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Background: Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing., Objective: The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sperm freezability, using two-dimensional difference gel electrophoresis (2D-DIGE)., Materials and Methods: Epididymal spermatozoa from 4 stallions were incubated with SP (80%, v/v) or without SP (control) before freezing. Sperm parameters were evaluated after thawing (viability, chromatin condensation, acrosomal integrity, reactive oxygen species [ROS]) and SP composition: total antioxidant capacity (TAC), fatty acid composition, total protein concentration, and protein components by 2D-DIGE., Results: After thawing, the proportions of viable and acrosome-intact spermatozoa were higher than control when SP from two stallions was used (F and O). The SP of all stallions reduced ROS production in comparison with the control. After analyzing the SP components, it was found that total protein concentration, TAC, polyunsaturated fatty acids (PUFA), and eight specific proteins identified by 2D-DIGE were different between stallions., Discussion: These studies allow the identification of SP components that could be involved in sperm protection or cryotolerance. Use of this information could help in the selection of stallions according to their semen freezing capacity., Conclusion: The composition of the SP probably contributes to semen cryotolerance capacity. Total protein, TAC, PUFA, and some proteins such as cysteine-rich secreted protein 3 could be used as biomarkers for the selection for sperm cryotolerance., (© 2020 American Society of Andrology and European Academy of Andrology.)

Skeletal muscle is the most abundant tissue in the adult body and plays an essential role in maintaining heat production for the entire body. Recently, muscle-derived non-shivering thermogenesis under cold conditions has received much attention. Zinc-α2-glycoprotein (ZAG) is an adipokine that was shown to influence energy metabolism in the adipose tissue. We used ZAG knock-out (ZAG KO) and wild-type (WT) mice to investigate the effect of ZAG on the lipid metabolism of skeletal muscle upon exposure to a low temperature (6°C) for one week. The results show that cold stress significantly increases the level of lipolysis, energy metabolism, and fat browning-related proteins in the gastrocnemius muscle of WT mice. In contrast, ZAG KO mice did not show any corresponding changes. Increased expression of β3-adrenoceptor (β3-AR) and protein kinase A (PKA) might be involved in the ZAG pathway in mice exposed cold stress. Furthermore, expression of lipolysis-related proteins (ATGL and p-HSL) and energy metabolism-related protein (PGC1α, UCP2, UCP3 and COX1) was significantly enhanced in ZAG KO mice after injection of ZAG-recombinant plasmids. These results indicate that ZAG promotes lipid-related metabolism in the skeletal muscle when the animals are exposed to low temperatures. This finding provides a promising target for the development of new therapeutic approaches to improve skeletal muscle energy metabolism.

Background: Members of the cysteine-rich secretory proteins (CRISPS), antigen 5 (Ag5) and pathogenesis-related 1 (Pr-1) (CAP) superfamily of proteins are found across the bacterial, fungal, plant and animal kingdoms. Although many CAP superfamily proteins remain poorly characterized, over the past decade evidence has accumulated, which provides insights into the functional roles of these proteins in various processes, including fertilization, immune defence and subversion, pathogen virulence, venom toxicology and cancer biology., Objective and Rationale: The aim of this article is to summarize the current state of knowledge on CAP superfamily proteins in mammalian fertility, organismal homeostasis and disease pathogenesis., Search Methods: The scientific literature search was undertaken via PubMed database on all articles published prior to November 2019. Search terms were based on following keywords: 'CAP superfamily', 'CRISP', 'Cysteine-rich secretory proteins', 'Antigen 5', 'Pathogenesis-related 1', 'male fertility', 'CAP and CTL domain containing', 'CRISPLD1', 'CRISPLD2', 'bacterial SCP', 'ion channel regulator', 'CatSper', 'PI15', 'PI16', 'CLEC', 'PRY proteins', 'ASP proteins', 'spermatogenesis', 'epididymal maturation', 'capacitation' and 'snake CRISP'. In addition to that, reference lists of primary and review article were reviewed for additional relevant publications., Outcomes: In this review, we discuss the breadth of knowledge on CAP superfamily proteins with regards to their protein structure, biological functions and emerging significance in reproduction, health and disease. We discuss the evolution of CAP superfamily proteins from their otherwise unembellished prokaryotic predecessors into the multi-domain and neofunctionalized members found in eukaryotic organisms today. At least in part because of the rapid evolution of these proteins, many inconsistencies in nomenclature exist within the literature. As such, and in part through the use of a maximum likelihood phylogenetic analysis of the vertebrate CRISP subfamily, we have attempted to clarify this confusion, thus allowing for a comparison of orthologous protein function between species. This framework also allows the prediction of functional relevance between species based on sequence and structural conservation., Wider Implications: This review generates a picture of critical roles for CAP proteins in ion channel regulation, sterol and lipid binding and protease inhibition, and as ligands involved in the induction of multiple cellular processes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The seminal fluid proteins (SFPs) transferred to mating partners along with sperm often play crucial roles in mediating post-mating sexual selection. One way in which sperm donors can maximize their own reproductive success is by modifying the partner's (sperm recipient's) post-copulatory behaviour to prevent or delay re-mating, thereby decreasing the likelihood or intensity of sperm competition. Here, we adopted a quantitative genetic approach combining gene expression and behavioural data to identify candidates that could mediate such a response in the simultaneously hermaphroditic flatworm Macrostomum lignano. We identified two putative SFPs-Mlig-pro46 and Mlig-pro63-linked to both mating frequency and 'suck' frequency, a distinctive behaviour, in which, upon ejaculate receipt, the worm places its pharynx over its female genital opening and apparently attempts to remove the received ejaculate. We, therefore, performed a manipulative experiment using RNA interference-induced knockdown to ask how the loss of Mlig-pro46 and Mlig-pro63 expression, singly and in combination, affects mating frequency, partner suck propensity and sperm competitive ability. None of the knockdown treatments impacted strongly on the mating frequency or sperm competitive ability, but knockdown of Mlig-pro63 resulted in a significantly decreased suck propensity of mating partners. This suggests that Mlig-pro63 may normally act as a cue in the ejaculate to trigger recipient suck behaviour and-given that other proteins in the ejaculate have the opposite effect-could be one component of an ongoing arms race between donors and recipients over the control of ejaculate fate. However, the adaptive significance of Mlig-pro46 and Mlig-pro63 from a donor perspective remains enigmatic., (© 2020 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

