Pairs of mature bovine testes with intact epididymides were obtained from a local slaughter house (Nicholas Meat, LLC, Loganton, PA, United States). Testes were transported to the laboratory in ice-filled coolers and processed within 2 h to collect spermatozoa, fluid, and tissues from the testes and epididymides (see following sections for specific collection procedures).

For the experiments that evaluated PRAMEY expression in spermatozoa, fluid, and tissue, samples were collected from five pairs of mature testes with intact epididymides (n = 5). For the experiments that evaluated the PRAMEY expression in sperm head and tail, as well in the sperm subcellular fraction, spermatozoa were collected from three pairs of mature testes with intact epididymides (n = 3). In the experiments where mRNA expression was analyzed, the samples (testis, caput, corpus, and cauda epididymis) were collected from three animals (n = 3). For the experiments using freshly ejaculated semen and seminal plasma, the samples were collected from nine yearling beef bulls (n = 9) from the Pennsylvania Department of Agriculture Livestock Evaluation Center.

Epididymides were removed from the testis and dissected into caput and cauda segments. The PRAMEY mRNA expression experiment was the only one to include the corpus epididymal segment. Sperm collection procedures were adapted from protocols previously established in our laboratory (Liu et al., 2017). Briefly, the isolated testicles were washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, and 1.8 mM KH2PO4, pH 7.4) and opened by a sagittal cut using a scalpel. By applying light pressure to the testis, the testicular fluid containing spermatozoa was collected, transferred to a 2 ml Eppendorf tube, and centrifuged at 1,500 x g for 10 min at 4 °C to remove the testicular spermatozoa (the supernatant was reserved). The testicular sperm pellet was washed twice in 2 ml PBS at 1,500 x g for 10 min at 4°C and the resulting sperm pellet was stored at −80°C. The dissected caput or cauda epididymides were washed in PBS and placed in a Petri dish. The epididymal tissue was cut with a scalpel and the fluid containing the spermatozoa was collected by applying pressure on the caput or cauda epididymides. The epididymal fluid was transferred to a 2 ml Eppendorf and centrifuged at 1,500 x g for 10 min at 4°C to collect the epididymal spermatozoa (the supernatant was reserved). The caput and cauda sperm pellets were washed twice in 2 ml PBS at 1,500 x g for 10 min at 4°C and the resulting sperm pellets were stored at −80°C. For the sperm head and tail separation portion of the project, the caput or cauda epididymal spermatozoa were isolated by slicing the epididymal tissue in a Petri dish with PBS. The isolated spermatozoa were centrifuged at 1,500 x g for 10 min at 4°C, and the caput and cauda sperm pellets were used to fractionate the spermatozoa into head and tail portions by sonication.

After collecting spermatozoa from the bovine testis and epididymis as described above, the reserved supernatants were transferred to new 2 ml Eppendorf tubes and centrifuged at 4,000 x g for 20 min at 4°C to remove cellular debris. The testis, caput and cauda fluids were stored at −80°C.

Tissue collection procedures were adapted from protocols previously established in our laboratory (Liu et al., 2017). Tissues from the testis and caput and cauda segments of the epididymis were collected using a scalpel, cut into small pieces, and washed twice in PBS. A portion of the tissues were snap frozen in liquid nitrogen and stored at −80°C until further use for protein and RNA extraction.

Ejaculated semen was obtained from yearling beef bulls at the Pennsylvania Department of Agriculture Livestock Evaluation Center from animals within the Bull Testing Program. The bulls were assessed through breeding soundness evaluation and electroejaculation was used to obtain semen by a certified veterinarian. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committees of the Pennsylvania State University. The collected semen was donated for our research. Semen was collected in 15 ml tubes, kept on ice for the duration of collection, and was transported to the laboratory in ice-filled coolers.

The sperm head and tail separation procedure was adapted from Tateno and co-workers (Tateno et al., 2000). The isolated spermatozoa from epididymal fluid were washed three times in PBS (containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, and 1.8 mM KH2PO4, pH 7.4) with centrifugation at 1,100 x g for 3 min at 4°C. Each sample was sonicated for 30 s at 8 W of power (Fisher Scientific, Sonic Dismembrator Model 100) to break the sperm heads from the tails. To separate the head and tail fractions of the sonicated mixture, 500 uL of Tris Buffer Saline (TBS) (containing 50 mM Tris-Cl, 150 mM NaCl, pH 7.6) was added to each sperm sample and then mixed by vortexing. The diluted spermatozoa (200 uL) were added to 1.0 ml of a 90% Percoll solution (GE Healthcare, product no. 17-0891-02) and the samples were centrifuged at 15,000 x g for 15 min at room temperature (RT). After centrifugation, the sperm tails were located in a thin band near the top of the 1.5 ml tube, and the sperm heads were located in a pellet at the bottom of the tube. The sperm heads and tails were removed separately from the 90% Percoll gradient and put into new 1.5 ml tubes to wash. The sperm heads and tails were washed in 500 uL of TBS (containing 50 mM Tris-Cl, 150 mM NaCl, pH 7.6) at 9,000 x g for 5 min at RT and were immediately frozen in liquid nitrogen.

