Mimicking the temperature gradient between the sow's oviduct and uterus improves in vitro embryo culture output.

This work was designed to determine temperature conditions within the reproductive tract of the female pig and study their impact on ARTs. Temperatures were recorded using a laparo-endoscopic single-site surgery assisted approach and a miniaturized probe. Sows and gilts were used to address natural cycle and ovarian stimulation treatments, respectively. According to in vivo values, IVF was performed at three temperature conditions (37.0°C, 38.5°C and 39.5°C) and presumptive zygotes were cultured in these conditions for 20 h, while further embryo culture (EC) (21-168 h post-insemination) was maintained at 38.5°C. After 20 h, different fertility parameters were assessed. During EC, cleavage and blastocyst stages were evaluated. Sperm membrane fluidity at the experimental temperatures was studied by using differential scanning calorimetry and fluorescence recovery after photobleaching techniques. An increasing temperature gradient of 1.5°C was found between the oviduct and uterus of sows (P < 0.05) and when this gradient was transferred to pig in vitro culture, the number of poly-nuclear zygotes after IVF was reduced and the percentage of blastocysts was increased. Moreover, the temperature transition phase for the boar sperm membrane (37.0°C) coincided with the temperature registered in the sow oviduct, and sperm membranes were more fluid at 37.0°C compared with those of sperm incubated at higher temperatures (38.5°C and 39.5°C). These data suggest that there may be an impact of physiological temperature gradients on human embryo development.

Transcriptomic and computational analysis identified LPA metabolism, KLHL14 and KCNE3 as novel regulators of Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a complex biological program between physiology and pathology. Here, amniotic epithelial cells (AEC) were used as in vitro model of transiently inducible EMT in order to evaluate the transcriptional insights underlying this process. Therefore, RNA-seq was used to identify the differentially expressed genes and enrichment analyses were carried out to assess the intracellular pathways involved. As a result, molecules exclusively expressed in AEC that experienced EMT (GSTA1-1 and GSTM3) or when this process is inhibited (KLHL14 and KCNE3) were identified. Lastly, the network theory was used to obtain a computational model able to recognize putative controller genes involved in the induction and in the prevention of EMT. The results suggested an opposite role of lysophosphatidic acid (LPA) synthesis and degradation enzymes in the regulation of EMT process. In conclusion, these molecules may represent novel EMT regulators and also targets for developing new therapeutic strategies.

Characterization, GFP gene Nucleofection, and allotransplantation in injured tendons of ovine amniotic fluid-derived stem cells.

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ∼63% and ∼37%, while cell recoveries were ∼10% and ∼22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achille's tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.

Quantitative assessment of bronchiolar smooth muscle in healthy and diseased porcine lungs.

Smooth muscle cells are major components of bronchiolar wall. Bronchiolar smooth muscle is reported to increase in some veterinary pulmonary disorders, but such assumption is not supported by detailed morphometric analyses. The present investigation aimed at quantitatively evaluating bronchiolar smooth muscle in healthy and diseased pig lungs. Our results suggest that bronchiolar smooth muscle cells significantly modify in size and number under different disease conditions, namely parasitic bronchopneumonia and Mycoplasma hyopneumoniae-induced enzootic pneumonia. Further studies are needed in order to understand the pathogenesis and the functional impact of such changes.

Achilles tendon regeneration can be improved by amniotic epithelial cell allotransplantation.

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14(-), CD31(-), CD45(-), CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial-temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-ß1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

Role of TRPV1 channels during the acquisition of fertilizing ability in boar spermatozoa.

