Spermatozoa and seminal plasma proteomics: Too many molecules, too few markers. The case of bovine and porcine semen.

The study of the protein composition of semen (i.e., spermatozoa and seminal plasma) is not new. However, with development of proteomics technologies, our understanding of the roles of cellular and fluid proteins has expanded enormously. Today, several seminal proteins have already been suggested as biomarkers associated with semen traits (e.g., sperm motility and integrity) and fertility. Also, many others were associated with infertility, being identified in humans and domestic animals with poor semen quality (e.g., oligozoospermia) and fertility impairment. These proteins not only might explain the causes of fail in fertilization but also have potential as diagnostic tools, improving traditional semen analyses. However, despite characterization of thousands of seminal proteins, to date, few commercial kits based on protein biomarkers are available. In this article, not only the advances and advantages of semen proteomics will be discussed, but also limitations in its application in a commercial AI centre.

Post-thaw dilution of Rhamdia quelen sperm improves the reproductive success.

The aim was to evaluate the effect of a post-thaw dilution of Rhamdia quelen sperm in 1.1% NaCl (325 mOsm kg-1; pH 7.6; 24 °C) solution on the quality and reproductive capacity. Sperm from eight males were cryopreservation in nitrogen vapor at - 170 °C for 18 h in 0.25 mL straws in a freezing medium containing 5% fructose, 5% Powdered milk, and 10% methanol. The samples were thawed and post-thaw diluted (1:20) in NaCl solution or not (control). The higher spermatozoa velocities were observed in the post-thaw diluted samples (curvilinear (VCL) - 69 ± 11 µm s-1; average path (VAP) - 45 ± 8 µm s-1; straight-line (VSL) - 43 ± 8 µm s-1) compared to the control (VCL - 47 ± 10 µm s-1; VAP - 31 ± 6 µm s-1; VSL - 30 ± 6 µm s-1). Greater straightness (STR), progression (PROG), and beat cross frequency (BCF) were observed in the post-thaw diluted samples (STR - 96 ± 7%; PROG - 666 ± 128 µm; BCF - 42 ± 2 Hz) than in control (STR - 95 ± 5%; PROG - 463 ± 92 µm; BCF - 40 ± 2 Hz). The strongly curled tail was the only morphology change that differ between the post-thaw diluted (5 ± 2%) and control (2 ± 1%). Membrane integrity, mitochondrial activity, and normal larvae rate were not different between treatments. Fertilization and hatching were higher in the post-thaw diluted sperm (93 ± 3%; 82 ± 9%) when compared to control samples (65 ± 13%; 55 ± 17%). Were used oocytes from one female, limiting these results. The post-thaw dilution improved the sperm kinetics and reproductive parameters. Thus, this methodology can be included in the sperm cryopreservation protocol for R. quelen.

The sperm mitochondria: clues and challenges.

Sperm cells rely on different substrates to fulfil thei energy demand for different functions and diverse moments of their life. Species specific mechanism involve both energy substrate transport and their utilization: hexose transporters, a protein family of facilitative passive transporters of glucose and other hexose, have been identified in spermatozoa of different species and, within the species, their localization has been identified and, in some cases, linked to specific glycilitic enzyme presence. The catabolism of hexose sources for energy purposes has been studied in various species, and recent advances has been made in the knowledge of metabolic strategies of sperm cells. In particular, the importance of aerobic metabolism has been defined and described in horse, boar and even mouse spermatozoa; bull sperm cells demonstrate to have a good adaptability and capacity to switch between glycolysis and oxidative phosphorylation; finally, dog sperm cells have been demonstrated to have a great plasticity in energy metabolism management, being also able to activate the anabolic pathway of glycogen syntesis. In conclusion, the study of energy management and mitochondrial function in spermatozoa of different specie furnishes important base knowledge to define new media for preservation as well as newbases for reproductive biotechnologies.

Structural modelling of the equine protein disulphide isomerase A1 and its quantification in the epididymis and seminal plasma.

The protein disulphide isomerase A1 (PDIA1) is an important chaperone involved in protein quality control and redox regulation. Also, the ability of PDIA1 to bind to oestrogens suggests that it may play a role in epididymal maturation and male fertility. The goals of this study were to (a) verify the possible interaction between 17ß-estradiol and equine PDIA1 using bioinformatics; (b) identify and quantify PDIA1 protein in equine cauda epididymis throughout peripuberty; and (c) determine whether the amounts of PDIA1 in equine seminal plasma and spermatozoa are associated with fertility. Using in silico analysis, we were able to predict the tertiary structure of equine PDIA1 and to demonstrate the interaction between 17ß-estradiol and the putative binding site in domains b and b'. Colts under 24 months of age had lower relative amounts of PDIA1 in cauda epididymal fluid in comparison with older males (p < .01). No difference was observed in seminal plasma PDIA1 between fertile and subfertile stallions. Our study demonstrates that PDIA1 expression in the epididymis increases during peripuberty. However, in the adult stallion, its quantity in seminal plasma is not associated with fertility.

