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Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

The release of extracellular proteins is a part of the sperm capacitation process; this allows the sperm surface reorganization that enables the sperm to fertilize an oocyte. Some of the components released are 'decapacitation factors', an uncoordinated or early release of which may cause inappropriate surface destabilization and premature capacitation. We studied the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in sperm capacitation, and reported that CD52 and CD55 exhibit bicarbonate-dependent release during in vitro sperm capacitation. Treating sperm with phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the enzymatic cleavage of CD55, in both capacitating and noncapacitating conditions. Moreover, PIPLC treatment in noncapacitating conditions caused surface reorganization events that included exposure of the ganglioside GM1, aggregation of flotillin-1, and the swelling of the apical acrosome region; all of which have been reported to be associated with sperm capacitation. The acrosomal swelling was monitored using wet mount atomic force microscopy, a new imaging technique that allows nanometer-level sperm surface measurements in samples hydrated with physiological buffer rather than dried. Despite these surface changes, PIPLC treatment in identical incubation conditions did not stimulate hyperactive sperm motility or protein tyrosine phosphorylation (other hallmarks of sperm capacitation in vitro). In full capacitating conditions (i.e., the presence of bicarbonate and albumin), PIPLC treatment caused sperm deterioration. The possible role of GPI-APs removal from the sperm surface during sperm capacitation is discussed.

The discovery of induced pluripotent stem cells (iPSCs) has the potential to revolutionize the field of regenerative medicine. In the past few years, iPSCs have been the subject of intensive research towards their application in disease modeling and drug screening. In the future, these cells may be applied in cell therapy to replace or regenerate tissues by autologous transplantation. However, two major hurdles need to be resolved in order to reach the later goal: the low reprogramming efficiency and the safety risks, such as the integration of foreign DNA into the genome of the cells and the tumor formation potential arising from transplantation of residual undifferentiated cells. Recently, aging emerged as one of the barriers that accounts, at least in part, for the low reprogramming efficiency of bona fide iPSCs. Here, we review the molecular pathways linking aging and reprogramming along with the unanswered questions in the field. We discuss whether reprogramming rejuvenates the molecular and cellular features associated with age, and present the recent advances with iPSC-based models, contributing to our understanding of physiological and premature aging.

Melatonin may play an important role in protecting gametes and embryos from the potential harmful effects of oxidative stress. In this study, we first examined two different heat stress (HS) treatments for in vitro oocyte maturation (Experiment 1: 38.5 vs 41.0°C, during the first 20 h; Experiment 2: 38.5 vs 41.5°C, during the entire period) on bovine oocyte maturation and embryo development. Second, we tested different melatonin concentrations added to the maturation and culture medium (Experiment 3: 0, 10(-12) , 10(-9) , 10(-4) m; Experiment 4: 0, 10(-3) m), both with and without HS (38.5 or 41.5°C, respectively). In Experiment 1, the HS treatment resulted in a lower maturation rate and number of cells/blastocyst (C/B) and a higher blastocyst rate than that in the control group. In Experiment 2, oocytes/embryos from heat-stressed oocytes (HSO) had a lower maturation, cleavage and blastocyst rates, as well as a lower C/B compared with the control. In Experiment 3, in HSO groups, 10(-4) m melatonin resulted in an increased blastocyst rate compared with 0 m melatonin, with a similar blastocyst rate to the non-HSO without melatonin. Melatonin did not have any effect in embryos from non-HSO groups compared with the control. In Experiment 4, 10(-3) m melatonin produced lower cleavage and blastocyst rates in HSO and lower blastocyst rate in non-HSO when compared to melatonin-untreated oocytes/embryos. In conclusion, 10(-4) m melatonin was found to alleviate bovine oocytes from the harmful effects of HS.

Semen cryopreservation and artificial insemination (AI) are potentially valuable methods for supporting the breeding management of endangered species like the Asian elephant. Cryopreservation of Asian elephant semen has however proven problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. In this study, nine ejaculates from three adult bulls were used to compare the influence of extender (TEST versus INRA96®) and penetrating cryoprotectants (3% glycerol, 5% glycerol and 4% methylformamide) on post-thaw semen quality. We demonstrate that not only the freezing process, but also the quality of the semen before freezing, significantly influences the freezability of Asian elephant semen. Pre-freeze motility, viability, semen volume, semen pH, sperm concentration and the incidence of sperm mid-piece and tail abnormalities all significantly (p < 0.05) affected post-thaw semen quality. While extender and cryoprotectant did not significantly affect any of the above semen quality parameters post-thaw, the skim-milk based extender (INRA96®) preserved DNA integrity better (p < 0.05) than the egg yolk extender (TEST). Considerable between-ejaculate variation in all post-thaw semen quality parameters was also noted. It is concluded that strict criteria for semen quality is essential for the selection of Asian elephant bull ejaculates suitable for cryopreservation; stricter initial selection should improve the mean post-thaw quality.

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.Keywords: extracellular vesicles; microvesicles; microparticles; exosomes; ectosomes; extracellular RNA(Published: 22 December 2014)Citation: Journal of Extracellular Vesicles 2014, 3: 26913 - http://dx.doi.org/10.3402/jev.v3.26913

BACKGROUND: Validation of a method for the minimally-invasive measurement of physiological stress will help understanding of risk factors that may contribute to stress-associated events including recrudescence of Equid herpesvirus (EHV), which is anecdotally associated with sales consignment of pregnant Thoroughbred mares. In this study we compared two similar groups of late-gestation Thoroughbred broodmares on the same farm: a consigned Sales group (N = 8) and a non-consigned Control group (N = 6). The Sales mares were separated from their paddock companions and grouped prior to their preparation for, transport to, and return from the sales venue. Both groups were monitored by sampling at regular intervals from 5 days prior to until 14 days after the sales date (D0) to measure physiological stress in terms of changes in faecal glucocorticoid metabolite (FGM) concentrations, and for event-related viral recrudescence via daily body temperature measurements and periodic nasal swabs for PCR analysis for EHV-1 and -4 DNA. RESULTS: In both groups, FGM levels increased post-sales before returning to pre-sales levels. Specifically, FGM concentrations in the Sales mares were significantly higher on D + 3 and D + 10 than on D-4 and D-3 (F = 12.03, P

Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients’ exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human. Electronic supplementary material The online version of this article (doi:10.1186/1477-7827-12-117) contains supplementary material, which is available to authorized users.