Subcellular fractionation was performed following the Abcam subcellular fractionation protocol (https://www.abcam.com/protocols/subcellular-fractionation-protocol). Briefly, sperm cells from caput and cauda epididymides were resuspended in 500 μl fractionation buffer and (20 mM HEPES, 10 mM KCL, 2 mM MgCl2, 1 mM EDTA, and 1 MM EGTA supplemented with 1 mM DTT), protease and phosphatase inhibitor cocktails. They were homogenized and incubated for 20 min on ice and were then centrifuged at 720 x g for 5 min. The supernatant containing the sperm cytoplasm, plasma membrane, and mitochondria was transferred into a fresh tube and kept on ice, while the pellet containing nuclei was washed with 500 μl fractionation buffer. The nuclear pellet was dispersed by vortexing, and it was then centrifuged again at 720 x g for 10 min. The resulting pellet was resuspended in TBS with 0.1% SDS, sonicated briefly to shear genomic DNA and homogenize the lysate (3 s on ice at a power setting of 2-continuous), and stored at −80°C. The supernatant containing the sperm cytoplasm, membrane, and mitochondria was centrifuged at 10,000 x g for 5 min at 4°C. The supernatant containing the cytoplasm and membrane was transferred into a fresh tube and kept on ice, while the pellet containing the mitochondria was processed as described for the nuclear pellet to obtain mitochondrial lysate in TBS/0.1% SDS. The supernatant containing the cytoplasm and membrane was centrifuged at 50,000 x g for 2 h. The supernatant containing the cytosol was stored at −80°C, while the pellet was resuspended in 400 μl of fractionation buffer supplemented with 1% Triton and re-centrifuged for 45 min under the same conditions. The resulting membrane pellet was processed as described for the nuclear pellet.

Protein extraction procedures were adapted from protocols previously established in our laboratory (Liu et al., 2017). Protein was extracted from testicular and epididymal spermatozoa, fluids, and tissues, and as well as from sperm heads and tails for this project. Protein was extracted using CelLytic buffer (Sigma, product no. C3228) added with protease inhibitor cocktail (Thermo Scientific, product no. 1860932) and phosphatase inhibitor cocktail (Thermo Scientific, product no. 1862495). The testes and epididymal sperm, fluid, and tissues, and sperm head and tail pellets were removed from the −80°C freezer and ice-cold extraction buffer (supplemented with protease inhibitor and phosphatase inhibitor cocktails) was immediately added to the pellets. The mixture was homogenized on ice and then incubated on ice for 10 min at 4°C on a shaker. The samples were centrifuged for 10 min at 13,200 x g at 4°C. The supernatants were removed and were referred to as the respective protein for each testis and epididymal sperm, fluid, tissue, and sperm head, and tail samples in this study.

The following kits were used for RNA extraction from the testis, and the caput, corpus, and cauda epididymal tissues: The miRNAeasy Mini Kit (Qiagen cat no. 217004), QIAshredder spin columns (Qiagen cat no. 79654), and the RNase-free DNase set (Qiagen cat no. 79254). The protocol for RNA extraction was performed following the “Purification of Total RNA, Including Small RNAs, from Animal Tissues” described in the miRNeasy Mini Handbook (file:///C:/Users/wul12/AppData/Local/Temp/HB-1253003_HB_miRNeasy_96_1,120_%20WW.pdf). cDNA was synthesized using the Superscript III First-Strand Synthesis System for reverse transcription (RT)-PCR (Invitrogen product no. 18080051). The cDNA synthesis was performed following the protocol under “First-strand cDNA synthesis” within the product handbook. RT-PCR was performed using the cDNA samples, The PCR protocol was as follows: each 20-μl reaction contained 13.76 μl of distilled water, 0.5 μl of each primer (10 pmol/μl), 4 μL of Bioline 5× buffer (Bioline United States Inc., Taunton, MA, including 200 μM deoxyribonucleotide triphosphates), 0.24 μl of Bioline Taq DNA polymerase (Bioline United States Inc.), and 1 μl of either genomic DNA (10 ng/μl) or water. Thermocycling for the gene of interest, PRAMEY (forward, 5′- TCA​GGA​CCT​GGA​GGT​CAA​C-3’; reverse, 5′-TGT​GGC​AAT​ATG​TGG​ATG​CG-3′), consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of at 94°C for 30 s, at 65°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. Thermocycling for the control gene, GADPH (forward, 5′- AAC​GGA​TTT​GGC​CGT​ATT​GG-3’; reverse, 5′-CAT​TCT​CGG​CCT​TGA​CTG​TG-3′), consisted of an initial denaturation at 95°C for 5 min, followed by 35 cycles of at 94°C for 30 s, at 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min.