During mammalian fertilization, sperm is fused with the oocyte's membrane, triggering the resumption of meiosis from the metaphase II arrest, the extrusion of the second polar body, and the exocytosis of cortical granules; these events are collectively called 'oocyte activation.' In all species studied to date, the transient rise in the cytosolic level of calcium (in particular, the repeated calcium increases called 'calcium oscillations' in mammals) is required for these events. Researchers have focused on identifying the factor(s) that can induce calcium oscillations during fertilization. Sperm-specific phospholipase C, i.e., PLC zeta (PLCζ), is a strong candidate of the factor(s), and several research groups using different species obtained evidence that PLCζ is a sperm factor that can induce calcium oscillations during fertilization. However, postacrosomal sheath Tryptophan-Tryptophan (WW)-domain-binding protein (PAWP) was recently shown to have a pivotal role in inducing calcium oscillations in some species. In this review, we focus on PLCζ and PAWP as sperm factors, and we discuss this controversy: Which of these two molecules survives as a sperm factor?, (© 2020 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Zinc-α2-glycoprotein (ZAG), a recently identified adipokine, is a multidisciplinary protein, which is secreted in various body fluids. The ZAG plays roles in lipolysis, regulation of metabolism, cell proliferation and differentiation, regulation of melanin synthesis, cell adhesion, and immunoregulation. The aim of this study is to estimate serum and tissue levels of ZAG in patients with vitiligo. The study included 30 vitiligo patients and 30 healthy controls. Lesional skin biopsy was performed, and blood sample was retrieved to determine the level of ZAG in blood using ELISA kit. In this study, the mean level of ZAG was found to be significantly lower in the vitiligo patients' tissue in comparison with the healthy control subjects' tissue (  p =0.001); the level of ZAG was also lower in vitiligo patients' serum in comparison with the healthy control subjects' serum (  p =0.001). A highly significant correlation was observed between the duration of the disease and the level of ZAG in the tissue of patients ( r  =0.9; p =0.001). Also a highly significant positive correlation was observed between the age of patients and the level of ZAG in the tissue ( r  =0.5; p =0.006). Diminishing of ZAG in serum and tissue of vitiligo patients is another important player sharing in the complex pathogenesis of vitiligo.

Infertile couples with low oocyte yield in combination with abnormal semen parameters may experience intra-cytoplasmic sperm injection (ICSI) failure. An established factor associated with ICSI failure is oocyte activation deficiency (AOD). The latter originates from seminal contributors, such as phospholipase C-zeta (PLCζ) that is not adequate to produce calcium (Ca 2+ ) oscillations for oocyte activation. Apart from this natural activator, other stimulants, such as A23187, ionomycin, strontium chloride or even electric pulses, have been used in embryological laboratories to overcome AOD and ICSI failure. The aim of the present narrative review is to discuss the role of Ca +2 oscillations in oocyte activation and summarize the evidence concerning the use of oocyte activators as agents for artificial oocyte activation (AOA). Studies in humans and animals have emerged many physiological, pathophysiological and ethical aspects of AOA. In conclusion, in mammalian eggs, the cytosolic Ca +2 oscillations derive from a periodic release of Ca +2 from intracellular pools. PLCζ, as well as artificial stimulants, have been used to produce Ca +2 oscillations for AOA. As the latter may increase the risk of epigenetic induced malformations, further studies are required to clarify whether AOA constitutes an effective and safe method to overcome ICSI failure. Abbreviations: AOA: artificial oocyte activation; AOD: oocyte activation deficiency; Ca +2 : Calcium; CAMKII: Ca +2 /calmodulin-dependent protein kinase II; CICR: calcium-induced calcium-release; DAG: diacylglycerol; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICSI: intra-cytoplasmic sperm injection; InsP 3 R: inositol-trisphosphate receptor; IP 3 : inositol 1,4,5-trisphosphate; IVF: in vitro fertilization; MAP: mitogen-activated protein; MII: metaphase II; NADP: nicotinic acid adenine dinucleotide phosphate; NO: nitric oxide; PAWP: post-acrosomal WW-binding domain protein; PIP 2 : phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase C; PLCζ: phospholipase C-zeta; SOAFs: spermatozoon-released oocyte-activating factors; Sr +2 : strontium; TFF: total fertilization failure.

Enkurin was identified initially in mouse sperm where it was suggested to act as an intracellular adaptor protein linking membrane calcium influx to intracellular signaling pathways. In order to examine the function of this protein, a targeted mutation was introduced into the mouse Enkurin gene. Males that were homozygous for this mutated allele were subfertile. This was associated with lower rates of sperm transport in the female reproductive tract, including reduced entry into the oviduct and slower migration to the site of fertilization in the distal oviduct, and with poor progressive motility in vitro. Flagella from wild-type animals exhibited symmetrical bending and progressive motility in culture medium, and demembranated flagella exhibited the "curlicue" response to Ca2+ in vitro. In contrast, flagella of mice homozygous for the mutated allele displayed only asymmetric bending, nonprogressive motility, and a loss of Ca2+-responsiveness following demembrantion. We propose that Enkurin is part of a flagellar Ca2+-sensor that regulates bending and that the motility defects following mutation of the locus are the proximate cause of subfertility.

