Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions.

Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma.

BACKGROUND: Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). METHODS: Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90ß was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. RESULTS: Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90ß differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90ß-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04-98.50%). CONCLUSIONS: This study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.

Small and Large Extracellular Vesicles of Porcine Seminal Plasma Differ in Lipid Profile.

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

Seminal Extracellular Vesicles and Their Involvement in Male (In)Fertility: A Systematic Review.

Seminal plasma contains numerous extracellular vesicles (sEVs). Since sEVs are apparently involved in male (in)fertility, this systematic review focused on studies specifically investigating such relationship. Embase, PubMed, and Scopus databases were searched up to 31 December 2022, primarily identifying a total of 1440 articles. After processing for screening and eligibility, 305 studies were selected as they focused on sEVs, and 42 of them were considered eligible because they included the word fertility or a related word such as infertility, subfertility, fertilization, and recurrent pregnancy loss in the title, objective(s), and/or keywords. Only nine of them met the inclusion criteria, namely (a) conducting experiments aimed at associating sEVs with fertility concerns and (b) isolating and adequately characterizing sEVs. Six studies were conducted on humans, two on laboratory animals, and one on livestock. The studies highlighted some sEV molecules, specifically proteins and small non-coding RNAs, that showed differences between fertile and subfertile or infertile males. The content of sEVs was also related to sperm fertilizing capacity, embryo development, and implantation. Bioinformatic analysis revealed that several of the highlighted sEV fertility-related proteins would be cross-linked to each other and involved in biological pathways related to (i) EV release and loading and (ii) plasma membrane organization.

The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation.

Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p < 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SRF. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility.

Seminal Plasma: Relevant for Fertility?

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA-the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model.

A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify-in parallel-genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14-15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.

The Transcriptome of Pig Spermatozoa, and Its Role in Fertility.

In the study presented here we identified transcriptomic markers for fertility in the cargo of pig ejaculated spermatozoa using porcine-specific micro-arrays (GeneChip® miRNA 4.0 and GeneChip® Porcine Gene 1.0 ST). We report (i) the relative abundance of the ssc-miR-1285, miR-16, miR-4332, miR-92a, miR-671-5p, miR-4334-5p, miR-425-5p, miR-191, miR-92b-5p and miR-15b miRNAs, and (ii) the presence of 347 up-regulated and 174 down-regulated RNA transcripts in high-fertility breeding boars, based on differences of farrowing rate (FS) and litter size (LS), relative to low-fertility boars in the (Artificial Insemination) AI program. An overrepresentation analysis of the protein class (PANTHER) identified significant fold-increases for C-C chemokine binding (GO:0019957): CCR7, which activates B- and T-lymphocytes, 8-fold increase), XCR1 and CXCR4 (with ubiquitin as a natural ligand, 1.24-fold increase), cytokine receptor activity (GO:0005126): IL23R receptor of the IL23 protein, associated to JAK2 and STAT3, 3.4-fold increase), the TGF-receptor (PC00035) genes ACVR1C and ACVR2B (12-fold increase). Moreover, two micro-RNAs (miR-221 and mir-621) were down- and up-regulated, respectively, in high-fertility males. In conclusion, boars with different fertility performance possess a wide variety of differentially expressed RNA present in spermatozoa that would be attractive targets as non-invasive molecular markers for predicting fertility.

Achievements and future perspectives of embryo transfer technology in pigs.

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.

Cryopreservation Differentially Alters the Proteome of Epididymal and Ejaculated Pig Spermatozoa.

Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.

New In-Depth Analytical Approach of the Porcine Seminal Plasma Proteome Reveals Potential Fertility Biomarkers.