The aim of this analysis was to determine whether pregnancy loss (PL) after embryo transfer (ET) in cattle was related to maternal progesterone (P4) concentrations during and shortly after ET, and maternal bovine pregnancy-associated glycoprotein-1 (bPAG-1) concentrations in plasma at days 25–35 of gestation. Embryos (n = 260) were produced either in vivo after superovulation (n = 115), or in vitro from oocytes (obtained with ovum pick-up) in co-culture (n = 44) or cultured in a synthetic medium (n = 101). Overall, PL was 56.9% (148) and no significant differences occurred in calving rate among the three embryo production groups. There was no difference in P4 concentrations on days 7–14 of gestation in the three groups, nor between ongoing and interrupted pregnancies. Between days 25 and 35 of pregnancy, bPAG-1 concentrations were unaffected by embryo production, but in cattle that had PL between days 26 and 120, four bPAG-1 profiles could be detected. Between days 25 and 32, bPAG-1 concentrations were influenced by PL, and concentrations were significantly lower in animals in which PL occurred between days 26 and 120 than in those animals that aborted later or calved at term. Early P4 concentrations suggested that maternal luteal factors were not responsible for PL which appeared to be caused by impaired conceptus development (regardless of embryo type) as reflected by low maternal bPAG-1 concentrations prior to embryonic death.

Evaluation of radiographic and genetic aspects of hereditary subluxation of the radial head in Bouviers des Flandres. Summary Objective-To study radiographic and genetic aspects of hereditary radial head subluxation in Bouviers des Flandres. Animals-26 related Bouviers des Flandres affected with bilateral subluxation of the radial head, 10 unaffected related dogs, and 29 unrelated Bouviers des Flandres with diagnoses of nonskeletal diseases. Procedures-All dogs were radiographically studied, and their DNA was analyzed with a genome-wide screen of 1,536 single nucleotide polymorphisms. In addition, karyotyping was performed in an unaffected dam and its affected offspring. Results-Both forelimbs of affected dogs were disproportionately short with caudolateral subluxation or luxation of the radial head. Angulation of the radial axis at the mid-diaphysis ranged from 9.3 degrees to 30.3 degrees (mean +/- SD, 14.9 +/- 6.1 degrees ), with an estimated age of onset from 0 to 4 months. Poorly defined medial coronoid processes and osteoarthritis of the elbow joint, cranial bowing of the olecranon, and disturbed growth in length of the ulna with sharply demarcated spurs were noticed on radiographs of affected dogs. Genealogical analysis indicated that most affected dogs were closely related, but the mode of inheritance was not clear. The DNA analysis found that 205 single nucleotide polymorphisms were monomorphic in the affected dogs. Conventional chromosome staining revealed no numerical chromosomal aberration. Conclusions and Clinical Relevance-Congenital radial head luxation and subluxation in the studied Bouviers des Flandres were characterized by angulation of the radial axis leading to caudolateral subluxation of the radial head and insufficient growth of the distal portion of the ulna together with cranial bowing of the olecranon. Affiliation Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, 3584 CM, Utrecht, The Netherlands. Journal Details Name: American journal of veterinary research ISSN: 0002-9645 Pages: 884-90

Porcine circovirus type-2 (PCV2) is widespread in domestic pig populations. It can be shed with boar semen, but the role boars have in epidemiology is still unclear. Vaccinating boars against PCV2 can reduce disease and virus load in semen, but may have unwanted side effects, that is, impairment of spermatogenesis. Therefore, the aim of this study was to investigate the effect and impact of two different PCV2 vaccines on boar semen quality and quantity. Healthy normospermic Large White boars in three groups of 12 each were vaccinated with either Circovac, Ingelvac CircoFLEX, or received NaCl. Eight ejaculates were collected starting 1 week after vaccination and assessed for quantitative traits. In general, sperm quantity and quality parameters did not change due to the vaccination (P > 0.05). Only DNA integrity between the Circovac and control group was P < 0.05 but remained at a low level (

Lecanicillium fungicola causes dry bubble disease and is an important problem in the cultivation of Agaricus bisporus. Little is known about the defense of mushrooms against pathogens in general and L. fungicola in particular. In plants and animals, a first attack by a pathogen often induces a systemic response that results in an acquired resistance to subsequent attacks by the same pathogen. The development of functionally similar responses in these two eukaryotic kingdoms indicates that they are important to all multi-cellular organisms. We investigated if such responses also occur in the interaction between the white button mushroom and L. fungicola. A first infection of mushrooms of the commercial A. bisporus strain Sylvan A15 by L. fungicola did not induce systemic resistance against a subsequent infection. Similar results were obtained with the A. bisporus strain MES01497, which was demonstrated to be more resistant to dry bubble disease. Apparently, fruiting bodies of A. bisporus do not express induced resistance against L. fungicola.

Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13.2%-19.3% vs. 26.4%). The presence of cysteamine during IVM of OPU-derived COCs also significantly increased the embryo production rate (34.4% vs. 23.4%). The higher number of embryos was again totally due to an increased number of blastocysts, whereas cryotolerance was not affected. The relative increase in embryo production rate was higher with OPU-derived oocytes compared with slaughterhouse-derived COCs (47% vs. 24%). This improvement resulted in a mean of 1.73 transferable embryos per OPU session compared with 1.06 in the absence of cysteamine. The presence of cysteamine did not affect pregnancy rate, gestation length, birth weight, perinatal mortality, and sex of calves born from either fresh or frozen-thawed embryos. This study reported that cysteamine supplementation during IVM greatly improved the efficiency and affectivity of an OPU-IVP program.

