Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status.

Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30-400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility.

Follicular-fluid proteomics during equine follicle development.

The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR <1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

In-depth proteomic analysis of boar spermatozoa through shotgun and gel-based methods.

BACKGROUND: Mature spermatozoa contain numerous epididymal and seminal plasma proteins, which full identification through high-throughput technologies may allow for a better understanding of the sperm biology. Therefore, we conducted a global proteomic analysis of boar spermatozoa through shotgun and gel-based methodologies. RESULTS: The total proteins were extracted from mature spermatozoa and subjecsted to proteome analyses. Functional analyses of gene ontology representations and pathway enrichments were conducted on the shotgun dataset, followed by immunology and gene expression validations. Shotgun and gel-based approaches allowed the detection of 2728 proteins and 2123 spots, respectively. Approximately 38% and 59% of total proteins were respectively fully and partially annotated, and 3% were unknown. Gene ontology analysis indicated high proportions of proteins associated with intracellular and cytoplasm localizations, protein and nucleic acid binding, hydrolase and transferase activities, and cellular, metabolic, and regulation of biological processes. Proteins associated with phosphorylation processes and mitochondrial membranes, nucleic acid binding, and phosphate and phosphorous metabolics represented 77% of the dataset. Pathways associated with oxidative phosphorylation, citrate cycle, and extra-cellular matrix-receptor interaction were significantly enriched. Protein complex, intracellular organelle, cytoskeletal parts, fertilization and reproduction, and gap junction pathway were significantly enriched within the top 116 highly abundant proteins. Nine randomly selected protein candidates were confirmed with gel-based identification, immunofluorescence detection, and mRNA expression. CONCLUSIONS: This study offers an in-depth proteomic mapping of mature boar spermatozoa that will enable comparative and discovery research for the improvement of male fertility.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium.

BACKGROUND: Synthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes. RESULTS: Measurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction. CONCLUSIONS: This study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

Progressive erosion of the Relaxin1 gene in bovids.

The relaxin/insulin-like (RLN/INSL) gene family is a group of genes that encode peptide hormones involved in a variety of physiological functions related to reproduction. Previous studies have shown that relaxin plays a key role in widening of the pubic bone during labor and in gamete maturation. Because of these functions, studying the evolution of RLN1, the gene encoding for relaxin, is relevant in livestock species, most of which belong in the group Laurasiatheria, which includes cow, pig, horse, goat, and sheep in addition to bats, cetaceans and carnivores. Experimental evidence suggests that cows do not synthesize relaxin, but respond to it, and sheep apparently have a truncated RLN1 gene. Thus, we made use of genome sequence data to characterize the genomic locus of the RLN1 gene in Laurasiatherian mammals to better understand how cows lost the ability to synthesize this peptide. We found that all ruminants in our study (cow, giraffe, goat, sheep and Tibetan antelope) lack a functional RLN1 gene, and document the progressive loss of RLN1 in the lineage leading to cows. Our analyses indicate that 1 - all ruminants have lost all key regulatory elements upstream of the first exon, 2 - giraffe, goat, sheep and Tibetan antelope have multiple inactivating mutations in the RLN1 pseudogene, and 3 - the cow genome has lost all traces of RLN1. The 5' regulatory sequence plays a key role in activating expression, and the loss of this sequence would impair synthesis of mRNA. Our results suggest that changes in regulatory sequence preceded mutations in coding sequence and highlight the importance of these regions in maintaining proper gene function. In addition, we found that all bovids examined posses copies of the relaxin receptors, which explains why they are able to respond to relaxin despite their inability to produce it.

Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa.

BACKGROUND: Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-QDs for bioimaging, there are strong concerns about QD nanocomposites containing cadmium which exhibits potential cellular toxicity. RESULTS: In this study, bioluminescent composites comprised of magnetic nanoparticles and firefly luciferase (Photinus pyralis) are examined as potential light-emitting agents for imaging, detection, and tracking mammalian spermatozoa. Characterization was carried out using infrared spectroscopy, TEM and cryo-TEM imaging, and ζ-potential measurements to demonstrate the successful preparation of these nanocomposites. Binding interactions between the synthesized nanoparticles and spermatozoon were characterized using confocal and atomic/magnetic force microscopy. Bioluminescence imaging and UV-visible-NIR microscopy results showed light emission from sperm samples incubated with the firefly luciferase-modified nanoparticles. Therefore, these newly synthesized luciferase-modified magnetic nanoparticles show promise as substitutes for QD labeling, and can potentially also be used for in vivo manipulation and tracking, as well as MRI techniques. CONCLUSIONS: These preliminary data indicate that luciferase-magnetic nanoparticle composites can potentially be used for spermatozoa detection and imaging. Their magnetic properties add additional functionality to allow for manipulation, sorting, or tracking of cells using magnetic techniques.

Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa.

BACKGROUND: Relaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars. METHODS: Spermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA. RESULTS: Both receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa. CONCLUSIONS: Immunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.

Beneficial effects of relaxin on motility characteristics of stored boar spermatozoa.

BACKGROUND: Relaxin is detected in seminal plasma of many species and its association with sperm motility may be beneficial in some aspects of assisted reproduction. Here, we immunolocalized relaxin receptors and investigated the effects of exogenous relaxin on motility characteristics, viability, and cAMP content of boar spermatozoa after storage. METHODS: Commercial doses of boar semen were obtained on the collection day (Day 0) and kept in shipping containers at room temperature for up to 4 days (Day 4). On Day 0, spermatozoa were fixed for immunofluorescence detection of relaxin receptors RXFP1 and RXFP2 (Experiment 1). Semen aliquots were taken from the same dose at Day 0, Day 1, and Day 2 (Experiment 2a), and Day 2 and Day 4 (Experiment 2b) for analyses. Alive spermatozoa were purified and incubated (1 h-37°C) with 0, 50, or 100 ng relaxin/ml (Experiment 2a) and 0, 100, or 500 ng relaxin/ml (Experiment 2b). Afterward, aliquots of each treatment group were subjected to motility (Experiments 2), viability (Experiment 3) analyses, and cAMP quantification (Experiment 4). Data (3-4 independent replicates) were statistically analyzed (ANOVA followed by pairwise comparisons) and p values less or equal to 0.05 was set for significant difference. RESULTS: Both RXFP1 and RXFP2 receptors were immunolocalized on the entire spermatozoon. Relaxin concentration of 100 ng/ml significantly improved the proportions of motile, progressive, and rapid spermatozoa up to Day 2. Only 500 ng relaxin/ml provided beneficial effects on Day 4. The viability of spermatozoa was not affected by relaxin (100 ng/ml) during storage, but the extent of mitochondria membrane damages was significantly decreased. Furthermore, relaxin did not affect the cAMP contents of spermatozoa during storage, in our conditions. CONCLUSIONS: Relaxin could be a valuable motility booster of stored- or aged-spermatozoa for assisted reproduction techniques. However, the related-intracellular signaling cascades of relaxin in boar spermatozoa remain undetermined.

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.

BACKGROUND: The fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes. METHODS: Freshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD-) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulus-oocyte complexes (COCs) were matured in the presence of QD- or QD+. Ovarian follicles were microinjected with QD- or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences. RESULTS: All labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD- or QD+. The QD- fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection. CONCLUSION: Findings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.

The use of a whole animal biophotonic model as a screen for the angiogenic potential of estrogenic compounds.

BACKGROUND: Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor) have estrogenic potential that can initiate inappropriate physiological responses in estrogenic-sensitive tissues following exposure in vivo. Thus, the current study was designed to determine whether the VEGFR-2-Luciferase (Luc) reporter transgenic mouse is a useful model for evaluating estrogenic tendencies of methoxychlor by monitoring wound healing via VEGFR-2-mediated gene expression using bioluminescence and real-time imaging technology. RESULTS: VEGFR-2-Luc gene activity peaked by d 7 (P<0.001) in all groups but was not different (P>0.05) between control and estrogen/methoxychlor exposed mice. CONCLUSIONS: Changes in VEGFR-2-Luc gene activity associated with the dermal wound healing process were able to be measured via photonic emission. The increase in vasculature recruitment and formation is paralleled by the increase of VEGFR-2-Luc activity with a peak on day 7. However, estrogen/methoxychlor did not significantly alter wound healing mediated VEGFR-2-Luc gene expression patterns compared to controls. This suggests that the VEGFR-2-Luc transgenic mouse wound model tested in this study may not be optimal for use as a screen for the angiogenic potential of estrogenic compounds.

L-Arginine enhances cell proliferation and reduces apoptosis in human endometrial RL95-2 cells.