Sexual conflict is acknowledged as pervasive, with the potential to generate and maintain genetic variation. Mechanistic studies of conflict have been important in providing direct evidence for the existence of sexual conflict. They have also led to the growing realization that there is a striking phenotypic diversity of adaptations whose evolution can be shaped by sexually antagonistic selection. The mechanisms involved range from the use of genital spines, claspers, songs, and smells to ejaculate molecules. In one well-studied example, sexual conflict can occur over the sexually antagonistic effects of seminal fluid proteins in Drosophila melanogaster. However, an important puzzle remains, namely, why seminal fluid proteins are so numerous and complex, hence whether all or some are involved in mediating sexual conflict. I hypothesize that this rich diversity and the complexity of traits subject to sexually antagonistic selection in general may arise, at least in part, due to the deployment of sexually antagonistic adaptations in males in a way that lessens the probability of broadscale, strong resistance evolution in females. In elaborating this hypothesis, I explore how research into the evolution of resistance to insecticides, antimicrobials, and vaccines might be used to provide insights into the evolution of female resistance to the effects of sexually antagonistic manipulative traits of males. In this manner, the manipulative traits of males can be resistance-proofed.

The objective of the present study was to describe the proteins from the seminal plasma of buffalo and correlate these proteins with sperm motility. Ejaculates from sixteen Murrah buffalo were used. Semen collection was performed by electroejaculation, and the ejaculate was evaluated by macroscopic (volume) and microscopic analysis (subjective motility and vigor, as well as sperm concentration). After the analysis, the samples were centrifuged (800g for 10 min and 10,000 for 30 min at 4 °C), and the supernatant (seminal plasma) was used to determine total protein concentration by the Bradford method. Based on total protein concentration, an aliquot (50 μg) was taken to conduct protein in-solution digestion for nano-LC-ESI-Q-TOF mass spectrometry analysis. Samples were divided into two groups, minimal (little sperm motility) and greater (typical sperm motility), based on non-hierarchical clustering considering motility and emPAI protein value. The data were analyzed by multivariate statistical analysis using principal component analysis (PCA) and partial analysis of minimum squares discrimination (PLS-DA). Forty-eight proteins were detected in the seminal plasma, and fifteen were common to two groups. There were six proteins that were significantly different between the groups. The main functions of proteins in seminal plasma were catalytic and binding activity. Spermadhesin protein, ribonuclease, 14-3-3 protein zeta/delta and acrosin inhibitor were in greater amounts in seminal plasma from the group with greater sperm motility; prosaposin and peptide YY were in greater amounts in the group with little sperm motility. The proteins detected in the greater motility group were correlated with sperm protection, including protection against oxidative stress, lipid peroxidation, protease inhibition and prevention of premature capacitation and acrosome reaction. In the group with little sperm motility, one of the identified proteins is considered to be an antifertility factor, whereas the function of other identified protein is not definitive. Results from the present study add to the knowledge base about the molecular processes related with sperm motility, and these findings can be used for determining potential markers of semen quality., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amyloid fibrils are linear polypeptide aggregates with a cross-β structure. These fibrils are best known for their association with neurodegenerative diseases, such as Alzheimer's or Parkinson's, but they may also be used by living organisms as functional units, e.g. in the synthesis of melanin or in the formation of bacterial biofilms. About a decade ago, in a search for semen factors that modulate infection by HIV-1 (a sexually transmitted virus and the causative agent of the acquired immune deficiency syndrome (AIDS)), it was demonstrated that semen harbors amyloid fibrils capable of markedly increasing HIV infection rates. This discovery not only created novel opportunities to prevent sexual HIV-1 transmission but also stimulated research to unravel the natural role of these factors. We discuss here the identification of these intriguing structures, their molecular properties, and their effects on both sexually transmitted diseases and reproductive health. Moreover, we review strategies to antagonize semen amyloid to prevent sexual transmission of viruses.

HSP-1/2 and PDC-109 belong to a family of fibronectin type II proteins, present in high concentrations in bovine and equine seminal plasma, respectively. These proteins act as extracellular small heat shock proteins and protect target/client proteins against various kinds of stress. They also exhibit characteristic binding to choline phospholipids present on the sperm plasma membrane and cause efflux of choline phospholipids and cholesterol, resulting in sperm capacitation. The current study demonstrates that hypersaline conditions decrease the chaperone-like activity (CLA) of HSP-1/2. On the other hand, lipoprotein aggregates formed by the binding of choline phospholipids to this protein exhibit higher CLA than HSP-1/2 alone in vitro; the increased CLA can be correlated to the increased surface hydrophobicity of the lipoprotein aggregates. Presence of cholesterol in the membrane was found to decrease such enhancement in the CLA. We have also observed that salinity of the medium affects the chaperone activity by altering the polydisperse nature of the HSP-1/2. Together these results indicate that hydrophobicity and polydispersity are important for the chaperone-like activity of HSP-1/2 and factors that can alter these properties of HSP-1/2 can modulate its CLA. Further, studies on PDC-109 show that the chaperone-like and membrane-destabilizing activities of this protein are differentially affected by change in pH.

Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium., (© 2016 by the Society for the Study of Reproduction, Inc.)

