Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF).

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45 h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.

Copy number variation of PRAMEY across breeds and its association with male fertility in Holstein sires.

Multi-copy gene families are especially prevalent in the male-specific region (MSY) of the mammalian Y chromosome. Copy number variations (CNV) of these Y-linked gene families have been shown to affect human and animal fertility. The PRAMEY (Preferentially expressed antigen in melanoma, Y-linked) gene family is a newly identified, bovid-specific Y-linked gene family, which codes for a cancer/testis antigen that is expressed predominantly in testis and various tumors. The PRAMEY gene family is believed to play an important role in spermatogenesis and male fertility in cattle. The objective of this study was to investigate the CNV of PRAMEY within and across breeds and to determine whether CNV was associated with reproductive traits in Holstein bulls. A quantitative real-time PCR method was applied to measure the copy number of PRAMEY among 460 bulls using a Y-linked single copy gene, DDX3Y (DEAD box polypeptide 3, Y-linked), as a reference. The median copy number of PRAMEY was 13, ranging from 2 to 31. Significant variations in PRAMEY copy number were observed among 15 breeds investigated. Holstein bulls had the lowest median copy number (12), whereas Limousin bulls possessed the highest median copy number (26). Furthermore, bulls in the taurine lineage (13) had a significantly lower median copy number than those bulls in the indicine lineage (20). Association analysis revealed that PRAMEY copy number was correlated negatively with scrotal circumference (SC), relative scrotal circumference (RLSC), percentage of normal sperm (PNS), and nonreturn rate (NRR), but had no significant association with postthaw motility (PTM), incubated motility (IM), percentage of intact acrosome (PIA), sire conception rate (SCR), or relative breeding efficiency (RBE). The data from this study indicate that CNV of the PRAMEY gene family is associated with male reproductive traits and may serve as a valuable marker for sire fertility selection at an early age in cattle.

The Dynamic of PRAMEY Isoforms in Testis and Epididymis Suggests Their Involvement in Spermatozoa Maturation.

The preferentially expressed antigen in melanoma, Y-linked (PRAMEY) is a cancer/testis antigen expressed predominantly in bovine spermatogenic cells, playing an important role in germ cell formation. To better understand PRAMEY's function during spermatogenesis, we studied the dynamics of PRAMEY isoforms by Western blotting (WB) with PRAMEY-specific antibodies. The PRAMEY protein was assessed in the bovine testicular and epididymal spermatozoa, fluid and tissues, and as well as in ejaculated semen. The protein was further examined, at a subcellular level in sperm head and tail, as well as in the subcellular components, including the cytosol, nucleus, membrane, and mitochondria. RNA expression of PRAMEY was also evaluated in testis and epididymal tissues. Our WB results confirmed the previously reported four isoforms of PRAMEY (58, 30, 26, and 13 kDa) in the bovine testis and spermatozoa. We found that testicular spermatozoa expressed the 58 and 30 kDa isoforms. As spermatozoa migrated to the epididymis, they expressed two additional isoforms, 26 and 13 kDa. Similarly, the 58 and 30 kDa isoforms were detected only in the testis fluid, while all four isoforms were detected in fluid from the cauda epididymis. Tissue evaluation indicated a significantly higher expression of the 58 and 13 kDa isoforms in the cauda tissue when compared to both the testis and caput tissue (p < 0.05). These results indicated that testis samples (spermatozoa, fluid, and tissue) expressed predominantly the 58 and 30 kDa PRAMEY isoforms, suggesting their involvement in spermatogenesis. In contrast, the 26 kDa isoform was specific to epididymal sperm and the 13 kDa isoform was marked in samples derived from the cauda epididymis, suggesting their involvement in sperm maturation. Results from the sperm head and tail experiments indicated that the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda, suggesting this isoform may have a significant role in tail function. Additionally, the 13 kDa isoform increased significantly (p < 0.05) in the cytosol during epididymal passage and tended to increase in other subcellular components. The expression of PRAMEY in the sperm subcellular components during epididymal maturation suggests the involvement of PRAMEY, especially the 13 kDa isoform, in sperm motility.

PRAMEY: A Bovid-Specific Y-Chromosome Multicopy Gene Is Highly Related to Postnatal Testicular Growth in Hu Sheep.

PRAMEY (preferentially expressed antigen in melanoma, Y-linked) belongs to the cancer-testis antigens (CTAs) gene family and is predominantly expressed in testis, playing important roles in spermatogenesis and testicular development. This study cloned the full-length cDNA sequence of ovine PRAMEY using the rapid amplification of cDNA ends (RACE) method and analyzed the expression profile and copy number variation (CNV) of PRAMEY using quantitative real-time PCR (qPCR). The results revealed that the PRAMEY cDNA was 2099 bp in length with an open reading frame (ORF) of 1536 bp encoding 511 amino acids. PRAMEY was predominantly expressed in the testis and significantly upregulated during postnatal testicular development. The median copy number (MCN) of PRAMEY was 4, varying from 2 to 25 in 710 rams across eight sheep breeds. There was no significant correlation between the CNV of PRAMEY and testicular size, while a significant positive correlation was observed between the mRNA expression and testicular size in Hu sheep. The current study suggests that the expression levels of PRAMEY were closely associated with testicular size, indicating that PRAMEY may play an important role in testicular growth.