BACKGROUND: L-arginine is considered to be one of the most versatile amino acids due to the fact that it serves as a precursor for many important molecules in cellular physiology. When supplemented in the diet, L-arginine can increase the number of implantation sites in mice and rats, suggesting an effect at the level of the endometrium. To this end, this study determined the effect that L-arginine has on apoptosis and cell proliferation in human endometrial RL95-2 cells. RESULTS: L-arginine at physiological (200 micromol/L) and supra-physiological (800 micromol/L) concentrations increased cell proliferation at days 2 and 4 post-treatment with a dose-dependent effect being observed on day 2. Additionally, inhibition of nitric oxide (NO) synthase and arginase, which are responsible for the conversion of L-arginine to NO and polyamines, respectively, reduced the proliferative effect of L-arginine. L-arginine also decreased the proportion of cells with TUNEL positive nuclei and increased the ratio of cells with healthy mitochondria compared to cells with a disrupted mitochondrial membrane potential, indicating that L-arginine prevents mitochondrial mediated apoptosis in endometrial RL95-2 cells. Furthermore, exposure to L-arginine did not affect total BAD protein expression; however, L-arginine increased the abundance of phosphorylated BAD protein. CONCLUSIONS: In summary, L-arginine added to the culture media at physiological (200 micromol/L) and supraphysiological concentrations (800 micromol/L) enhanced endometrial RL95-2 cell proliferation through mechanisms mediated by NO and polyamine biosynthesis. In addition, L-arginine reduced endometrial RL95-2 mitochondrial mediated apoptosis through increased phosphorylation of BAD protein.

Does Equine Interaction Facilitate Emotional Safety and Learning for College Students within an Agricultural-Based Classroom?

Effective teaching requires an educational environment that promotes learning, and yet, developing such an environment can be challenging within today's agricultural-based classroom for educators due to the trend to a more virtual teaching format and less hands-on learning. Animal interaction, particularly equine activities, has been shown to assist educators in the development of an emotionally safe environment for promoting learning. However, research is lacking as to whether the interaction with the animal needs to be direct or indirect within the collegiate educational environment to observe benefits. Therefore, the objective of this study was to determine the impact of equine interaction, both direct and indirect, within an educational environment on the emotional safety and learning for the college student within the agricultural-based classroom. Three course types were observed within the agricultural-based educational environment that included courses with no equine interaction (Group A) and courses with equine interaction, both direct (Group B) and indirect (Group C) interaction with the horse. Indirect interaction included items such as observation of equine handling via a video or gaining knowledge from reading online materials, but not engaging in direct, hands-on activities with the horse. Development of emotional safety within the students enrolled within these courses was measured using a self-reporting emotional safety evaluation. Due to the structure of the scale, a decrease in emotional safety indicated a positive change. Learning, both development of semantic and procedural memory, was measured using a student-completed knowledge examination and an instructor-completed skill evaluation, respectively. While significant improvement in emotional safety was not observed within any of the course types, a weak negative correlation was found between emotional safety and semantic memory for students enrolled in equine courses, both direct (R = -0.55, R2 = 0.28) and indirect (R = -0.25, R2 = 0.06) interaction, finding as emotional safety scores lowered to the ideal range that knowledge improved. In addition, students within equine courses showed semantic memory development in specific areas of equine sciences (Group B: Grooming/Tacking, p = 0.03; Group C: Equine Behavior, p = 0.04) and direct equine interaction resulted in development of equine-based procedural memory in all four skill areas measured within the study (p = 0.00). As such, learning is promoted through equine interaction, whether direct or indirect interaction, within the agricultural-based classroom, suggesting that both forms of equine interaction can be a valuable educational tool for the instructor within the collegiate setting.

Dietary L-arginine supplementation during gestation in mice enhances reproductive performance and Vegfr2 transcription activity in the fetoplacental unit.

