index (DFI) and reactive oxygen species (ROS)

Sperm Chromatin Dispersion Test (SCD) showed that leptin (5 ng/mL) treatment sperms had significantly lower DFI than that of control group (0 ng/mL) (P = 0.004) (Figs. 6A, 6B). The H₂DCFDA test showed that leptin treated sperms had a significantly lower ROS than that of control (P = 0.0059) (Figs. 6C, 6D).

Effect of leptin supplementation on Spermatozoon AI and AR rate

No significant difference was found in sperm acrosome integrity between leptin treated sperms and the controls (P = 0.852) (Figs. 7A, 7C) and the same was in spermatozoa between the groups. (P = 0.852) (Figs. 7B, 7C).