Effect of scrotal insulation on sperm quality and seminal plasma proteome of Brangus bulls.

Seminal plasma (SP) contributes to sperm physiology and metabolism, prevents premature capacitation, and protects sperm against oxidative stress. In order to evaluate the impact of heat stress in the semen of tropically adapted Brangus breed and in their seminal plasma proteome, we studied the effects of scrotal insulation for 72 h. Semen samples from six bulls, between 7 and 8 years of age, were collected prior to scrotal insulation (pre-insulation), and at 4 and 11 wk after insulation. Seminal plasma samples were analyzed by 2D SDS-PAGE and liquid chromatography coupled with mass spectrometry (LC-MS/MS). Insulation caused decrease in vigour, gross and total motility after 4 wk of scrotal insult (P < 0.001). Total defects in sperm were higher after 4 wk compared to pre-insulation and 11 wk after scrotal insulation (P < 0.001). The analysis of the 2D protein profile of the SP resulted in the identification 183 unique protein spots in all gels evaluated. There was no difference in mean number of protein spots amongst time points. Eight protein spots were more abundant in SP after scrotal insulation, returning to the same expression level at 11 wk post-insulation. One spot had higher abundance at 11 wk post-insulation, and one spot had decreased abundance 4 wk after insulation. The ten protein spots with differential abundance amongst time points were identified as Seminal plasma protein PDC-109, Seminal plasma protein A3, Seminal plasma protein BSP-30 kDa, Spermadhesin-1 and Metalloproteinase inhibitor 2. The validation of these five proteins as biomarkers for thermal testicular stress in Brangus breed would allow the development of new biotechnologies that could improve bovine semen analysis in breeding systems in tropical and subtropical conditions. A close association between the identified BSP and Spermadhesin-1 was evidenced in protein-protein interaction analysis. Based on gene ontology analysis, variation in sperm function after insulation could be explained by variation in the expressed proteins in the SP. Further studies are required to verify if these proteins could be used as biomarkers for the identification of bulls with increased seminal resistance to heat stress in Brangus breed.

Differential seminal plasma proteome signatures of boars with high and low resistance to hypothermic semen preservation at 5°C.

BACKGROUND: Hypothermic storage at 5°C has been investigated as an alternative to promote the prudent use of antibiotics for boar artificial insemination doses. However, this temperature is challenging for some ejaculates or boars. OBJECTIVE: The present study aimed to identify putative biomarkers for semen resistance to hypothermic storage at 5°C by comparing the seminal plasma proteomes of boars with high and low seminal resistance to preservation at 5°C. MATERIALS AND METHODS: From an initial group of 34 boars, 15 were selected based on the following criteria: ejaculate with ≤20% abnormal spermatozoa and at least 70% progressive motility at 120 hours of storage at 17°C. Then, based on the response to semen hypothermic storage at 5°C, boars were classified into two categories: high resistance-progressive motility of >75% in the three collections (n = 3); and low resistance-progressive motility of <75% in the three collections (n = 3). Seminal plasma proteins were analyzed in pools, and differential proteomics was performed using Multidimensional Protein Identification Technology. RESULTS: Progressive motility was lower at 120 hours of storage in low resistance, compared to high resistance boars (P < .05). Acrosome and plasma membrane integrity were not affected by the boar category, storage time, or their interaction (P ≥ .104). Sixty-five proteins were considered for differential proteomics. Among the differentially expressed and exclusive proteins, the identification of proteins such cathepsin B, legumain, and cystatin B suggests significant changes in key enzymes (eg, metalloproteinases) involved in spermatogenesis, sperm integrity, and fertilizing potential. DISCUSSION AND CONCLUSION: Differences in the seminal plasma suggest that proteins involved in the proteolytic activation of metalloproteinases and proteins related to immune response modulation could disrupt key cellular pathways during spermatogenesis and epididymal maturation, resulting in altered resistance to chilling injury. Further in vivo studies focusing on the immunological crosstalk between epithelial cells and gametes might explain how the immune regulators influence sperm resistance to hipothermic storage.