The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cysteine-rich secretory protein 3 (CRISP-3) is upregulated in prostate cancer as compared to the normal prostate tissue. Higher expression of CRISP-3 has been linked to poor prognosis and hence it has been thought to act as a prognostic marker for prostate cancer. It is proposed to have a role in innate immunity but its role in prostate cancer is still unknown. In order to understand its function, its expression was stably knocked down in LNCaP cells. CRISP-3 knockdown did not affect cell viability but resulted in reduced invasiveness. Global gene expression changes upon CRISP-3 knockdown were identified by microarray analysis. Microarray data were quantitatively validated by evaluating the expression of seven candidate genes in three independent stable clones. Functional annotation of the differentially expressed genes identified cell adhesion, cell motility, and ion transport to be affected among other biological processes. Prostate-specific antigen (PSA, also known as Kallikrein 3) was the top most downregulated gene whose expression was also validated at protein level. Interestingly, expression of Annexin A1 (ANXA1), a known anti-inflammatory protein, was upregulated upon CRISP-3 knockdown. Re-introduction of CRISP-3 into the knockdown clone reversed the effect on invasiveness and also led to increased PSA expression. These results suggest that overexpression of CRISP-3 in prostate tumor may maintain higher PSA expression and lower ANXA1 expression. Our data also indicate that poor prognosis associated with higher CRISP-3 expression could be due to its role in cell invasion.

The objective was to determine the relationship between seminal plasma proteins and sperm morphology in Bos indicus bulls of the Brahman breed. Fifty-six 24-month-old Australian Brahman bulls were electroejaculated and samples were examined to determine the percentage of morphologically normal sperm (PNS24) and the seminal plasma protein composition was identified and quantified by 2-D gel electrophoresis. The total integrated optical density of 152 seminal plasma protein spots (SPPs) across all gels was determined using the PDQuest software version 8.0 (Bio Rad, USA). Using a single regression mixed model with the density of individual spots as a covariate for PNS24, 17 SPPs were significantly associated with PNS24 (p<0.05). A multiple regression analyses of these SPPs, using three models; non-parametric Tree Model, Generalized Additive Model, and a step-wise selection method were conducted, and 6 SPPs could be used to predict PNS24; four SPPs had positive and two had negative association with PNS24. Together these spots explained 35% of the phenotypic variation in PNS24. Using mass spectrometry (MALDI-ToF and TripleToF-MS) the SPPs with positive relationship contained mainly apolipoprotein A-I (1310), protein DJ-1 and glutathione peroxidase 3 (2308), phosphoglycerate kinase 1 (6402) and apolipoprotein A-I and secretoglobin family 1D member (8008). The SPPs inversely associated with PNS24 were clusterin/seminal plasma protein A3 (1411) and epididymal secretory protein E1 (8108). This is the first comprehensive report on the association between seminal plasma protein composition in Bos indicus Brahman bulls and sperm morphology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca(2+) oscillations in mammalian eggs but also intracellular Ca(2+) release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca(2+) oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization., (© 2015 by the Society for the Study of Reproduction, Inc.)

The acrosome is a specialized organelle that covers the anterior region of the sperm nucleus, and plays an essential role in mammalian fertilization. Although acrosome biogenesis is an important aspect of spermiogenesis, the molecular mechanism that regulates this event remains unknown. In the present study, we identified a novel gene, Fam170b (family with sequence similarity 170, member B), exclusively expressed in mouse testes. Fam170b expression first started at postnatal week 3, and increased in an age-dependent manner until plateauing in adulthood. Immunofluorescence staining revealed its enrichment in round spermatids, and redistribution to a perinuclear spot adjacent to the Golgi and the acrosome of elongating spermatids and spermatozoa; this localization was shared between mouse and human spermatozoa. Anti-FAM170B antibody was remarkably found to inhibit murine in vitro fertilization, specifically blocking the acrosome reaction. We further determined that FAM170B interacts with GOPC (Golgi-associated PDZ and coiled-coil motif containing protein) during acrosome formation, as verified by immunofluorescence and co-immunoprecipitation assays. Thus, we document the expression and function for the endogenous acrosomal protein FAM170B during spermiogenesis and fertilization., (© 2015 Wiley Periodicals, Inc.)

The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

The aim of this study was to elucidate the role of llama seminal plasma in the formation of oviductal sperm reservoirs. Female llamas with follicles in the mature phase were mated with a bulbourethral glands-removed male. Females mated with nonbulbourethral glands-removed males were used as control. Oviducts were obtained by surgery 24 h after mating. The uterotubal junction and isthmus were examined by scanning electron microscopy, and mucopolysaccharides were identified by Alcian blue staining. To know the proteins probably involved in sperm reservoir formation, SDS-PAGE of seminal plasma (8% and 18% resolving gel) was made. Spermatozoa only adhered to the oviductal mucosa surface of uterotubal junction of females mated with nonbulbourethral glands-removed males confirming that seminal plasma and, in particular, bulbourethral secretions are related with the oviductal sperm reservoir formation. Histological sections showed sperm in the lumen, immersed in substance, positive for acid mucopolysaccharides. Alcian blue staining of seminal plasma proteins SDS-PAGE showed a band of high molecular weight containing mucopolysaccharides, only present in nonbulbourethral glands-removed males. Bulbourethral glands would secrete at least eight different proteins that most likely participate in the process of sperm storage in the oviduct., (© 2013 Blackwell Verlag GmbH.)

Proteins of the Binder of SPerm superfamily are known to bind choline phospholipids on sperm membrane and promote sperm capacitation. The current study focuses on the biochemical and functional characterization of the murine Binder of SPerm homolog 2 (BSPH2). A recombinant protein (rec-BSPH2) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS cells using pET32a vector. It was purified by immobilized metal ion affinity chromatography and refolded on column using a decreasing urea gradient. Rec-BSPH2 was found to share some binding characteristics with other BSP proteins, such as binding to gelatin, heparin, and epididymal sperm. Rec-BSPH2 as well as murine recombinant BSPH1 were found to have different immunofluorescence patterns when bound to uncapacitated versus capacitated sperm, indicating a rearrangement of these proteins on sperm surface during or following capacitation. Surprisingly, rec-BSPH2 was unable to bind phosphorylcholine liposomes or promote sperm capacitation. It is the first time that such results are reported for proteins of the BSP family. The results indicate that murine BSPH1 and BSPH2 might not have redundant functions, as is the case with bovine BSPs. This study could lead to a better understanding of the role of BSP proteins in sperm functions and the existence of redundant BSP proteins in the reproductive tract.