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple “transportomics” approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2-/- mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.—Krumpochova, P., Sapthu, S., Brouwers, J. F., de Haas, M., de Vos, R., Borst, P., van de Wetering, K. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

The term ‘prostasomes’ is generally used to classify the extracellular vesicles (EVs) released into prostatic fluid by prostate epithelial cells. However, other epithelia within the male reproductive tract also release EVs that mix with ‘true’ prostasomes during semen emission or ejaculation. Prostasomes have been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, as well as to stimulate sperm motility where all three are prerequisite processes for spermatozoa to attain fertilising capacity. Other proposed functions of prostasomes include interfering with the destruction of spermatozoa by immune cells within the female reproductive tract. On the other hand, it is unclear whether the distinct presumed functions are performed collectively by a single type of prostasome or by separate distinct sub-populations of EVs. Moreover, the exact molecular mechanisms through which prostasomes exert their functions have not been fully resolved. Besides their physiological functions, prostasomes produced by prostate tumour cells have been suggested to support prostate cancer spread development, and prostasomes in peripheral blood plasma may prove to be valuable biomarkers for prostate cancer.

Mobilization of fatty acids from adipose tissue during metabolic stress increases the amount of free fatty acids in blood and follicular fluid and is associated with impaired female fertility. In a previous report we described the effects of the three predominant fatty acids in follicular fluid (saturated palmitate and stearate; unsaturated oleate) on oocyte maturation and quality. In the current study the effects of elevated fatty acid levels on cumulus cells were investigated. The three fatty acids dose-dependently induced lipid storage in cumulus cells accompanied by an enhanced immune labeling of perilipin-2, a marker for lipid droplets. Lipidomic analysis confirmed incorporation of the administered fatty acids into triglyceride, resulting in a 3-6 fold increase of triglyceride content. In addition, palmitate selectively induced ceramide formation, which has been implicated in apoptosis. Indeed, of three fatty acids tested, palmitate induced reactive oxygen species formation, caspase 3 activation, and mitochondria deterioration, leading to degeneration of the cumulus cell layers. This effect could be mimicked by addition of ceramide C2 analog and could be inhibited by the ceramide synthase inhibitor fumonisin B1. Interfering with the intactness of the cumulus cell layers, either by mechanical force or by palmitate treatment, resulted in enhanced uptake of lipids in the oocyte and increased radical formation. Our results show that cumulus cells act as a barrier, protecting oocytes from in vitro induced lipotoxic effects. We suggest that this protective function of the cumulus cell layers is important for the developmental competence of the oocyte. The relevance of our findings for assisted reproduction technologies is discussed.

This study was conducted to evaluate the relationship between boar and semen related parameters and the variation in field fertility results. In 8 years time semen insemination doses from 110 186 ejaculates of 7429 boars were merged to fertility parameters of inseminations of 165 000 sows and these records were used for analysis. From all ejaculates boar and semen related data were recorded at the artificial insemination (AI) centers. Fertility parameters, such as farrowing rate (FR), ranging between 80.0% and 84.0%, and the total number of piglets born (TNB), ranging between 12.7 and 13.1, were recorded and from these the least square means per ejaculate were calculated. Only 5.9% of the total variation in FR was due to boar and semen variability of which 21% (P = 0.0001) was explained by genetic line of the boar, 11% (P = 0.047) was explained by laboratory technician, and 7% (P = 0.037) was explained by the AI center. For TNB the total variation was 6.6% boar and semen related of which 28% (P < 0.0001) was explained by genetic line of the boar and 7% (P = 0.011) was explained by the AI center. Only 4% of the boar and semen related variation was caused by sperm motility (microscopically assessed at collection, ranging from 60% to 90%). Other variation in FR and TNB was explained by management and semen related parameters (age of boar, 3%; P = 0.009; and 8%; P = 0.031, respectively), days between ejaculations (1%; P < 0.0001 of FR), number of cells in ejaculate (1%; P = 0.042 of TNB), year (9%; P = 0.032), and 13%; P = 0.0001, respectively), and month (11%; P = 0.0001; and 5%; P = 0.0001, respectively). Although semen motility is considered an important parameter to validate the quality of the ejaculate processed, it only minimally relates to fertility results under the current Dutch AI practice. Other boar and semen related parameters, like genetic line of the boar, are more relevant factors to select boars for AI purposes.

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.

Oleic acid protects oocytes against the detrimental effects of saturated free fatty acids During the last four decades, the fertility of high-producing dairy cows has declined dramatically. This decline in fertility has been linked to the equally marked increase in milk production, and the consequent more severe negative energy balance (NEB) that dairy cows experience during the early post-partum period. This NEB results from the high energy costs of milk production, which cannot be compensated by energy intake from food in the initial weeks after calving. A major metabolic characteristic of a NEB is an elevation in free fatty acid (FFA) concentrations in the blood, due to the mobilization of body fat reserves. The released FFAs serve as an alternative energy supply for aerobically functional tissues. However, marked elevations of the saturated FFA level in particular can become toxic to these tissues. In this thesis the impact of an elevated level of FFAs on the bovine oocyte and its immediate microenvironment was investigated, as the potential link between NEB and reduced fertility. The three dominating FFAs in blood and follicular fluid were the saturated palmitic and stearic acids, and mono-unsaturated oleic acid. Elevated levels of the two saturated FFAs had detrimental effects on the in vitro developmental competence of maturing oocytes, as evidenced by inhibited blastocyst formation. By contrast, the mono-unsaturated FFA was harmless to oocytes. Interestingly, the mono-unsaturated oleic acid also prevented the negative impact of the saturated FFAs on oocyte developmental competence. Remarkably, oocytes exposed to a nearly two-fold increase of the FFA concentration in follicular fluid during maturation in vivo, triggered by inducing metabolic stress in non-pregnant cows by restricted feeding, did not show any signs of reduced developmental competence. The follicular fluid appeared to have a relatively high oleic acid, but low stearic acid, content in comparison to blood; moreover, the cumulus cells that surround the oocyte incorporated, and thereby effectively sequestered, large amounts of the FFAs into their lipid droplets. Stimulating FFA storage is a protective mechanism by which oleic acid is known to help prevent lipotoxic effects on somatic cell types like skeletal muscle. The combination of a relatively high oleic acid concentration in follicular fluid, and the capacity of the surrounding cumulus cell layer to store FFAs, appears to protect the oocyte against lipotoxicity. In conclusion, elevated levels of saturated FFAs can have lipotoxic effects on the maturing oocyte; however, lipotoxicity is prevented by oleic acid and the cumulus cells that surround the oocyte. The protective environment provided by follicular fluid and the multiple cumulus cell layers surrounding the oocyte are absent during early follicular growth. Future research should examine whether elevated levels of FFAs are lipotoxic to oocytes during the early stages of follicular development.