Recently, the transient receptor potential vanilloid type 1 (TRPV1) channel was shown to be involved in capacitation, the process that allows mammalian spermatozoa to acquire their fertilizing ability within the female genital tract. Unfortunately, the role of TRPV1 in this process is still unclear. Thus, the aims of the present work were to 1) investigate the function of TRPV1 in the male gamete signaling system and 2) modulate TRPV1 activity by administering a specific activator, capsaicin, or a specific inhibitor, capsazepin, to spermatozoa during in vitro capacitation. Using confocal microscopy, cellular responses were assessed in terms of changes in 1) cell membrane resting potential, 2) intracellular calcium concentrations, and 3) actin polymerization dynamics. As a result, TRPV1 channels were shown to act as specific cationic channels: their activation led to membrane depolarization and, consequently, the opening of voltage-gated calcium channels and an increase in intracellular calcium concentrations. These ionic events promote actin cytoskeletal depolymerization and a loss of acrosome structure integrity. In contrast, TRPV1 inhibition caused a slowing of the capacitation-dependent increase in intracellular calcium concentrations, a reduction in actin polymerization, and acrosome rupture. In conclusion, these results suggest that TRPV1 channels modulate the major pathways involved in capacitation.

Role of TRPV1 channels in boar spermatozoa acquisition of fertilizing ability.

Recently the transient receptor potential vanilloid type 1 (TRPV1) has been described to be involved in the capacitation, the process leading mammalian spermatozoa to acquire full fertilizing ability within the female genital tract. TRPV1 immunolocalization during capacitation and the effect of TRPV1 inhibition by the capsazepin (CPZ) or activation by the capsaicin (CPS) on membrane resting potential, calcium clearance and actin polymerization have been investigated. It was found that the capacitation promoted the translocation of TRPV1 from the post-acrosomal to the apical region of sperm head. Moreover the CPZ induced the progressive drop in intracellular Ca2+ levels during capacitation and the inhibition of actin polymerization in the acrosomal region. On the contrary, the CPS caused the sperm membrane depolarization due to the Na+ influx and the consequent voltage gated calcium channels (VGCC) opening. In conclusion it was suggested that TRPV1 channels modulate the major pathways involved in capacitation.

Extremely low frequency electromagnetic field exposure affects fertilization outcome in swine animal model.

Modern society continuously exposes the population to electromagnetic radiation, the effects of which on human health, in particular reproduction, are still unknown. The aim of this research was to assess the effect of acute (1h) exposure of boar spermatozoa to a 50 Hz extremely low frequency electromagnetic field (ELF-EMF) on early fertility outcome. The effect of intensities ranging from 0 to 2 mT on morpho-functional integrity of capacitated spermatozoa was examined in vitro. The oviducts containing or without spermatozoa were then exposed to the minimum in vivo, TD(50,) and maximum intensities determined in vitro, 4h before ovulation. The effects of ELF-EMF on spermatozoa in terms of early embryo development were evaluated after 12h and 6 days. It was found that in vitro ELF-EMF > 0.5 mT induced a progressive acrosome damage, thus compromising the ability of spermatozoa to undergo acrosomal reaction after zona pellucida stimulation and reducing the in vitro fertilization outcome. These effects became evident at 0.75 mT and reached the plateau at 1 mT. Under in vivo conditions, the ELF-EMF intensity of 1 mT was able to compromise sperm function, significantly reducing the fertilization rate. In addition, the exposure of oviducts to fields > or = 0.75 mT in the absence of spermatozoa was able to negatively affect early embryo development. In fact, it was found to cause a slowdown in the embryo cleavage. In conclusion, it was demonstrated how and at which intensities ELF-EMF negatively affect early fertility outcome in a highly predictive animal model.

Vascular supply as a discriminating factor for pig preantral follicle selection.

This research analyses how somatic and vascular compartments change during preantral follicle growth. To address this aim, theca-granulosa (somatic) proliferation indexes (PIs), proportion of proliferating endothelial cells (PE), vascular area (VA) and vascular endothelial growth factor A (VEGFA) expression were simultaneously recorded on single healthy preantral follicles, classified into six different stages on the basis of the diameter and the granulosa layers. An autonomous blood vessel network starts to appear only in class 3. Vascular remodelling requires VEGFA expression, and VEGFA mRNA and VA significantly increase between class 3 and classes 4 and 5 and, further, in class 6. In addition, a positive correlation exists between these parameters in classes 3-5. Despite variation in angiogenesis results from classes 3 to 5, the statistical analysis reveals that the vascular parameters are positively and strictly correlated with somatic PIs. Conversely, class 6, also characterized by higher values of somatic PIs, displays a stable proportion of PEs ( congruent with 40%) without showing any correlation among the different parameters analysed. To identify follicular subpopulations within different classes, a multivariate hierarchical cluster analysis was performed. This analysis reveals that the majority of classes 3 and 4 are quiescent follicles or structures that grow very slowly. Class 5 represents a transitory category, where half of the follicles maintain a low activity and the remaining express significantly higher levels of granulosa PI and VA. The follicles with this high activity are probably able to reach class 6 becoming dominant structures where somatic and vascular parameters are constantly on high levels and the VA remains the unique differentiating element.