Obesity becomes pandemic, predisposing these individuals to great risk for lung injury. In this review, we focused on the anti-inflammatories and addressed the following aspects: adipocytokines and obesity, inflammation and other mechanisms, adipocytokines and lung injury in obesity bridged by inflammation, and potential therapeutic targets. To sum up, the majority of evidence supported that adiponectin, omentin, and secreted frizzled-related protein 5 (SFRP5) were reduced significantly in obesity, which is associated with increased inflammation, indicated by increase of TNF α and IL-6, through activation of toll-like receptor (TLR4) and nuclear factor light chain κ B (NF- κ B) signaling pathways. Administration of these adipocytokines promotes weight loss and reduces inflammation. Zinc-α 2-glycoprotein (ZAG), vaspin, IL-10, interleukin-1 receptor antagonist (IL-1RA), transforming growth factor β (TGF-β1), and growth differentiation factor 15 (GDF15) are also regarded as anti-inflammatories. There were controversial reports. Furthermore, there is a huge lack of studies for obesity related lung injury. The effects of adiponectin on lung transplantation, asthma, chronic obstructive pulmonary diseases (COPD), and pneumonia were anti-inflammatory and protective in lung injury. Administration of IL-10 agonist reduces mortality of acute lung injury in rabbits with acute necrotizing pancreatitis, possibly through inhibiting proinflammation and strengthening host immunity. Very limited information is available for other adipocytokines.

Drosophila seminal fluid proteins elicit physiological and behavioral changes in the female after mating. For example, the seminal protein sex peptide (SP) causes females to lay more eggs, reduce receptivity to re-mating, consume more food and produce more concentrated excreta upon mating. It has been reported that SP indirectly increases food consumption as a result of its stimulation of egg production, but its role in producing more concentrated excreta in the mated female was reported to be independent of egg production. Additionally, it has been shown that SP's effect on food consumption persists for several days after mating, while it is unknown whether this is true for its effect on excretion. SP can have both transient and long-term effects on mated females; the latter occur because of the peptide's binding to, and slow release from, sperm in the female. Here we used timed measures of excretion by female flies that had mated to males mutant in SP or in its regulators, to test the duration of SP's effect on excretion. We found that SP's effect on excretion persists for at least ~1 week after mating, and that this persistence requires that SP bind to and be released from sperm. Although these binding/release requirements of SP are similar to those for increased egg production (and consequent increased food intake) following mating, we find that the long-term change in excretion phenotype is only partially dependent on the presence of eggs in the female. Our data indicate that a change in intestinal transit is part of the long-term post-mating response elicited by the gradual release of sperm-bound SP in the female after mating, even though it is not fully dependent on other long-term responses elicited by SP., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Glycosaminoglycans (GAGs) have crucial roles in cell-cell interaction and communication. The communication between sperm and egg during fertilization is the finest example of intercellular communication involving a protein-carbohydrate recognition system. GAGs, especially heparin, are implicated in various processes, such as capacitation, acrosome reaction (AR), and sperm nuclei decondensation by interacting with a wide range of proteins, leading to fertilization. Seminal plasma (SP) comprises of multiple proteins that bind to heparin and related GAGs. Heparin binding proteins (HBPs) originating from secretions of the male accessory sex glands are known to play a vital role during fertilization events. They interact with GAGs present in the female genital tract and enhance the subsequent zona pellucida-induced AR, and thus have been correlated with fertility in some species. Several carbohydrate and zona pellucida-binding proteins, many of which belong to the spermadhesin family, are identified as HBPs. Many studies have been documented about the potential physiological role of some HBPs in various steps of fertilization. However, there is insufficient knowledge about functions executed by various HBPs and their exact mechanism and pathways. An in-depth knowledge of HBPs and their role in fertilization is of fundamental importance to resolve biological pathways and protein interactions at the molecular level. This review surveys some of the relevant findings supporting the potential role of heparin and HBPs in reproduction. It also describes consensus heparin binding sites emerging from known literature on HBPs that interact with heparin.

Females frequently mate with several males, whose sperm then compete to fertilize available ova. Sperm competition represents a potent selective force that is expected to shape male expenditure on the ejaculate. Here, we review empirical data that illustrate the evolutionary consequences of sperm competition. Sperm competition favors the evolution of increased testes size and sperm production. In some species, males appear capable of adjusting the number of sperm ejaculated, depending on the perceived levels of sperm competition. Selection is also expected to act on sperm form and function, although the evidence for this remains equivocal. Comparative studies suggest that sperm length and swimming speed may increase in response to selection from sperm competition. However, the mechanisms driving this pattern remain unclear. Evidence that sperm length influences sperm swimming speed is mixed and fertilization trials performed across a broad range of species demonstrate inconsistent relationships between sperm form and function. This ambiguity may in part reflect the important role that seminal fluid proteins (sfps) play in affecting sperm function. There is good evidence that sfps are subject to selection from sperm competition, and recent work is pointing to an ability of males to adjust their seminal fluid chemistry in response to sperm competition from rival males. We argue that future research must consider sperm and seminal fluid components of the ejaculate as a functional unity. Research at the genomic level will identify the genes that ultimately control male fertility.