Experimental studies and field surveys suggest that embryonic loss during the first 6 weeks of gestation is a common occurrence in the mare. During the first 2 weeks of development, a number of important cell differentiation events must occur to yield a viable embryo proper containing all three major germ layers (ectoderm, mesoderm, and endoderm). Because formation of the mesoderm and primitive streak are critical to the development of the embryo proper, but have not been described extensively in the horse, we examined tissue development and differentiation in early horse conceptuses using a combination of stereomicroscopy, light microscopy, and immunohistochemistry. Ingression of epiblast cells to form the mesoderm was first observed on day 12 after ovulation; by Day 18 the conceptus had completed a series of differentiation events and morphologic changes that yielded an embryo proper with a functional circulation. While mesoderm precursor cells were present from Day 12 after ovulation, vimentin expression was not detectable until Day 14, suggesting that initial differentiation of mesoderm from the epiblast in the horse is independent of this intermediate filament protein, a situation that contrasts with other domestic species. Development of the other major embryonic germ layers was similar to other species. For example, ectodermal cells expressed cytokeratins, and there was a clear demarcation in staining intensity between embryonic ectoderm and trophectoderm. Hypoblast showed clear α1-fetoprotein expression from as early as Day 10 after ovulation, and seemed to be the only source of α1-fetoprotein in the early conceptus.

A newly uncovered means of communication between cells involves intercellular transfer of nano-sized extracellular vesicles (EV), composed of lipids, proteins, and genetic material. EV released by cells of the immune system can play a regulatory role in the induction and suppression of immune responses. These functions may be mediated not only by the bioactive lipids and proteins present in EV but also by EV-associated RNAs. The RNA in EV mainly consists of microRNAs and a large range of other small non-coding RNA species. Since many of these small RNAs have the potential to regulate gene expression, intercellular transfer of these RNAs via EV may cause long-term changes in the function of EV-targeted cells. Several types of innate immune cells release EV that affect innate immune responses and other (patho)physiological processes. Additionally, the innate immune system is influenced by EV released by non-immune cells and EV found in body fluids. In this review, we focus on how EV-associated RNAs contribute to these immune regulatory processes.

REASONS FOR PERFORMING STUDY: Equine embryogenesis post implantation is not well studied, and only two-dimensional illustrations are available. A thorough appreciation of the complex three-dimensional relationship between tissues and organs and their development is, however, crucial for understanding physiological and pathological mechanisms. OBJECTIVES: The goals were 2-fold: 1) to establish a freely accessible online atlas as a reference tool for the scientific and pedagogic communities; and 2) to create a framework for integration of data with known spatiotemporal distribution, such as gene expression or cell lineage. STUDY DESIGN: Descriptive anatomical study. METHODS: Magnetic resonance microscopy was performed on embryos of 28, 32, 35, 37, 39, 40, 42, 45, 50 and 65 days gestation using a 9.4 T magnet. Equine embryos were staged according to the Carnegie system. Acquired images were optimised using histogram optimisation and processed for easy online access. RESULTS: Magnetic resonance microscopy protocols for imaging of equine embryos and fetuses were developed. The wider spread of signal intensity values achieved by histogram equalisation increased visual contrast considerably. Despite their longer gestation, equine conceptuses appeared to reach the various Carnegie staging benchmarks earlier than human embryos. CONCLUSIONS: The equine atlas is designed to serve as an online reference tool for research and teaching. POTENTIAL RELEVANCE: The equine atlas may serve as a foundation and scaffold for improved anatomical labelling, spatial and temporal data integration and further understanding of physiological and pathophysiological processes involved in development and disease.

Description This book contains 19 chapters that discuss theoretical and applied andrology for domestic, zoo and wild animals. Topics include semen and its constituents; sperm production and harvest; determinants of sperm morphology; sperm preparation for fertilization; practical aspects of semen cryopreservation; evaluation of semen in the andrology laboratory; genetic aspects of male reproduction; emerging techniques and future development of semen evaluation and handling and applied andrology in cats, dogs, fowls, turkeys, sheep, goats, cervids, horses, cattle, zebu, buffaloes, pigs, camelids, zoo animals and wild animals. It will be of use for those teaching animal physiology at a tertiary level and a reference for those interested in male animal reproductive evaluation, performance and in semen evaluation, handling and use for artificial breeding.

OBJECTIVE: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined. METHODS: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes. RESULTS: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR. CONCLUSIONS: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated.

Understanding the links between genetic, epigenetic and non-genetic factors throughout the lifespan and across generations and their role in disease susceptibility and disease progression offer entirely new avenues and solutions to major problems in our society. To overcome the numerous challenges, we have come up with nine major conclusions to set the vision for future policies and research agendas at the European level.

Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca2+, and albumin; however, these conditions are insufficient in the horse. We hypothesized that binding to the oviduct epithelium is an essential requirement for the induction of capacitation in stallion spermatozoa. Sperm-oviduct binding was established by coincubating equine oviduct explants for 2 h with stallion spermatozoa (2 × 106 spermatozoa/ml), during which it transpired that the highest density (per mm2) of oviduct-bound spermatozoa was achieved under noncapacitating conditions. In subsequent experiments, sperm-oviduct incubations were performed for 6 h under noncapacitating versus capacitating conditions. The oviduct-bound spermatozoa showed a time-dependent protein tyrosine phosphorylation response, which was not observed in unbound spermatozoa or spermatozoa incubated in oviduct explant conditioned medium. Both oviduct-bound and unbound sperm remained motile with intact plasma membrane and acrosome. Since protein tyrosine phosphorylation can be induced in equine spermatozoa by media with high pH, the intracellular pH (pHi) of oviduct explant cells and bound spermatozoa was monitored fluorometrically after staining with BCECF-AM dye. The epithelial secretory cells contained large, alkaline vesicles. Moreover, oviduct-bound spermatozoa showed a gradual increase in pHi, presumably due to an alkaline local microenvironment created by the secretory epithelial cells, given that unbound spermatozoa did not show pHi changes. Thus, sperm-oviduct interaction appears to facilitate equine sperm capacitation by creating an alkaline local environment that triggers intracellular protein tyrosine phosphorylation in bound sperm.

Before the sperm cell can reach the oocyte it needs to be activated and to undergo a series of preparative steps. The sperm surface dynamics was studied in relation to this activation process and the modifications and removal of sperm surface components havebeen investigated. Bicarbonate-induced radical dependent formation of oxysterols caused efficient free sterol depletion from responsive sperm cells. This formation of oxysterols sperm cells was shown to be functional for making sperm cells competent in fertilizing the oocyte. Bicarbonate enriched media also caused a spontaneous removal of extracellular matrix components in activated sperm cells. The removal of glycosylphophatidylinositol anchored proteins was shown to be a preparative step required for sperm cells to acquire receptivity for recognizing the zona pellucida. A novel wet mount atomic force microscopic technique showed the specific removal of extracellular matrix components in the anterior part of equatorial segment of the sperm head. This is the specific site of the sperm surface involved in binding to the oocyte and in the fertilization fusion. This removal allowed the exposure membrane particles which were strictly arranged in a 17 nanometer hexagonal pattern. During the acrosome reaction this specific area fused with the underlying outer acrosomal membrane to form 1 micrometer long and 50 nanometer in diameter membrane tubules attached to the equatorial sub segment. We hypothesize that the described capacitation-dependent sperm surface changes are critical for obtaining fertilization capacity. It is matter for future research to identify the key membrane proteins involved in the fertilization fusion structures that appear on the sperm surface.

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defense and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. These small RNA molecules play an important role in defending the germline against transposons, which are selfish DNA elements that can move and/or multiply themselves to new positions in the genome, thereby endangering the genomic stability of an organism piRNAs discriminate themselves from most other small RNA molecules by carrying a 2’O methylgroup at their 3’ end. The protein that methylates piRNA is the methyltransferase Hen1. We studied the role of Hen1 in the zebrafish D. rerio and the nematode C. elegans. Zebrafish Hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line. In hen1 mutant testes, piRNAs become unstable and transposons are mildly upregulated. Hen1 protein localizes to nuage through its C-terminal domain, which is a peri-nuclear structure consisting of RNA and many proteins related to RNA-interference. The C. elegans methyltransferase HENN-1 is responsible for the methylation of two germline expressed small RNA species, the 26G siRNA and piRNAs. We show that the stability of piRNAs is hardly affected in henn-1 mutant animals. However, most 26G RNA display reduced stability and have additional uracil and adenines at their 3' end. The affected 26G RNAs are involved in the endogenous RNAi pathway and influence gene expression during zygotic development. However, loss of henn-1 leads only to mild effects on germline expressed genes. In addition, the HENN-1 protein does not form a stable complex with the Piwi protein that bind piRNAs. However, HENN-1 localizes close to the regions that also host Piwi proteins. Small RNAs not only influence expression of proteins at the RNA level, they can also induce silencing at the DNA level by preventing transcription of RNA. We showed that in C. elegans, the Piwi pathway can initiate gene silencing by inducing heterochromatin formation. More specifically, the piRNA pathway initiate an extremely stable form of gene silencing on a transgenic, single-copy target. This type of silencing is faithfully maintained over tens of generations by factors known to act in the nuclear RNAi pathway. This nuclear pathway induces the tri-methylation of lysine 9 of histone 3 - a repressive chromatin mark- on their target loci. In conclusion, we studied two 'methylation steps' of the Piwi-pathway, starting with the 3' end methylation of piRNAs by Hen1 and moving towards the histone methylation initiated by the C elegans specific piRNA-pathway.