Ready for the journey: a comparative proteome profiling of porcine cauda epididymal fluid and spermatozoa.

In the present study, we describe the proteome of porcine cauda epididymis fluid and spermatozoa by means of Multidimensional Protein Identification Technology (MudPIT). Ten sexually mature healthy boars were surgically castrated and epididymides were dissected to obtain the cauda epididymal content. Polled protein extracts of cauda epididymal fluid (CEF) and spermatozoa (CESperm) were loaded in an Agilent 1100 quaternary HPLC and peptides eluted from the microcapillary column were electro-sprayed directly into a LTQ Orbitrap XL mass spectrometer. Using bioinformatics, identified proteins were classified by their molecular functions, involvement in biological processes and participation in relevant metabolic pathways associated with spermatozoa physiology, fertility potential and protection. A total of 645 proteins were identified in the CEF, with epididymal-specific lipocalin-5, beta-hexosaminidase subunit beta precursor and phosphatidylethanolamine-binding protein 4 being the most abundant proteins found. A total of 2886 proteins were identified in the CESperm proteome with 81 proteins being considered more abundant (spectral counts > 100). CEF and CESperm data were compared and 345 proteins were present in both proteomes. Phosphatidylethanolamine-binding protein 4 precursor was the only protein found most abundant in both CEF and CESperm proteomes. Based on Gene Ontology analysis, we identified CEF and CESperm proteins associated with sperm protection against ROS and immune mediated response, glycosaminoglycan degradation, ubiquitin-proteasome system, metabolic process and maturation, modulation of acrosome reaction and ZP binding and oocyte penetration. These results provide a better comprehension about the molecular process and biological pathways involved in sperm epididymis maturation and establishment of the cauda epididymis sperm reservoir.

Proteomic identification of boar seminal plasma proteins related to sperm resistance to cooling at 17 °C.

The modern pig industry relies on extensive use of artificial insemination with cooled semen. It is important that semen doses maintain their quality during processing, transport and storage before insemination to guarantee maximum fertility rates. However, ejaculates may respond differently to liquid preservation at 17 °C, despite the optimal quality assessed before cooling. Thus, the aim of this study was to identify differences in seminal plasma proteome of ejaculates with a higher or lower seminal resistance to storage at 17 °C. A total of 148 ejaculates from 65 sexually mature healthy boars were classified as: High Resistance to cooling (HR, total motility > 60% at 144h) and Low resistance to cooling (LR, total motility <60 at 72h). To identify differentially expressed seminal plasma proteins between HR and LR ejaculates, ten ejaculates of each group were analyzed by 2D SDS-PAGE and ESI-Q-TOF mass spectrometry. The proteins associated with HR ejaculates were cathepsin B (spot 2803 and 6601, p < 0.01); spermadhesin PSP-I (spots 3101 and 3103, p < 0.05); epididymal secretory protein E1 precursor (spot 2101, p < 0.05) and IgGFc binding protein (spot 1603, p < 0.01). The protein associated with LR group was the Major seminal plasma PSPI (spot 9103, p < 0.01). To our knowledge, this is the first report of the association of boar seminal plasma proteins to semen resistance to cold storage at 17 °C. These results suggest the use of these proteins as biomarkers for semen resistance to preservation at 17 °C.

Essential oils rich in monoterpenes are unsuitable as additives to boar semen extender.

Despite the development of efficient boar semen extenders, there is still room for improvement of new formulas using new molecules that could increase fertilisation outcomes and substitute cryoprotectants and antibiotics. The goal of this work was to evaluate if the essential oils from the leaves of Myrrhinium atropurpureum and Cymbopogon citratus are suitable as additives in boar semen extender. The major compounds found in the essential oils from M. atropurpureum were 1,8-cineole (37.37%) and terpinolene (19.18%); and geranial (49.8%) and neral (33.24%) in essential oil of C. citratus. The addition of 1% and 0.1% of both essential oils to extended semen had immediate spermicidal effects (p < 0.05). Lower concentrations were tested and no cytotoxic effect was observed when M. atropurpureum essential oil was added at 0.001%. Differently, essential oil from C. citratus reduced sperm motility, membrane functionality and integrity and mitochondrial membrane potential even in concentrations as low as 0.001%. Also, addition of essential oils in low concentrations had no inhibitory effect against Escherichia coli and Pseudomonas aeruginosa growth. We conclude that the essential oils from C. citratus and M. atropurpureum, rich in monoterpenes, are cytotoxic to swine spermatozoa, therefore unsuitable as semen extender additives.