A complete characterization of the proteome of seminal plasma (SP) is an essential step to understand how SP influences sperm function and fertility after artificial insemination (AI). The purpose of this study was to identify which among characterized proteins in boar SP were differently expressed among AI boars with significantly different fertility outcomes. A total of 872 SP proteins, 390 of them belonging specifically to Sus Scrofa taxonomy, were identified (Experiment 1) by using a novel proteomic approach that combined size exclusion chromatography and solid-phase extraction as prefractionation steps prior to Nano LC-ESI-MS/MS analysis. The SP proteomes of 26 boars showing significant differences in farrowing rate (n = 13) and litter size (n = 13) after the AI of 10 526 sows were further analyzed (Experiment 2). A total of 679 SP proteins were then quantified by the SWATH approach, where the penalized linear regression LASSO revealed differentially expressed SP proteins for farrowing rate (FURIN, AKR1B1, UBA1, PIN1, SPAM1, BLMH, SMPDL3A, KRT17, KRT10, TTC23, and AGT) and litter size (PN-1, THBS1, DSC1, and CAT). This study extended our knowledge of the SP proteome and revealed some SP proteins as potential biomarkers of fertility in AI boars.

Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study.

Owing to the quick genetic turnover of the pig industry, most AI-boar sires live 2-3 yr, a period during which for 1-2 yr their semen is extended and used in liquid form for AI. Despite showing low cryosurvival, affecting fertility after AI, boar semen is frozen for easiness of transport overseas and reposition of valuable genetics. For the latter, semen is stored in liquid nitrogen (LN2, cryostorage) for many years, a controversial practice. Here we studied how length of cryostorage could affect sperm quality. Straws (0.5 mL) frozen using the same cryopreservation protocol at one specific location from AI- sires of proven fertility were stored in LN2 for up to 8 yr. Post-thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, always compared to early thawing (15 d after freezing). Sperm motility and kinematics were evaluated post-thaw using CASA and sperm viability was cytometrically evaluated using specific fluorophores. Sperm viability was not affected by length of cryostorage, but total and progressive sperm motility were lower (p < 0.01) in sperm samples cryostored for 4 or 8 yr compared to those thawed 15 d after freezing. Cryostorage time affected sperm kinetics, but with greater intensity in the samples cryostored for 4 yr (p < 0.001) than in those for 2 yr (p < 0.01). The fact that the major phenotypic characteristic of boar spermatozoa, motility, is constrained by time of cryostorage should be considered when building cryobanks of pig semen. Attention should be placed on the finding that >2 yr of cryostorage time can be particularly detrimental for the post-thaw motility of some sires, which might require increasing sperm numbers for AI.

Active paraoxonase 1 is synthesised throughout the internal boar genital organs.

The paraoxonase type 1 (PON1) is an enzyme with antioxidant properties recently identified in the seminal plasma (SP) of several species, including the porcine. The aims of the present study were to (1) describe the immunohistochemical localisation of PON1 in the genital organs of fertile boars and (2) evaluate the relationship among PON1 activity and high-density lipoprotein cholesterol (HDL-C) concentration in fluids of the boar genital organs. Immunohistochemical analysis demonstrated that PON1 was present in testis (specifically in Leydig cells, blood vessels, spermatogonia and elongated spermatids), epididymis (specifically in the cytoplasm of the principal epithelial cells, luminal secretion and in the surrounding smooth muscle) and the lining epithelia of the accessory sexual glands (cytoplasmic location in the prostate and membranous in the seminal vesicle and bulbourethral glands). The Western blotting analysis confirmed the presence of PON1 in all boar genital organs, showing in all of them a band of 51 kDa and an extra band of 45 kDa only in seminal vesicles. PON1 showed higher activity levels in epididymal fluid than those in SP of the entire ejaculate or of specific ejaculate portions. A highly positive relationship between PON1 activity and HDL-C concentration was found in all genital fluids. In sum, all boar genital organs contributing to sperm-accompanying fluid/s were able to express PON1, whose activity in these genital fluids is highly dependent on the variable HDL-C concentration present.

Developmental competence of porcine genome-edited zygotes.

Genome editing in pigs has tremendous practical applications for biomedicine. The advent of genome editing technology, with its use of site-specific nucleases-including ZFNs, TALENs, and the CRISPR/Cas9 system-has popularized targeted zygote genome editing via one-step microinjection in several mammalian species. Here, we review methods to optimize the developmental competence of genome-edited porcine embryos and strategies to improve the zygote genome-editing efficiency in pigs.

Seminal plasma affects sperm sex sorting in boars.