Optimization of bovine ovum pick up (OPU) followed by in vitro embryo production (IVP) has been driven by the desire of both beef and dairy cattle breeders to enhance genetic improvement. The work presented in this thesis focuses on optimizing the efficiency and efficacy of the OPU-IVP program. Attention has been paid to both genetic and non-genetic factors affecting the outcome of OPU-IVP. Moreover, by performing embryo transfer experiments, it was possible to ensure that effects and resulting conclusions were not limited to the embryo production stage, but also encompassed post-transfer embryo survival and calf characteristics. The optimal maturation culture period for OPU-derived cumulus oocyte complexes (COCs) in relation to their post-fertilization developmental capacity was determined in a retrospective study covering the analyses of OPU, IVP and calving results from commercial program over a 10 year period. In vitro maturation (IVM) culture periods within 16-28 h range had no detrimental effect on embryo production rate, calving rate, birth-weight, gestation length or proportion of male calves. The presence of cysteamine during IVM of OPU-derived COCs significantly increased the embryo production rate (34.4% with cysteamine vs. 23.4% without cysteamine). The improved embryo production was due to an increased percentage of blastocysts, whereas cryotolerance was not affected. This improvement resulted in a mean of 1.73 transferable embryos per OPU session after IVM in the presence of cysteamine compared to 1.06 in the absence of cysteamine. The presence of cysteamine did not affect post-transfer embryo survival or calf characteristics. The presence of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) did not affect embryo production at Day 7 nor embryo stage or quality. However, the pregnancy rate was improved for both fresh (46.3% vs. 41.0%) and frozen/thawed embryos (40.8% vs. 35.6%). Genetic factors influencing the outcome of bovine OPU-IVP and its relation to female fertility were also investigated. For the first time, genetic parameters were estimated for the number of COCs (Ncoc), quality of COCs (Qcoc) and the number and proportion of embryos at Day 4 (NcleavD4, PcleavD4) and Day 7 of culture (NembD7, PembD7 and NTembD7, PTembD7). Estimates of heritability were 0.25 for Ncoc, 0.09 for Qcoc, 0.19 for NcleavD4, 0.21 for NembD7, 0.16 for NTembD7, 0.07 for PcleavD4, 0.12 for PembD7, and 0.10 for PTembD7. Genetic correlation between Ncoc and Qcoc was close to zero, whereas genetic correlations between Ncoc and the number of embryos were positive ranging from moderate to high. These results suggest that COC quality is independent of the total number of COCs collected via OPU and that in general, a higher number of COCs will lead to a higher number of embryos produced. This study identified OPU-IVP traits that could be of potential value for genetic selection. In conclusion, the results presented in this thesis provide a source of useful information for future attempts to improve both the efficiency and efficacy of OPU-IVP programs in commercial breeding enterprises

Recent findings have refined our thinking on sperm interactions with the cumulus–oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona–cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm–ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.

This study demonstrates for the first time that porcine and mouse sperm incubated in capacitation media supplemented with bicarbonate produce oxysterols. The production is depen- dent on a reactive oxygen species (ROS) signaling pathway that is activated by bicarbonate and can be inhibited or blocked by addition of vitamin E or vitamin A or induced in absence of bicarbonate with pro-oxidants. The oxysterol formation was required to initiate albumin dependent depletion of 30% of the total free sterol and >50% of the formed oxysterols. Incubation of bicarbonate treated sperm with oxysterol-binding proteins (ORP-1 or ORP-2) caused a reduction of >70% of the formed oxysterols in the sperm pellet but no free sterol depletion. Interestingly, both ORP and albumin treatments led to similar signs of sperm capacitation: hyperactivated motility, tyrosin phosphorylation, and aggregation of flotillin in the apical ridge area of the sperm head. However, only albumin incubations led to high in vitro fertilization rates of the oocytes, whereas the ORP-1 and ORP-2 incubations did not. A pretreatment of sperm with vitamin E or A caused reduced in vitro fertilization rates with 47% and 100%, respectively. Artificial depletion of sterols mediated by methyl-beta cyclodextrin bypasses the bicarbonate ROS oxysterol signaling pathway but resulted only in low in vitro fertilization rates and oocyte degeneration. Thus, bicarbonate- induced ROS formation causes at the sperm surface oxysterol formation and a simultaneous activation of reverse sterol transport from the sperm surface, which appears to be required for efficient oocyte fertilization. © 2013 by the Society for the Study of Reproduction, Inc.

Special Issue: FROM GENOME TO PROTEOME:OPEN INNOVATIONS; Mouse embryonic stem cells (mESC) and mouse epiblast stem cells (mEpiSC) share similar pluripotency factors like NANOG or POU5F1, however, their state of pluripotency differs significantly. mESC and mEpiSC can be derived from embryos generated by fertilization (FT) or by somatic cell nuclear transfer (NT). In this study we performed a 4-plex iTRAQ LC-MS/MS based approach, facilitating the multiplexed comparison of the four indicated types of stem cells. From four replicates of each cell type, 1650 proteins were quantified. 234 non redundant proteins with significant abundance alterations between FT/NT-mESC and FT/NT-mEpiSC, and 44 between FT and NT derived cells were detected. Bioinformatic analysis revealed that several pluripotency associated proteins, among them POU5F1, DNMT3L, TIF1B, and proteins involved in DNA repair like MSH2 and MSH6, are more abundant in mESC compared to mEpiSC. The abundance level of these proteins is not affected by the mode of embryo generation, whereas several cytoskeleton proteins show a higher abundance in NT-mESC compared to FT-mESC. In addition, a number of cytoskeletal proteins are enriched in mEpiSC, e.g., myosins, filamins and intermediate filament proteins, reflecting the progressed differentiation state of epiblast derived versus inner cell mass derived murine pluripotent stem cells. This study aims to get new insights in the pluripotency state of stem cells and to deepen the knowledge of early cell differentiation. In an iTRAQ MS approach, we quantitatively compared proteomes of inner cell mass derived stem cells (mESC) with epiblast derived stem cells (mEpiSC). These stem cell types are derived from embryos of different developmental stages, and therefore vary considerably in their state of pluripotency and reflect different stages of early differentiation. The proteins which show significant abundance differences between the two stem cell lines represent (i) promising targets to further decipher molecular processes during early embryo development and (ii) useful molecular markers to monitor early differentiation events of stem cells by targeted approaches. This article is part of a Special Issue entitled: From Genome to Proteome: Open Innovations.