Effects of 50 Hz extremely low frequency magnetic field on the morphology and function of boar spermatozoa capacitated in vitro.

The aim of this study was to evaluate the effect of an acute exposure to a sinusoidal MF-ELF (50 Hz, 1mT) on the ability of boar mature spermatozoa to acquire the fertilizing competence in vitro. The spermatozoa exposed during the 4h of incubation to the MF-ELF were evaluated for morphological (surface morphology and acrosome integrity) and functional parameters (cell viability, motility, induction of acrosomal reaction, AR, and the ability to in vitro fertilize oocytes). In parallel, the intracellular Ca(2+) levels as well as the major mechanisms of Ca(2+) clearance were assessed: (45)Ca intakes and intracellular Ca(2+) sequestration by analyzing intracellular Ca(2+) elevation induced by thapsigargin or studying mitochondrial function with Mito-Tracker. The MF-ELF exposure did not affect sperm viability and morphology during the first h of incubation when sperm Ca(2+) homeostasis were already compromised. First of all, MF-ELF treated spermatozoa showed resting intracellular Ca(2+) levels significantly lower than those recorded in controls. This result was dependent on a lower extracellular Ca(2+) intake and from the inhibitory role exerted on both intracellular Ca(2+) storages. As a consequence, after 1h of incubation MF-ELF exposed cells displayed a reduced motility, a modest reactivity when coincubated with solubilized zonae pellucidae and a reduction in oocyte penetrating ability. After 2 or 4h of incubation, in addition, signs of morphological damage appeared on plasma membrane and at acrosomal level. In conclusion, MF-ELF influence negatively spermatozoa first by impairing cell Ca(2+) homeostasis then by dramatically affecting sperm morphology and function.

Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period.

Vascular endothelial growth factor (VEGF) expression pattern and blood vessel remodelling were evaluated during the transition from the preovulatory follicle to the corpus luteum (CL). To this end, prepubertal gilts were treated with equine chorionic gonadotrophin (eCG) to collect preovulatory follicles (60 h after eCG) and with human chorionic gonadotrophin (hCG) to obtain periovulatory follicles 18 h and 36 h later. The VEGF mRNA content was analysed by in situ hybridization, while protein localization in follicular fluid (FF) and in granulosa and theca compartments was evaluated by ELISA, immunohistochemistry or western blot. Blood vessel architecture and vascular area (VA) were investigated using immunohistochemistry for von Willenbrand Factor, a specific endothelial marker. Vascular remodelling was finally tested using Ki-67 immunocytochemistry as a proliferation marker, or alpha-smooth muscle actin (alpha-SMA) as a specific mural cell marker. eCG-treated follicles showed high VEGF levels and two concentric blood vessel networks composed of proliferating endothelial cells without any association with mural components. hCG injection inhibited VEGF synthesis in the granulosa compartment and, as a consequence, the protein fell within the FF. In parallel, endothelial cell proliferation stopped and the VA decreased. Close to ovulation, VEGF production restarted in both follicular compartments and VEGF mRNA content significantly increased in the theca layer. Changes in follicular VEGF secretion were observed; the protein disappeared from FF and was observed in the extracellular matrix. An active angiogenesis characterized the follicle; endothelial cell proliferation was associated with a recruitment of alpha-SMA-positive mural cells. The data presented in this work showed that, in the phases preceding ovulation, a complete vascular remodelling occurs, characterized by both an evident neovascularization and the appearance of blood vessels presenting smooth musculature which could be involved in CL formation after ovulation.