Regarded as one of the most versatile amino acids, arginine serves as a precursor for many molecules and has been reported to improve the reproductive performance of rats and pigs. To this end, we sought to determine if dietary L-arginine alters fetoplacental vascular endothelial growth factor receptor-2 (Vegfr2) transcription activity. Eighteen wild-type FVB/N female mice were bred to homozygous FVB/N-Tg(Vegfr2-luc)-Xen male mice. Bred female mice received 1 of 2 experimental diets: one supplemented with 2.00% (wt:wt) L-arginine (+Arg) or 1 supplemented with 4.10% (wt:wt) alanine (+Ala) to serve as an isonitrogenous control for +Arg. In addition, 6 mice were fed a nonsupplemented control (Con) diet to normalize bioluminescent imaging data. All data were analyzed using ANOVA followed by Fisher's least significant difference. Total feed intake did not differ between groups; however, mice in the +Arg group consumed more arginine (P < 0.05). Arginine supplementation increased weight gain during the latter one-third of gestation (d 12- 18), total litter size, number of pups born alive, number of placental attachment sites, litter birth weight, and litter weight of pups born alive but decreased the individual birth weights (P < 0.05). During d 12-18, arginine supplementation increased (P < 0.05) the mean total Vegfr2 transcription activity and Vegfr2 transcription activity corrected for fetoplacental mass. Moreover, mice in the +Arg group had an earlier rise in Vegfr2 transcription activity. In conclusion, our results demonstrate that the beneficial effect of dietary L-arginine supplementation on mammalian reproduction is associated with enhanced Vegfr2 transcription activity in fetoplacental tissues.

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.

BACKGROUND: Various obstacles are encountered by mammalian spermatozoa during their journey through the female genital tract, and only few or none will reach the site of fertilization. Currently, there are limited technical approaches for non-invasive investigation of spermatozoa migration after insemination. As the knowledge surrounding sperm behavior throughout the female genital tract still remains elusive, the recent development of self-illuminating quantum dot nanoparticles may present a potential means for real-time in vitro and in vivo monitoring of spermatozoa. RESULTS: Here, we show the ability of boar spermatozoa to harmlessly interact and incorporate bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles. The confocal microscope revealed in situ fluorescence of BRET-QD in the entire spermatozoon, while the ultra-structural analysis using the transmission electron microscope indicated BRET-QD localization on the sperm plasma membrane and intracellular compartment. In controlled-in vitro assays, bioluminescent imaging demonstrated that spermatozoa incubated with BRET-QD and luciferase substrate (coelenterazine) emit light (photons/sec) above the background, which confirmed the in situ fluorescence imaging. Most importantly, sperm motility, viability, and fertilizing potential were not affected by the BRET-QD incorporation when used at an appropriated ratio. CONCLUSIONS: Our results demonstrate that pig spermatozoa can incorporate BRET-QD nanoparticles without affecting their motility and capacity to interact with the oocyte when used at an appropriated balance. We anticipate that our study will enable in-depth exploration of the male components of in vivo migration, fertilization, and embryonic development at the molecular level using this novel approach.

Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock.

Despite the progress in assisted reproductive techniques, there is still a lack of rapid and minimally invasive in situ approaches for further enhancements of female fertility. Therefore, we synthesized clinically relevant liposome nanoparticles for ovarian intrafollicular injection to allow in vivo cellular imaging for future drug delivery, using the mare as an animal model. Ovarian follicles of living mares were injected in vivo with fluorescently labeled liposomes. Samples of the follicular wall (mural granulosa, theca interna, and theca externa), granulosa cells, and follicular fluid were harvested 24 h post-injection through the follicle wall biopsy (FWB), flushing, and aspiration techniques, respectively, using a transvaginal ultrasound-guided approach. In parallel, post-mortem dissected, and cultured porcine antral follicles were microinjected with doxorubicin-encapsulated liposomes to assess intracellular delivery potential. All injected mare and pig follicles were macroscopically healthy, and fluorescence imaging revealed successful intrafollicular binding to mural granulosa cells and progressive migration of liposomes to other follicle cell layers (theca interna, and theca externa), regardless of the follicle size. Intracellular delivery of doxorubicin was confirmed in all porcine follicle wall cell types. We conclude that the intrafollicular injection of nanomolecules is a promising approach for real-time monitoring of intrafollicular processes and potential utilization of in vivo cellular drug delivery to assist in follicle disease treatments and fertility improvement.

In vitro effects of relaxin on gene expression in porcine cumulus-oocyte complexes and developing embryos.