The uptake of Ag–Ab immune complexes (IC) after the ligation of activating FcγR on dendritic cells (DC) leads to 100 times more efficient Ag presentation than the uptake of free Ags. FcγRs were reported to facilitate IC uptake and simultaneously induce cellular activation that drives DC maturation and mediates efficient T cell activation. Activating FcγRs elicit intracellular signaling via the ITAM domain of the associated FcRγ-chain. Studies with FcRγ-chain knockout (FcRγ−/−) mice reported FcRγ-chain ITAM signaling to be responsible for enhancing both IC uptake and DC maturation. However, FcRγ-chain is also required for surface expression of activating FcγRs, hampering the dissection of ITAM-dependent and independent FcγR functions in FcRγ−/− DCs. In this work, we studied the role of FcRγ-chain ITAM signaling using DCs from NOTAM mice that express normal surface levels of activating FcγR, but lack functional ITAM signaling. IC uptake by bone marrow–derived NOTAM DCs was reduced compared with wild-type DCs, but was not completely absent as in FcRγ−/− DCs. In NOTAM DCs, despite the uptake of ICs, both MHC class I and MHC class II Ag presentation was completely abrogated similar to FcRγ−/− DCs. Secretion of cytokines, upregulation of costimulatory molecules, and Ag degradation were abrogated in NOTAM DCs in response to FcγR ligation. Cross-presentation using splenic NOTAM DCs and prolonged incubation with OVA-IC was also abrogated. Interestingly, in this setup, proliferation of CD4+ OT-II cells was induced by NOTAM DCs. We conclude that FcRγ-chain ITAM signaling facilitates IC uptake and is essentially required for cross-presentation, but not for MHC class II Ag presentation.

Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCE Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.

Transvaginal ultrasound-guided pregnancy reduction (TUGR) is a procedure described for the management of twins post-fixation in the horse. Success rates are often disappointing but are reported to be more favorable for bilaterally situated twins and when intervention takes place before day 35 of gestation. This study aimed to determine whether stabbing the embryo/fetus rather than aspirating conceptus fluids improved the likelihood of success, measured as the birth of a normal live singleton foal. Data from 103 TUGR interventions were analyzed by logistic regression analysis; method of treatment, relative conceptus location (i.e., uni- vs. bilateral), and stage of gestation were included as interdependent factors that potentially influence the outcome. Overall, 34/103 (33%) TUGR interventions resulted in a single live foal. There was no significant difference (P = 0.14) in the outcome between TUGR based on fetal stabbing (12/28: 42.9%) versus fluid aspiration (22/75: 29.3%). There was also no significant influence (P = 0.11) of the conceptuses being located unilaterally (19/65: 29.2%) versus bilaterally (15/38: 39.5%). However, TUGR was numerically more successful (P = 0.05) when performed ≤ Day 35 of gestation (21/53: 39.6%), as opposed to > Day 35 (13/50: 26%). Day 45 may represent an even more critical time point because only 2 out of 15 TUGRs (13.3%) performed beyond this day resulted in the birth of a live foal, compared with 11/35 (31.4%) performed between Days 36 and 45. Although the numbers are low, this suggests that TUGR is not the method of choice for reducing > Day 45 twins. Four pregnancy losses were recorded 1 to 7 months post-TUGR (4/38: 10.5%), and although it is tempting to attribute the losses to TUGR, this rate of late gestation pregnancy loss is normal. We conclude that TUGR by fetal stabbing does not offer significant advantages over fluid aspiration. However, TUGR should be performed before Day 35 of gestation and is considered primarily a salvage procedure to be used when re-breeding is not a viable alternative.

During the last years it has become evident that many different cell types can communicate with each other through intercellular transfer of extracellular vesicles. Such cell-derived membrane vesicles function in several physiological processes and also in disease. This thesis describes two subclasses of prostasomes, extracellular vesicles that are released by prostate epithelial cells and ultimately enter the seminal plasma. Prostasomes stimulate sperm cell motility and influence sperm cell capacitation and the acrosome reaction, all of which are prerequisites for successful fertilization. Furthermore, prostasomes suppress immune-mediated destruction of sperm cells in the female reproductive tract. However, the exact molecular mechanisms through which prostasomes exert such diverse functions remain largely unknown. In the studies described in this thesis, prostasomes were purified and their protein, lipid and nucleic acid contents characterized. This resulted in the definition of two distinct types of prostasomes, a smaller (50 nm) and a larger (100 nm) type, which may perform different functions. The two prostasome types have distinct protein and lipid compositions. Many other types of extracellular vesicles are known to contain RNA, which may elicit epigenetic effects in their target cells. However, the RNA content of prostasomes had not been explored. In the research described in this thesis, RNA was found associated with the large prostasomes. Deep sequencing of this RNA showed that the majority is composed of a type that had not been described before. Physiological target cells for the larger prostasomes, and hence for their RNA contents, remained unknown. Given its unique composition, prostasomal RNA may serve as a biomarker of prostate cancer. The blood of healthy men is devoid of prostasomes, but in case of prostate cancer prostasomes may reach the blood stream as well. Comparison of prostasomal RNA from prostate cancer patients with that of healthy men, either from blood, urine or seminal plasma, may provide new insights in its potential as cancer biomarker. The interactions between small prostasomes and sperm cells during sperm cell capacitation were studied in an equine model. Small prostasomes were found to bind to sperm cells, but only at conditions that favoured sperm cell capacitation. Moreover, recruited prostasomes reduced the degree of protein tyrosine phosphorylation in sperm cells, which is a hallmark of a late phase in sperm cell capacitation. Prostasomes may therefore help to prevent premature full capacitation of sperm cells and their acrosome reaction. Other studies showed that transfer of progesterone receptors to the sperm cells through fusion of prostasomes with their plasma membrane. Once the sperm cells have reached the progesterone-secreting cumulus cells that surround the oocyte, transfer of progesterone receptors by prostasomes may assist the progesterone-dependent stimulation of sperm cell hypermotility and the acrosome reaction. Thus, prostasomes may bind and accompany sperm cells on their journey to the oocyte, and fuse with the sperm cells only upon their approach of an oocyte, providing them with tools that are required for effective oocyte fertilization

