Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN

Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein

Semen amyloids participate in spermatozoa selection and clearance

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens

Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN

<div><p>Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins. </p> </div

Inhibition of sperm motility in male macaques with EP055, a potential non-hormonal male contraceptive

<div><p>Abstract</p><p>Men have two practical choices for contraception; the condom which has a high typical use failure rate or vasectomy. New male hormonal and non-hormonal contraceptives are under development that target either the production of sperm (spermatogenesis) or the delivery of sperm. One particular target is the sperm protein EPPIN, which is present on the surface of human spermatozoa. EP055 is a small organic compound that targets EPPIN on the surface of sperm and inhibits motility. EP055 was tested in cynomolgus (<i>Macaca fascicularis)</i> males to determine its plasma half-life after intravenous (i.v.) infusion of a single dose and for binding to its target tissues. Our initial study demonstrated a plasma half-life for EP055 of 10.6 minutes. In a second study examination of macaque testis, epididymis, and plasma after i.v. infusion of a single dose of compound EP055 (63.25 mg/kg) demonstrated that EP055 was detected in testis and epididymis two hours and six hours post-infusion. We initiated a trial in rhesus (<i>Macaca mulatta</i>) males to assess the availability of EP055 in semen and its effect on sperm motility as a measure of the drug's efficacy. Four macaques were infused with a low dose (75–80 mg/kg) followed by a recovery period and a subsequent high dose (125–130 mg/kg) of EP055. After high dose administration, sperm motility fell to approximately 20% of pretreatment levels within 6 hours post-infusion; no normal motility was observed at 30 hours post-infusion. Recovery of sperm motility was obvious by 78 hours post-infusion; with full recovery in all animals by 18 days post-infusion. EP055 has the potential to be a male contraceptive that would provide a reversible, short-lived pharmacological alternative.</p></div

Human semenogelin I (SEMG1) sequence.

<p><b>A</b>) SEMG1 primary sequence. Signal peptide is indicated by a dashed underline. <b>B</b>) Schematic representation of SEMG1 containing repeats Ia, Ib, IIa, IIb, IIIa, and IIIb. Recombinant SEMG1 fragments used in AlphaScreen assay and CASA experiments: G26-R281 (SEMG1^214-42), R165-R281 (SEMG1^74-42), R165-Q247 (SEMG1^74-8), Q207-R281 (SEMG1^32-42), Y220-L262 (SEMG1^19-22), E229-L269 (SEMG1^10-30), and Q207-Q247 (SEMG1^32-8) are shown. Cysteine residue at position 239 is indicated by a box in (<b>A</b>) and a vertical arrow in (<b>B</b>).</p

EP055 levels in rhesus macaque (Macaca mulatta) plasma and semen after infusion.

<p>EP055 levels in rhesus macaque (Macaca mulatta) plasma and semen after infusion.</p

Binding of recombinant SEMG1 fragments to EPPIN in the AlphaScreen assay.

<p><b>A</b>) Concentration-response curve for SEMG1^214-42, SEMG1^74-42, SEMG1^74-8, and SEMG1^32-8 in the presence of a constant concentration of EPPIN. <b>B</b>) Displacement of bt-SEMG1^214-42 from its binding site on EPPIN by SEMG1 constructs. Specific signal for each data point was normalized as percentage of specific signal in the absence of SEMG1 competitors. Data are expressed as mean ± SD from two independent experiments, each performed in four replicates. <b>C</b>) Concentration-response curve for SEMG1^214-42 (wild-type) and Cys239 mutants (C239G, C239D, C239H, C239R, C239S) in the presence of a constant concentration of EPPIN. Data points in (<b>A</b>) and (<b>C</b>) represent mean ± SD of specific signal from a representative experiment of (<b>A</b>) four (SEMG1^214-42 and SEMG1^74-42) and two (SEMG1^74-8) experiments; and (<b>C</b>) three experiments, each performed in four replicates. cps = counts per second.</p