Angiotensin II type 2 receptor is present in human semen and may be involved in the control of sperm motility. In-depth understanding of the proteins involved in sperm motility can help to elucidate the role of these proteins in male infertility as well as to establish new biomarkers for male infertility.

The phosphoproteome downstream KOR has been studied in human spermatozoa by applying quantitative MS-based proteomics. This studies have been combined with functional approaches to analyze the role of the receptor in the sperm physiology. Results reported phosphorylation changes in spermspecific proteins as well as the inhibition of sperm motility and acrosome after the addition of U50488H, the specific ligand. Graphical Abstract Highlights• Insights into molecular mechanisms underlying GPCR in human spermatozoa.• Quantitative phosphoproteomics downstream Kappa opioid receptor in human spermatozoa.• U50488H agonist induces phosphorylation changes in sperm-specific proteins.• U50488H inhibits human sperm motility and acrosome reaction.

Human sperm protein associated with the nucleus on the X chromosome (SpAnX) genes encode a protein family (SpAnX-A,-B,-c and-D), whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumour cells. present only in hominids, SpAnX-A/D is exclusively expressed in post-meiotic spermatids and mature spermatozoa. However, the biological role of the protein family in human spermatozoa is largely unknown. combining proteomics and molecular approaches, the present work describes the presence of all isoforms of SpAnX-A/D in human spermatozoa and novel phosphorylation sites of this protein family. In addition, we identify 307 potential SpAnX-A/D interactors related to nuclear envelop, chromatin organisation, metabolism and cilia movement. Specifically, SPANX-A/D interacts with fumarate hydratase and colocalises with both fumarate hydratase and Tektin 1 proteins, involved in meeting energy demands for sperm motility, and with nuclear pore complex nucleoporins. We provide insights into the molecular features of sperm physiology describing for the first time a multifunctional role of SPANX-A/D protein family in nuclear envelope, sperm movement and metabolism, considered key functions for human spermatozoa. SpAnX-A/D family members, therefore, might be promising targets for sperm fertility management. Currently, over 186 million people worldwide have infertility problems 1 , and male factors represent approximately 40-50% of clinical infertility cases 2. Among other factors, anatomical or genetic abnormalities, problems in spermatogenesis or environmental factors 3 , are responsible for abnormal sperm parameters which contribute to male infertility. However, approximately 20-30% of men with normal sperm parameters have impaired fertility suggesting that male infertility can be caused by different deficiencies not yet described. The sperm protein associated with the nucleus mapped to the X chromosome (SPANX) family is a testis-specific multigene family. SPANX genes encode proteins that belong to the so-called "cancer testis antigens" (CTA) family, a group of proteins whose expression is limited to the testis and spermatozoa in normal tissues and to a wide variety of tumours 4,5. The SPANX family includes two subfamilies: the SPANX-N subfamily, composed of SPANX-N1,-N2,-N3,-N4 (in the locus Xq27.3) and-N5 (in the locus Xp11.22) that share a high level of sequence homology 6 ; and the SPANX-A/D subfamily, which includes SPANX-A1,-A2,-C,-D (in the locus Xq27.2) and-B (in the locus Xq27.1) with high sequence homology 5,7. Evolved from a common ancestor, the SPANX-N gene family is present in mice, rats and all primates, while the SPANX-A/D family is expressed only in hominids, including humans, bonobos and chimpanzees 8. All SPANX proteins exhibit a similar post-meiotic expression pattern during spermatogenesis, appearing for the first time in haploid spermatids 4,7,9. In mature spermatozoa, SPANX-N proteins are localised on the acrosome 6 , while SPANX-A/D proteins are expressed as dots