BACKGROUND: Relaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig. METHODS: Immature cumulus-oocyte complexes (COCs) were obtained from ovarian follicles of sows. In experiment 1, COCs were matured in the presence of 0, 20, or 40 ng relaxin/ml, or 10% (v/v) porcine follicular fluid. In experiment 2, COCs were in vitro matured, fertilized and resulting embryos were cultured in the presence of 0, 20, or 40 ng relaxin/ml. In experiment 3, COCs were matured in the presence of 40 ng relaxin/ml, fertilized and zygotes were cultured as indicated in experiment 2. We evaluated the proportions of matured oocytes in experiment 1, cleaved and blastocysts on Day 2 and Day 7 post insemination in all experiments. The total cell number of blastocysts was also evaluated. In parallel, transcription levels of both relaxin and its receptors (RXFP1 and RXFP2), as well as a pro- (Bax) and anti- (Bcl2-like 1) apoptotic-related genes were determined. All data were analyzed by ANOVA and significant differences were fixed for P < 0.05. RESULTS: In experiment 1, relaxin significantly increased the proportions of matured oocytes and cleaved embryos, as well as the expression level of RXFP2 mRNA compared to RXFP1 (P < 0.05). There was no effect on endogenous expression of relaxin and Bcl2-like1/Bax ratios. In all experiments, relaxin did not affect the proportions of blastocysts, but did significantly increase their total cell numbers (P < 0.05). Furthermore, no effect of relaxin was observed on Bcl2-like1/Bax expression ratios, which were similar between groups. CONCLUSIONS: Exogenous relaxin influences its own receptors expression, improves oocyte nuclear maturation. Its beneficial effect on total cell number of blastocysts appears to be through a Bcl2-like1/Bax-independent mechanism.

In vivo monitoring of fetoplacental Vegfr2 gene activity in a murine pregnancy model using a Vegfr2-luc reporter gene and bioluminescent imaging.

BACKGROUND: Vascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is characterized by a marked increase in fetoplacental angiogenesis. Thus, the objective of this study was to determine the feasibility of monitoring fetoplacental Vegfr2 gene activity non-invasively using a Vegfr2-luc reporter transgenic mouse and bioluminescent imaging. METHODS: Imaging parameters were optimized using two wild-type (WT) females, bearing Vegfr2-luc fetuses. Then, seven WT females, bred to Vegfr2-luc males, were imaged from gestational day (GD) 12 to 18 to determine the usefulness of the Vegfr2-luc mouse as a model for studying fetoplacental Vegfr2 activity during pregnancy. Semi-quantitative RT-PCR of Vegfr2 was also performed on whole fetoplacental units during this time. Additionally, resultant neonates were imaged at postnatal day (PND) 7, 14 and 21 to monitor Vegfr2 activity during post-natal development. RESULTS: Fetoplacental Vegfr2 gene activity was detected as light emissions beginning on GD 12 of gestation and increased throughout the imaging period (P < 0.05), and this paralleled the Vegfr2 mRNA data obtained from RT-PCR analysis. A decline in fetoplacental light emissions was associated with a poor pregnancy outcome in one pregnancy, indicating that this approach has potential use for studies monitoring pregnancy well being. Additionally, neonatal Vegfr2 activity was detected at PND 7, 14 and 21 but declined with time (P < 0.0001). CONCLUSIONS: In utero fetoplacental Vegfr2 gene activity was monitored longitudinally in a quantitative manner using a luciferase reporter gene and bioluminescent imaging during the latter third of gestation. This study demonstrates the feasibility of using the Vegfr2-luc mouse to monitor late gestation fetoplacental angiogenic activity under normal and experimental conditions. Additionally, neonatal Vegfr2 gene activity was monitored for three weeks postpartum, allowing continuous monitoring of Vegfr2 activity during the latter third of gestation and postnatal development within the same animals.

Examination of relaxin and its receptors expression in pig gametes and embryos.

BACKGROUND: Relaxin is a small peptide also known as pregnancy hormone in many mammals. It is synthesized by both male and female tissues, and its secretions are found in various body fluids such as plasma serum, ovarian follicular fluid, utero-oviduct secretions, and seminal plasma of many mammals, including pigs. However, the presence and effects of relaxin in porcine gametes and embryos are still not well-known. The purpose of this study was to assess the presence of relaxin and its receptors RXFP1 and RXFP2 in pig gametes and embryos. METHODS: Immature cumulus-oocyte complexes (COCs) were aspirated from sows' ovaries collected at the abattoir. After in vitro-maturation, COCs were in vitro-fertilized and cultured. For studies, immature and mature COCs were separately collected, and oocytes were freed from their surrounding cumulus cells. Denuded oocytes, cumulus cells, mature boar spermatozoa, zygotes, and embryos (cleaved and blastocysts) were harvested for temporal and spatial gene expression studies. Sections of ovary, granulosa and neonatal porcine uterine cells were also collected to use as controls. RESULTS: Using both semi-quantitative and quantitative PCRs, relaxin transcripts were not detected in all tested samples, while RXFP1 and RXFP2 mRNA were present. Both receptor gene products were found at higher levels in oocytes compared to cumulus cells, irrespective of the maturation time. Cleaved-embryos contained higher levels of RXFP2 mRNA, whereas, blastocysts were characterized by a higher RXFP1 mRNA content. Using western-immunoblotting or in situ immunofluorescence, relaxin and its receptor proteins were detected in all samples. Their fluorescence intensities were consistently more important in mature oocytes than immature ones. The RXFP1 and RXFP2 signal intensities were mostly located in the plasma membrane region, while the relaxin ones appeared homogeneously distributed within the oocytes and embryonic cells. Furthermore, spermatozoa displayed stronger RXFP2 signal than RXFP1 after western-immunoblotting. CONCLUSION: All together, our findings suggest potential roles of relaxin and its receptors during oocyte maturation, early embryo development, and beyond.