Three Binder of SPerm proteins (BSP1, BSP3, BSP5) are secreted by bovine seminal vesicles into seminal plasma and adsorbed onto sperm. When sperm inseminated into the female reach the oviduct, the BSP proteins bind them to its epithelial lining, forming a sperm storage reservoir. Previously, we reported that binding of capacitated sperm to oviductal epithelium in vitro is lower than that of uncapacitated sperm and we proposed that reduced binding was due to loss of BSP proteins during capacitation. Because of differences in amino acid sequences, we predicted that each BSP would respond differently to capacitating conditions. To test whether all three BSP proteins were lost from sperm during capacitation and whether the kinetics of loss differed among the three BSP proteins, ejaculated bull sperm were incubated under various capacitating conditions, and then the amounts of BSP proteins remaining on the sperm were assayed by Western blotting. Capacitation was assayed by analysis of protein tyrosine phosphorylation. While loss of BSP1 was not detected, most of the BSP5 was lost from sperm during incubation in TALP medium, even without addition of the capacitation enhancers heparin and dbcAMP-IBMX. Surprisingly, a smaller molecular mass was detected by anti-BSP3 antibodies in extracts of incubated sperm. Its identity was confirmed as BSP3 by mass spectrometry, indicating that BSP3 undergoes modification on the sperm surface. These changes in the composition of BSP proteins on sperm could play a role in releasing sperm from the storage reservoir by modifying sperm interactions with the oviductal epithelium.

SPACA1 is a membrane protein that localizes in the equatorial segment of spermatozoa in mammals and is reported to function in sperm-egg fusion. We produced a Spaca1 gene-disrupted mouse line and found that the male mice were infertile. The cause of this sterility was abnormal shaping of the sperm head reminiscent of globozoospermia in humans. Disruption of Spaca1 led to the disappearance of the nuclear plate, a dense lining of the nuclear envelope facing the inner acrosomal membrane. This coincided with the failure of acrosomal expansion during spermiogenesis and resulted in the degeneration and disappearance of the acrosome in mature spermatozoa. Thus, these findings clarify part of the cascade leading to globozoospermia.

Male mosquitoes transfer seminal fluid proteins (hereafter 'SFPs') during mating. These proteins can have profound effects on female behavior in the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. SFPs are thought to be responsible for female refractoriness to mating in both species. However, only limited information is available about the duration of induced refractoriness or the quantity of SFPs required to be effective in Ae. albopictus. Here, we tested the duration of the effect of SFPs on female refractory behavior for both Aedes species. Additionally, we determined the lowest SFP dose required to induce female refractory behavior in Ae. aegypti. Virgin females were injected intra-thoracically with doses ranging from 0.25 to 0.008 equivalents of one male's SFP amount. Our results demonstrate high sensitivity of female Ae. aegypti and Ae. albopictus to SFPs of their own species, with the majority of females becoming refractory at doses ≥ 0.031 male-equivalents after injection into the hemocoel. This effect was long-lasting in both species; none of the injected females were inseminated when presented with males of their own species 30 to 34 days post-injection, whereas most saline-injected control females mated at this time point. These results will aid future work to characterize individual SFPs involved in post-mating refractoriness in these two species. Moreover, they show that as is the situation in the mosquito Anopheles gambiae, and unlike Drosophila melanogaster, sperm are not required for the maintenance of a sexual refractoriness response in Ae. aegypti and Ae. albopictus., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The biochemical composition of seminal plasma (SP) is very complex and variable among species. Advances in reproductive technologies reveal SP as a nutritive-protective medium for sperm cells suspended in it, and some SP components are very important for sperm metabolism, as well as sperm function, survival, and transport in the female reproductive tract. Biochemistry of SP is a relatively modern but rapidly expanding field of research, particularly concerning the biological significance of the various biochemical constituents of SP. However, identification of active factors in SP and mechanisms by which they act are not sufficiently understood in different species. Especially in camelids, the origin, composition, and function of the viscous component of SP remain a mystery, and only some biochemical and morphological characteristics of SP have been described. In this review, we focus on the current understanding of physiological and biochemical properties of SP in ruminants and camelids.

Seminal plasma (SP) is known to play an important role in mammalian fertilization. However, the variability found in its composition among species, males and even fractions of the same ejaculate has made difficult to completely understand its effect in sperm function. Proteins are one of the major SP components that modulate sperm functionality. During the last years, intensive work has been performed to characterize the role of these proteins. They have been found to influence sperm capacitation, formation of the oviductal sperm reservoir and sperm-oocyte interaction. Sperm biotechnologies, such as sperm cryopreservation and flow cytometric sex-sorting, that involve a substantial dilution of the SP are detrimental to sperm quality. Attempts to improve the outcome of these biotechnologies include the restoration of SP, which has produced contradictory results. To overcome this variability, different research groups have proposed the application of isolated SP proteins. Herein, we will review the current knowledge in the role of the major SP proteins as modulators of sperm functionality. Furthermore, we will discuss the possible applications of the SP proteins in sperm cryopreservation and flow cytometric sex-sorting., (© 2012 Blackwell Verlag GmbH.)

Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/or openers of these ion channels could interfere with sperm function. In this study, in vivo electroporation (EP) and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower (P<0.05) hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca(2+) peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hyperactivation.

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity., (2011 Elsevier Inc. All rights reserved.)

Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral postmating changes in females. These changes include decreasing receptivity to remating; affecting sperm storage parameters; increasing egg production; and modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have antimicrobial functions and induce expression of antimicrobial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the postmating processes of female insects.

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.