Enzymatic scavengers in the epididymal fluid: comparison between pony and miniature breed stallions.

The use of stallion semen collected from cauda epididymis for AI has increased due to the new protocols available for cryopreservation. Preserving the genetic material from valuable males that suffer sudden death or other events that prematurely end the stallion's reproductive life is an important strategy for Stud breeding management. While protecting spermatozoa from oxidative stress and infectious agents, the epididymis promotes the enhancement of sperm cell morphology and changes in membrane protein profile, increasing its fertility potential. The epididymal fluid must be a balanced redox environment to allow sperm preservation and protein-protein and protein-lipids interactions to quantify. The aim of this study was quantify the enzymatic ROS scavengers in epididymal fluid of pony and miniature breed stallions. Epididymides from 8 pony stallions and 12 miniature breed stallions were dissected and fluid from caput, corpus and cauda epididymis collected. Spermatozoa were separated of epididymal fluid by 2-step centrifugation. The activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured and compared between stallion groups and epididymal regions. The three enzymes were present in all epididymal regions tested, with higher activities of catalase and SOD in cauda epididymis in miniature breed stallions (P<0.05). GPx activity was higher in caput epididymis in pony stallions (P<0.05), however with no difference to fluid from cauda epididymis of both breeds. These results show a difference in antioxidant enzymatic scavengers between pony and miniature breed stallions. Also, our data confirm the protective role of cauda epididymis, preserving spermatozoa integrity from oxidative damage. As glutathione peroxidase is involved in several signaling pathways, its constant activity during epididymal transit corroborates the importance of this enzyme for spermatozoa maturation.

Criopreservação de espermatozóides eqüinos comparando duas curvas de congelamento combinadas com diluentes comerciais: uma análise laboratorial / Cryopreservation of equine spermatozoa comparing different freezing rates combined with commercial extenders: laboratorial analysis

Durante o processo de criopreservação de sêmen, os espermatozóides sofrem alguns danos que resultam na diminuição da fertilidade deste. O presente estudo foi realizado com o objetivo de avaliar os efeitos da utilização combinada de duas curvas de congelamento com dois diluentes comerciais (FR-5® e Botu-Crio®) sobre a criopreservação de sêmen eqüino. Foram analisados 20 ejaculados de dois garanhões. As amostras foram avaliadas por microscopia de contraste de fase e microscopia de epifluorescência, observando-se a motilidade progressiva e total do sêmen pós-descongelamento e a integridade e a funcionalidade da membrana dos espermatozóides. A combinação entre curva automatizada e Botu-Crio® apresentou as maiores médias nas análises de motilidade total e progressiva, após o descongelamento. O diluente Botu-Crio®, isoladamente, preservou também as membranas destes, quando foram realizadas as análises de integridade utilizando teste com diacetato de carboxifluoresceína e iodeto de propídio e funcionalidade de membrana pelo teste hiposmótico.

During semen cryopreservation, sperm cells were submitted to several deleterious events leading to membrane damage which result in fertility decrease. This study was designed to compare the effects of two freezing techniques (conventional and automated), and the use of two commercial extenders as cryoprotectants (FR-5® and Botu-Crio®) on total and progressive motility, integrity and functionality of spermatic membranes during the cryopreservation of equine semen. Twenty ejaculates from two stallions were analyzed. The total and progressive motility of fresh and post-thawing semen samples were evaluated by patterns assays. Function of plasmatic membrane was measured by the hipoosmotic swelling test. Integrity of plasmatic membrane was evaluated using carboxifluorescein diacatate and iodidium propide fluorescent probes. There were significant differences between the two freezing techniques and/or between cryoprotectants for all assessed parameters. The combination of Botu-Crio® and automated curves showed better results on total and progressive post-thawing motility. The extender Botu-Crio®, alone, showed to better preserve the membrane integrity and function.

Apoptose em espermatozóides: uma nova abordagem na avaliação da viabilidade espermática / Apoptosis in spermatozoa: a new approach in sperm viability evaluation

As biotécnicas aplicadas à reprodução animal desenvolvem-se a cada dia. Entretanto, as tecnologias empregadas e estudadas atualmente envolvem processos de grande estresse celular. A criopreservação é a técnica mais utilizada na conservação de gametas, embora esta metodologia ainda não esteja totalmente dominada em algumas espécies. A apoptose vem sendo vista como uma das causas da baixa viabilidade espermática pós-descongelação. A presente revisão aborda novos conceitos da espermatogênese e resultados de pesquisas deste tipo de morte celular em células espermáticas.