Gel electrophoresis and WB procedures were adapted from protocols previously established in our laboratory (Liu et al., 2017). The protein extracts were separated by a 4–12% acrylamide gel. The gels were electronically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, product no. IPVH00010). The membranes were blocked in 5% nonfat dried milk (NFDM) in tris buffered saline containing 0.05% Tween-20 (TBST). After being briefly washed in TBST, the membrane was incubated in the primary antibody, a PRAMEY-specific (Liu et al., 2017) or PRAMEYc custom antibody produced by New England Peptide, LLC (Gardner, MA, United States) at a dilution of 1:500, overnight at 4°C. The membranes were washed with TBST 3 times for 10 min each and then incubated in the secondary antibody, anti-rabbit IgG, HRP linked (Cell Signaling Technology, product no. 7074S, 1:5,000 dilution) for 1 h at room temperature. The membranes were washed 3 times for 10 min each and the reactive proteins were detected by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, product no. 34095). After PRAMEY or PRAMEYc antibody incubation, membranes were stripped with 15 ml of stripping buffer (10% SDS, 0.5 M Tris HCl, β-mercaptoethanol) for 30 min at 50°C. Membranes were washed with TBST 6 times for 5 min each and then re-blocked in 5% NFDM. Alpha tubulin (TUBA) primary antibody (Cell Signaling Technology, product no. 3873, 1:1,000 dilution) was used to probe the membranes again overnight at 4°C. The membranes were washed with TBST 3 times for 10 min each and then incubated in the secondary antibody, anti-mouse IgG, HRP linked (Cell Signaling Technology, product no. 7076, 1:5,000 dilution) for 1 h at room temperature. The membranes were washed 3 times for 10 min each and the reactive proteins were detected by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, product no. 34095).

WB images were analyzed using ImageJ software (Schneider et al., 2012). The data were analyzed by the normality test (Shapiro-Wilk test) and equal variance test (Brown-Forsythe) using SigmaPlot 12.0 (Statistical Software). After meeting the assumptions of normally distributed data and homogeneity of variance, the difference in treatment levels was compared by one-way ANOVA with the post-hoc Tukey test. Data are expressed as the mean ± SEM, and a value of p < 0.05 was considered statistically significant.

In a previous report, four different isoforms of the bovine PRAMEY protein including the 58, 30, 26, and 13 kDa isoforms were identified by WB with a PRAMEY-specific antibody (Liu et al., 2017). In the current study, all four isoforms of PRAMEY were found to be present in testicular, epididymal, and ejaculated spermatozoa of mature adult bulls (Figure 1A). The 58 kDa protein, which is the predicted molecular weight for the PRAMEY was detected, but at a low amount in spermatozoa (Table 1) from testis, caput, and cauda of the epididymis and ejaculated sperm (p > 0.05) (Figure 1A,B). The 30 kDa isoform had a higher expression in epididymal spermatozoa (Table 1) compared to testicular and ejaculated sperm (p < 0.05) (Figure 1A,B). As spermatozoa moved to the epididymis, two additional isoforms, 26 and 13 kDa were detected. Although there was no difference (p > 0.05) in the 26 kDa protein amount between caput and cauda sperm, a significant difference was found between epididymal and ejaculated sperm (P˂0.05). The protein level of the 13 kDa isoform was similar (p > 0.05) between cauda and caput sperm. Comparable to the 26 kDa isoform, the 13 kDa protein amount was higher in epididymal sperm compared to ejaculated sperm (Figure 1A,B). These results suggest that the two smaller isoforms of PRAMEY may be involved in sperm maturation and sperm fertilizing ability. However, it is unknown whether the 26 and 13 kDa proteins are new isoforms or they are products of the 58 and 30 kDa isoform cleavage/degradation.

Evaluation of the fluid collected from the testis, caput, and cauda segment of the epididymis and ejaculate (seminal plasma) revealed a different PRAMEY pattern from that observed for spermatozoa. As shown in Figure 2A, a weak expression of the 58 kDa isoform was observed in all fluid types (Table 1). Where the 58 kDa expression was similar among the fluids from testis and epididymis, its expression level was lower (p < 0.05) in seminal plasma (Figure 2A,B). The 30 kDa isoform was strongly detected compared to the 58 kDa and its expression was higher in fluid from testis and caput compared to seminal plasma (p < 0.05). Interestingly, the 26 kDa isoform was not observed in fluid from the testis, while it was hardly observed in caput epididymal fluid or seminal plasma, but was mainly observed in fluid from the cauda segment of the epididymis (p < 0.05). The 13 kDa isoform was weakly detected in fluid from the caput segment of the epididymis but had a higher (p < 0.05) protein concentration observed in fluid from the cauda epididymis and seminal plasma (Figure 2A,B) (Table 1).