The importance of white adipose tissue in the control of energy balance is now firmly recognized. In addition to fuel storage, adipocytes secrete an array of proteins factors (adipokines), which regulate multiple physiological and metabolic processes as well as influence body fat accumulation. Zinc-α2-glycoprotein (ZAG), a lipid mobilizing factor initially characterized as a tumor product associated with cachexia, has recently been identified as a novel adipokine. Although the exact role of ZAG in adipose tissue remains to be clarified, there is evidence that ZAG expression appears to be inversely related to adiposity, being upregulated in cachexia whereas reduced in obesity. Investigations on the regulation of ZAG give insights into its potential function in adipose tissue with a link to lipid mobilization and an anti-inflammatory action. Recent work shows that ZAG stimulates adiponectin secretion by human adipocytes. Data from genetic studies suggest that ZAG may be a candidate gene for body weight regulation; this is supported by the demonstration that ZAG-knockout mice are susceptible to weight gain, whereas transgenic mice overexpressing ZAG exhibit weight loss. The present review summarizes these new perspectives of ZAG and the potential mechanisms by which it might modulate adipose tissue mass and function.

Objective: To explore the effects of zinc-alpha2-glycoprotein (ZAG) on body weight and body fat in high-fat-diet (HFD)-induced obesity in mice and the possible mechanism., Methods: Thirty-six male mice were fed with standard food (SF) (n = 9) and HFD (n = 27), respectively. Five weeks later, 9 mice fed with HFD were subjected to ZAG expression plasmid DNA transfection by liposome transfection method, and another 9 mice to negative control plasmid transfection. Two weeks later, serum ZAG level in the mice was assayed by Western blot, and the effects of ZAG over-expression on body weight, body fat, serum biochemical indexes, and adipose tissue of obese mice were evaluated. The mRNA expressions of fatty acid synthase (FAS) and hormone-sensitive lipase (HSL) in liver tissue were determined by reverse transcription-polymerase chain reaction., Results: Serum ZAG level significantly lowered in simple HFD-fed mice in comparison to SF-fed mice (0.51 +/- 0.10 AU vs. 0.75 +/- 0.07 AU, P < 0.01). Further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r = -0.56, P < 0.001), epididymal fat mass (r = -0.67, P < 0.001), percentage of epididymal fat (r = -0.65, P < 0.001), and increased weight (r = -0.57, P < 0.001) in simple SF- and HFD-fed mice. ZAG over-expression in obese mice reduced body weight and the percentage of epididymal fat. Furthermore, FAS mRNA expression decreased (P < 0.01) and HSL mRNA expression increased (P < 0.001) in the liver in ZAG over-expressing mice., Conclusions: ZAG is closely related to obesity. Serum ZAG level is inversely correlated with body weight and percentage of body fat. The action of ZAG is associated with reduced FAS expression and increased HSL expression in the liver of obese mice.

We performed a series of comparative studies of bull and stallion seminal plasma (SP) and its role on sperm-neutrophil binding as well as the interaction between semen extender and seminal DNase. Because of contrasting roles of SP on sperm-neutrophil binding between horses and cattle, it was suspected there were some species-specific differences on sperm interaction with SP proteins due to the variations in the natural location of semen deposition (uterus compared to vagina). Bull frozen-thawed sperm removed from egg yolk extender showed similar results to fresh sperm, but this also caused extensive sperm agglutination unless SP or egg yolk was included. If similar agglutination occurs after AI with frozen bull semen, it could interfere with sperm transport or sperm functions. Commonly used bull semen extenders were poor media for seminal DNase activity on plasmid DNA degradation, raising the prospect that the same may be true with other SP factors important to fertility. DNase activity per mg SP protein of bulls was less than that of horses (P<0.05), but DNase activity associated with bull sperm was greater (P<0.05) indicating a different affinity of DNase to spermatozoa. This could be related to the fact that bull sperm naturally migrate from the vagina to the uterus leaving the bulk of SP behind. In such migration, sperm cells needed to carry DNase and other SP factors along. Incorporation of egg yolk in bull semen and introducing SP into the uterus of cattle with current AI protocols may contribute to reduced fertility. Modifications of semen extender and/or semen processing should be examined to allow sperm cells a maximum potential for fertilization., (2010 Elsevier B.V. All rights reserved.)

Background: Previously, we documented the presence of ovulation-inducing factor (OIF) in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s) in seminal plasma responsible for inducing ovulation., Methods: In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group) were treated i.m. with whole seminal plasma (positive control), phosphate-buffered saline (negative control), or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or < 5 kDa. In Experiment 2, female llamas (n = 7 per group) were given an i.m. dose of seminal plasma treated previously by: 1) enzymatic digestion with proteinase-K, 2) incubation with charcoal-dextran, 3) heating to 65 degrees C, or 4) untreated (control). In Experiment 3, female llamas (n = 10 per group) were given an i.m. dose of pronase-treated or non-treated (control) seminal plasma. In all experiments, llamas were examined by transrectal ultrasonography to detect ovulation and CL formation. Ovulation rate was compared among groups by Fisher's exact test and follicle and CL diameters were compared among groups by analyses of variance or student's t-tests., Results: In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each), but none ovulated in the other groups (P < 0.001). In Experiment 2, ovulations were detected in all llamas in each treatment group; i.e., respective treatments of seminal plasma failed to inactivate the ovulation-inducing factor. In Experiment 3, ovulations were detected in 0/10 llamas given pronase-treated seminal plasma and in 9/10 controls (P < 0.01)., Conclusions: We conclude that ovulation-inducing factor (OIF) in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.

Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipient's optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined.

In mammals, sperm ascension within the female reproductive tract involves a transient adhesion to the caudal isthmus of the oviduct. Sperm adhesion to this specialized region, which is termed the "oviductal reservoir", extends the sperm fertile life span by delaying capacitation until, around ovulation, specific signals induce sperm release. In vivo and in vitro studies demonstrated that carbohydrates on the oviductal cell apical membranes and lectin-like molecules on the rostral sperm surface are involved in adhesion in a species-specific way. In this respect, the most intensely studied species are pigs and cattle. On the other hand, less is known about molecules involved in sperm release. Direct evidence that molecules present in the oviductal fluid trigger the release of sperm bound to in vitro cultured oviductal epithelium has been provided only in cattle. However, the identity of sperm and/or oviductal molecules that respond to these releasing signals is still unknown. The comprehension of molecular mechanisms underlying sperm-oviduct interaction may advance our understanding of the behavior of sperm within the female reproductive tract and provide new tools for sperm selection, extension of fertile life and modulation of capacitation in the field of reproductive biotechnologies. The aim of the present paper is to review the available knowledge on molecules involved in sperm selection, storage and release from the oviductal reservoir., (2010 Elsevier Inc. All rights reserved.)