Two experiments were conducted in boar semen samples to evaluate how both holding time (24h) and the presence of seminal plasma (SP) before sorting affect sperm sortability and the ability of sex-sorted spermatozoa to tolerate liquid storage. Whole ejaculate samples were divided into three aliquots immediately after collection: one was diluted (1:1, v/v) in Beltsville thawing solution (BTS; 50% SP); the SP of the other two aliquots was removed and the sperm pellets were diluted with BTS + 10% of their own SP (10% SP) or BTS alone (0% SP). The three aliquots of each ejaculate were divided into two portions, one that was processed immediately for sorting and a second that was sorted after 24h storage at 15-17°C. In the first experiment, the ability to exhibit well-defined X- and Y-chromosome-bearing sperm peaks (split) in the cytometry histogram and the subsequent sorting efficiency were assessed (20 ejaculates). In contrast with holding time, the SP proportion influenced the parameters examined, as evidenced by the higher number of ejaculates exhibiting split and better sorting efficiency (P<0.05) in semen samples with 0-10% SP compared with those with 50% SP. In a second experiment, the quality (viability, total and progressive motility) and functionality (plasma membrane fluidity and intracellular generation of reactive oxygen species) of sex-sorted spermatozoa were evaluated after 0, 72 and 120h storage at 15-17°C (10 ejaculates). Holding time and SP proportion did not influence the quality or functionality of stored sex-sorted spermatozoa. In conclusion, a holding time as long as 24h before sorting did not negatively affect sex sorting efficiency or the ability of sorted boar spermatozoa to tolerate long-term liquid storage. A high proportion of SP (50%) in the semen samples before sorting reduced the number of ejaculates to be sorted and negatively influenced the sorting efficiency, but did not affect the ability of sex-sorted spermatozoa to tolerate liquid storage.

Measurement of activity and concentration of paraoxonase 1 (PON-1) in seminal plasma and identification of PON-2 in the sperm of boar ejaculates.

This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = -0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = -0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = -0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters.

The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa.

BACKGROUND: Sex allocation of offspring in mammals is usually considered as a matter of chance, being dependent on whether an X- or a Y-chromosome-bearing spermatozoon reaches the oocyte first. Here we investigated the alternative possibility, namely that the oviducts can recognise X- and Y- spermatozoa, and may thus be able to bias the offspring sex ratio. RESULTS: By introducing X- or Y-sperm populations into the two separate oviducts of single female pigs using bilateral laparoscopic insemination we found that the spermatozoa did indeed elicit sex-specific transcriptomic responses. Microarray analysis revealed that 501 were consistently altered (P-value < 0.05) in the oviduct in the presence of Y-chromosome-bearing spermatozoa compared to the presence of X-chromosome-bearing spermatozoa. From these 501 transcripts, 271 transcripts (54.1%) were down-regulated and 230 transcripts (45.9%) were up-regulated when the Y- chromosome-bearing spermatozoa was present in the oviduct. Our data showed that local immune responses specific to each sperm type were elicited within the oviduct. In addition, either type of spermatozoa elicits sex-specific signal transduction signalling by oviductal cells. CONCLUSIONS: Our data suggest that the oviduct functions as a biological sensor that screens the spermatozoon, and then responds by modifying the oviductal environment. We hypothesize that there might exist a gender biasing mechanism controlled by the female.

Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity.

The constitutive 70  kDa heat-shock protein, HSPA8, has previously been shown to contribute to the long-term survival of spermatozoa inside the mammalian female reproductive tract. Here, we show that a recombinant form of HSPA8 rapidly promotes the viability of uncapacitated spermatozoa, the ability of spermatozoa to bind to oviductal epithelial cells, enhances IVF performance, and decreases sperm mitochondrial activity. Fluorescence recovery after photobleaching revealed that the repair of membrane damage is achieved by an almost instantaneous increase in sperm membrane fluidity. The ability of HSPA8 to influence membrane stability and fluidity, as well as its conserved nature among mammalian species, supports the idea that this protein protects sperm survival through membrane repair mechanisms. Free Persian abstract A Persian translation of the abstract is freely available online at http://www.reproduction-online.org/content/147/5/719/suppl/DC1.

Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate.

Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24h at 15-17°C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.