The presence of PRAMEY in the fluids suggested that PRAMEY may have originated from the testicular and/or epididymal tissue. Therefore, we next evaluated the PRAMEY protein isoform expression in testicular and epididymal tissue (Figure 3A,B). This evaluation showed that the 58 kDa isoform was relatively weakly expressed in testicular and epididymal tissue. However, its expression level was higher in tissue from the cauda segment of the epididymis compared to tissue from the testis and the caput segment of the epididymis (p < 0.05) (Figure 3A,B). In contrast to the 58 kDa isoform, the 30 kDa isoform was heavily expressed in all three tissues studied (Table 1), however, no significant difference was observed among them (p > 0.05) (Figure 3A,B). Interestingly, the 26 kDa isoform was not observed in the testis or epididymal tissue (Figure 3A,B). The 13 kDa protein expression was weakly detected in caput tissue, while its expression level was higher in cauda tissue (p < 0.05) (Figure 3A,B). Moreover, the 13 kDa isoform was not observed in testicular tissue (Figure 3A,B) (Table 1).

To address the question of whether the PRAMEY gene is expressed in the epididymal tissue, RT-PCR was performed with a pair of PRAMEY gene-specific primers on testicular and epididymal tissues (Figure 4). We found that PRAMEY was expressed only in the testis, while no expression was detected in the caput, corpus, or cauda segments of the epididymis, and the negative control, i.e. the liver tissue (Figure 4), confirming our previous report that PRAMEY is germ cell-specific (Liu et al., 2017).

The PRAMEY antibody used in the above experiments was referred to as a N-terminus antibody, which produced four isoforms in the WB results. To determine whether a different antibody designed from the C-terminus of PRAMEY would detect the same isoforms, a custom-made C-terminal PRAMEY antibody (called PRAMEYc) was produced and used in this study. We found that the new PRAMEYc antibody detected the intact 58 kDa PRAMEY only, and the expression level of 58 kDa isoforms was low, but consistent, and no significant variations were found among spermatozoa (Figure 5A), fluid (Figure 5B), and tissue (Figure 5C) from testis and caput and cauda segments of the epididymis (p > 0.05). The finding indicated that the 30, 26, and 13 kDa PRAMEY isoforms were generated from the N-terminus of PRAMEY.

We next evaluated the dynamic of PRAMEY expression between sperm head and tail during maturation in the epididymis (Figure 6A). Among the four PRAMEY isoforms, the 58 kDa isoform was very weakly detected in the separated sperm head and tail, thus it was not evaluated in this experiment. When comparing the 30, 26, and 13 kDa isoforms individually (Figures 6A,B), we found a remarkable decrease of 10.9, 5.4, and 3.8-fold respectively, in protein expression from the caput to cauda epididymis in sperm heads, but there was a small decrease in the expression for both the 30 and 26 kDa isoforms in sperm tails (1.9 and 1.2-fold respectively). In contrast, the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda (Figures 6A,B), suggesting this isoform may have a significant role in tail function, but is likely not as important in sperm head function of cauda spermatozoa. Variations in protein expression were observed among bulls, particularly in the sperm head. For example, the 30 and 26 kDa isoforms in the sperm head of this animal shown in Figure 6A were not detected. When expression levels of all isoforms were combined, PRAMEY expression was nearly equivalent for both caput sperm head and tail (Figure 6B). Alternatively, cauda sperm tails had an expression 6-fold higher than cauda sperm heads, suggesting the role of PRAMEY in tail function (Figure 6B). It is worth noting that α-tubulin antibodies detected two bands from the sperm head and tail samples (Figure 6A). This phenomenon also occurred in α-tubulin detected in caput fluid (Figure 2A).

To better understand the PRAMEY functional dynamic between head and tail during sperm maturation, we further evaluated the PRAMEY expression in sperm cytosol, nucleus, membrane, and mitochondria during spermatozoal maturation in the epididymis (Figure 7A). Similar to the head and tail experiment, the 58 kDa isoform was very weakly detected, thus it was not evaluated in this experiment. During bovine sperm maturation in the epididymis, the 30 and 26 kDa isoforms tended to decrease in sperm cytosol, nucleus, plasma membrane, and mitochondria from caput to cauda. However, the 13 kDa isoform significantly increased from caput to cauda sperm cytosol (p < 0.05), while the nucleus, membrane, and mitochondria expression tended to increase from caput to cauda spermatozoa (Figure 7A,B). The dynamic of PRAMEY localization during sperm transit through epididymis possibly suggests the involvement of PRAMEY in sperm maturation, especially the 13 kDa isoform.