Immunomodulator expression in trophoblasts from the feline immunodeficiency virus (FIV)-infected cat.

BACKGROUND: FIV infection frequently compromises pregnancy under experimental conditions and is accompanied by aberrant expression of some placental cytokines. Trophoblasts produce numerous immunomodulators that play a role in placental development and pregnancy maintenance. We hypothesized that FIV infection may cause dysregulation of trophoblast immunomodulator expression, and aberrant expression of these molecules may potentiate inflammation and compromise pregnancy. The purpose of this project was to evaluate the expression of representative pro-(TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, IL-12p35, IL-12p40, IL-18, and GM-CSF) and anti-inflammatory cytokines (IL-4, IL-5, and IL-10); CD134, a secondary co-stimulatory molecule expressed on activated T cells (FIV primary receptor); the chemokine receptor CXCR4 (FIV co-receptor); SDF-1α, the chemokine ligand to CXCR4; and FIV gag in trophoblasts from early-and late-term pregnancy. METHODS: We used an anti-cytokeratin antibody in immunohistochemistry to identify trophoblasts selectively, collected these cells using laser capture microdissection, and extracted total RNA from the captured cell populations. Real time, reverse transcription-PCR was used to quantify gene expression. RESULTS: We detected IL-4, IL-5, IL-6, IL-1ß, IL-12p35, IL-12p40, and CXCR4 in trophoblasts from early-and late-term pregnancy. Expression of cytokines increased from early to late pregnancy in normal tissues. A clear, pro-inflammatory microenvironment was not evident in trophoblasts from FIV-infected queens at either stage of pregnancy. Reproductive failure was accompanied by down-regulation of both pro-and anti-inflammatory cytokines. CD134 was not detected in trophoblasts, and FIV gag was detected in only one of ten trophoblast specimens collected from FIV-infected queens. CONCLUSION: Feline trophoblasts express an array of pro-and anti-inflammatory immunomodulators whose expression increases from early to late pregnancy in normal tissues. Non-viable pregnancies were associated with decreased expression of immunomodulators which regulate trophoblast invasion in other species. The detection of FIV RNA in trophoblasts was rare, suggesting that the high rate of reproductive failure in FIV-infected queens was not a direct result of viral replication in trophoblasts. The influence of placental immune cells on trophoblast function and pregnancy maintenance in the FIV-infected cat requires additional study.

A comparative biomechanical analysis of term fetal membranes in human and domestic species.

OBJECTIVE: The purpose of this study was to biomechanically characterize and compare human, porcine, equine, and ovine fetal membranes. STUDY DESIGN: Noncontact metrology was used for topographic analyses. Uniaxial tensile testing was performed to resolve specific biomechanical values. Puncture force and radial stresses were determined with biaxial puncture testing. Microstructure and surface tortuosity were analyzed histologically. RESULTS: Equine and human membranes sustained larger magnitude loading, but ovine and porcine membranes exhibited stronger material properties. Biaxial puncture validated uniaxial results; human and equine groups accommodated the largest loads but lowest stresses. Equine membranes were mostly vascularized; tortuosity was highest in porcine membranes. Species' gestation length was correlated positively with membrane thickness. CONCLUSION: The anatomy of placentation and length of species gestation show distinct relationships to membrane biomechanics. Unlike other species, human fetal membranes do not compensate for structural weakness with a thicker membrane. This finding may explain the high incidence of preterm premature rupture of membranes in humans.