Life starts with fertilisation. Variation in fertility is caused by both farm and sow related parameters and boar and semen related parameters. Therefore, achieving high fertility is not obvious. Predominantly, artificial insemination (AI) is used for breeding pigs. The advantage of AI is that you can dilute semen from high fertile breeding boars and in this way inseminate many sows. The last years, the pig industry is increased in scale and evaluated in specialisation. Unfortunately there is no golden standard concerning the requirements for qualitative good semen. This thesis handles different semen quality characteristics in relation to fertility, showing that the effect of semen quality characteristics on the variation in fertility is relatively small. These effects seems small, but the economic value in a high producing pig industry is large, with the objective to minimise the variation between farms. Assessing semen quality characteristics objectively and relate them to field fertility leads to an efficient production of insemination doses, which results in an efficient spreading of the genetic genes. Semen motility assessed with CASA (Computer Assisted Semen Analysis) and analysing DNA damage are semen quality parameters which show a relation with fertility, in high fertile breeding boars. The value of these tests is possibly even higher in a population in which less selection is performed, added with other tested parameters from this thesis. The conclusions in this thesis contribute to the development of semen quality assessments which improve the prediction of porcine male fertility.

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.

The Argonaute protein family consists of two distinct sub-clades: AGO and PIWI proteins. AGO proteins can form RNA induced silencing complex (RISC) and are responsible for RNAi and miRNA pathways. PIWI proteins are mostly expressed in the germline and together with specific small RNA groups, named PIWI-interacting RNAs (piRNAs), they function in a germline-specific RNAi-like pathway. PIWI proteins are responsible for piRNA biogenesis and germline development/ function in many species. Zebrafish genome encodes two PIWI proteins: Ziwi and Zili. Loss of Ziwi or Zili function, leads to sterility and transposon de-repression in the germline. PIWI proteins in various species have been reported to interact with a special protein family, Tudor domain containing (Tdrd) proteins. Tdrd mutants often phenocopy piwi mutants, suggesting their roles in the PIWI-piRNA pathway. Tdrd proteins interact with proteins with methylated arginine residues in the RG motifs, which are also found in the N-terminus of PIWI proteins. Via reverse genetic screening, we obtained mutant zebrafish alleles in tdrd1, tdrd6 and tdrd9 genes. These three tdrd genes are germline-specific. We found that these three tdrd proteins are involved in the piRNA functions to different extent. Tdrd1 function as a scaffold to facilitate the piRNA pathway by recruiting PIWI proteins and their targets. Tdrd9 is important in maintaining the germ meiotic stem cells. Tdrd6 is a possible reservoir for Ziwi-specific piRNAs and assisting Ziwi targeting retrotransposons. Moreover, Tdrd6 is involved in germ plasm assembly in the early stage of embryogenesis.

In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 6 13 nm vs. 105 6 25 nm) but similar equilibrium buoyant density (;1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostatespecific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.

Mobilization of fatty acids from adipose tissue during metabolic stress will increase the amount of free fatty acids in blood and follicular fluid and, thus, may affect oocyte quality. In this in vitro study, the three predominant fatty acids in follicular fluid (saturated palmitic and stearic acid and unsaturated oleic acid) were presented to maturing oocytes to test whether fatty acids can affect lipid storage of the oocyte and developmental competence postfertilization. Palmitic and stearic acid had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets and a concomitant detrimental effect on oocyte developmental competence. Oleic acid, in contrast, had the opposite effect, causing an increase of lipid storage in lipid droplets and an improvement of oocyte developmental competence. Remarkably, the adverse effects of palmitic and stearic acid could be counteracted by oleic acid. These results suggest that the ratio and amount of saturated and unsaturated fatty acid is relevant for lipid storage in the maturing oocyte and that this relates to the developmental competence of maturing oocytes.

We report on the presence and formation of cholesterol oxidation products (oxysterols) in bovine sperm. Although cholesterol is the most abundant molecule in the membrane of mammalian cells and is easily oxidized, this is the first report on cholesterol oxidation in sperm membranes as investigated by state-of-the-art liquid chromatographic and mass spectrometric methods. First, oxysterols are already present in fresh semen samples, showing that lipid peroxidation is part of normal sperm physiology. After chromatographic separation (by high-performance liquid chromatography), the detected oxysterol species were identified with atmospheric pressure chemical ionization mass spectrometry in multiple-reaction-monitoring mode that enabled detection in a broad and linear concentration range (0.05–100 pmol for each oxysterol species detected). Second, exposure of living sperm cells to oxidative stress does not result in the same level and composition of oxysterol species compared with oxidative stress imposed on reconstituted vesicles from protein-free sperm lipid extracts. This suggests that living sperm cells protect themselves against elevated oxysterol formation. Third, sperm capacitation induces the formation of oxysterols, and these formed oxysterols are almost completely depleted from the sperm surface by albumin. Fourth, and most importantly, capacitation after freezing/thawing of sperm fails to induce both the formation of oxysterols and the subsequent albumin-dependent depletion of oxysterols from the sperm surface. The possible physiological relevance of capacitation-dependent oxysterol formation and depletion at the sperm surface as well as the omission of this after freezing/thawing semen is discussed.

The long-awaited second edition of the International Symposium on "Epigenome Network, Development and Reprogramming of Germ Cells" hosted by Hiroyuki Sasaki took place at the Kyushu University School of Medicine, in Fukuoka, Japan from 22 to 24 November 2010. This meeting brought together again the crème de la crème of the Japanese research community investigating germline development, reprogramming and genetic networks as well as eminent international scientists. Novel trend concepts including the "reprogramming expressway", "canalization", "licensing", "epigenetic barrier", "flex points" and "hydroxymethylation" were introduced and discussed in the context of development, reprogramming and pluripotency.