The effect of supplementation of sex-sorted and non-sorted spermatozoa with seminal plasma protein (SPP) on fertility after cervical insemination was examined in the present study. Spermatozoa were sorted into high purity X and Y chromosome-bearing spermatozoa or not sorted and then either supplemented with SPP (>10 kDa) before freezing and/or after thawing (non-sorted only) or processed without supplementation. Inseminations were performed over 2 days with ewes receiving 100 or 25 million motile non-sorted spermatozoa in the cervix or uterus, respectively, or two cervical inseminations of 3.5 million motile sorted spermatozoa. Pregnancy rates in cervically inseminated ewes were unaffected by supplementation of sorted or non-sorted spermatozoa with SPP before freezing compared with no supplementation. The effect of post-thaw supplementation of non-sorted spermatozoa with SPP on pregnancy rates after cervical insemination varied with the day of insemination (P < 0.05); fertility was similar to laparoscopic insemination on Day 1 (56.0 +/- 10.2% v. 58.6 +/- 10.1%), but not on Day 2 (23.1 +/- 7.4% v. 66.7 +/- 9.2%). In conclusion, under the conditions of the present study, SPP did not consistently improve pregnancy rates after cervical insemination with frozen-thawed ram spermatozoa. This is the first report of pregnancies (5/56 ewes inseminated) after cervical insemination with frozen-thawed sex-sorted ram spermatozoa. Although the success rate is low, the findings are encouraging because ewes inseminated with the sex-sorted spermatozoa received only 7% of the recommended dose (100 million motile) for cervical insemination of frozen-thawed spermatozoa.

Competing Interests: The author has declared that no competing interests exist.

PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma that is able to preserve, in vitro, the viability, motility and mitochondrial activity of highly-extended boar spermatozoa. However, a relationship between the protective effects of the heterodimer and sperm capacitation is still unclear. The present study investigated the effect of the PSP-I/PSP-II (1.5 mg/mL) on membrane stability, intracellular calcium concentration ([Ca(2+)](I)) and plasma membrane and acrosome integrity of highly extended boar spermatozoa. Boar spermatozoa were diluted to 1 x 10(6) spermatozoa/mL and incubated at 38 degrees C in Phosphate-buffered saline (PBS) for 10, 30, 60, 120 and 300 min or in modified Tris-buffered medium (mTBM) for 10, 20, 30, 60 and 120 min. After each incubation time, the membrane stability (using Merocyanine-540/Yo-Pro-1), elevation of [Ca(2+)](I) (using Fluo-3-AM/PI) and the sperm plasma membrane and acrosome integrity (using SYBR-14/PI/PE-PNA) were evaluated by flow cytometry. As expected, exposure of the spermatozoa to the PSP-I/PSP-II preserved the plasma membrane and acrosome integrity compared to non-exposed spermatozoa in both media PBS and mTBM (p < .01). The evaluation of membrane stability showed no differences in the percentages of viable sperm with instable plasma membrane in the presence of the PSP-I/PSP-II compared to controls irrespective of the dilution media. The evaluation of the [Ca(2+)](I) levels showed that while spermatozoa incubated in mTBM and exposed to PSP-I/PSP-II had lower [Ca(2+)](I) than controls (39.08% vs. 47.97%, respectively; p < .05), no differences were observed in those samples incubated in PBS. However, a temporal evaluation of the samples showed that a similar proportion of live spermatozoa were able to achieve high levels of [Ca(2+)](I) and membrane instability independent of the presence of PSP-I/PSP-II. In conclusion, PSP-I/PSP-II exert a non-permanent decapacitation effect on highly extended boar spermatozoa that is related with a delay in the increase of [Ca(2+)](I) levels.

Context: Zinc-alpha2-glycoprotein (ZAG) was found to influence lipolysis in adipose tissue and has recently been proposed as a candidate factor in the regulation of body weight., Objective: To elucidate the association of serum ZAG level with body weight and percentage of body fat in normal, obese subjects and high-fat diet (HFD)-induced obese mice., Design: The relationship between serum ZAG and obesity-related parameters was studied in 44 human subjects and 36 mice fed standard food and HFD. Furthermore, the effects of ZAG overexpression on adipose tissue of mice was also evaluated by using a liposome transfection method., Results: Serum ZAG level was significantly lower in obese patients and obese mice in comparison to that in people and mice with normal weight. The further statistical analysis demonstrated that ZAG level was negatively correlated with body weight (r=-0.62, P<0.001), body mass index (r=-0.64, P<0.001), waist circumference(r=-0.68, P<0.001), hip circumference (r=-0.60, P<0.001), percentage of body fat (r=-0.52, P=0.03) and fat mass(r=-0.59, P=0.01) in human subjects after adjustment for age and sex. Furthermore, ZAG overexpression in mice reduced body weight and the percentage of epididymal fat. The decreased FAS, ACC1 and DGAT mRNA and the increased HSL mRNA were also observed in epididymal adipose tissue in ZAG overexpression mice., Conclusion: ZAG is closely linked to obesity. Serum ZAG level is inversely associated with body weight and percentage of body fat. The action of ZAG is associated with downregulated lipogenic enzymes and upregulated lipolytic enzyme expressions in adipose tissue of mice.