Immature Porcine Research Articles

MiR-222 Suppresses Immature Porcine Sertoli Cell Growth by Targeting the GRB10 Gene Through Inactivating the PI3K/AKT Signaling Pathway

Sertoli cells are central and essential coordinators of spermatogenesis. Accumulating evidence has demonstrated that miRNAs participate in the regulation of Sertoli cell growth. However, the functions and the regulatory mechanisms of miRNAs in Sertoli cells of domestic animals remain largely unknown. Here we report that miR-222 overexpression repressed cell cycle progression and proliferation and promoted the apoptosis of immature porcine Sertoli cells, whereas miR-222 inhibition resulted in the opposite result. miR-222 directly targeted the 3′-UTR of the GRB10 gene and inhibited its mRNA abundance. An siRNA-induced GRB10 knockdown showed similar effects as did miR-222 overexpression on cell proliferation and apoptosis and further attenuated the role of miR-222 inhibition. Furthermore, both miR-222 overexpression and GRB10 inhibition repressed the phosphorylation of PI3K and AKT, the key elements of the PI3K/AKT signaling pathway, whereas GRB10 inhibition offsets the effects of the miR-222 knockdown. Overall, we concluded that miR-222 suppresses immature porcine Sertoli cell growth by targeting the GRB10 gene through inactivation of the PI3K/AKT signaling pathway. This study provides novel insights into the epigenetic regulation of porcine spermatogenesis by determining the fate of Sertoli cells.

MiR‐130a promotes immature porcine Sertoli cell growth by activating SMAD5 through the TGF‐β‐PI3K/AKT signaling pathway

Sertoli cells play vital roles in normal spermatogenesis, and microRNAs (miRNAs) participate in regulating Sertoli cell development. However, the functions and mechanisms of action of most identified miRNAs in porcine Sertoli cells remain largely unknown. Herein, we primarily explored the regulatory roles of miR-130a in immature porcine Sertoli cells using EdU-based high-content screening assay. The results demonstrated that 27 miRNAs have potential roles in the promotion of immature porcine Sertoli cell proliferation, and miR-130a was identified as a promising candidate. miR-130a promoted cell cycle progression and cell proliferation, whereas it impeded cell apoptosis in immature porcine Sertoli cells. It also contributed to Sertoli cell proliferation and testis development in vivo. A TMT-based proteomics approach revealed that miR-130a regulated the expression of 91 proteins and multiple pathways, including the TGF-β and PI3K/AKT signaling. miR-130a did not directly target the 3'-UTR of SMAD5; however, it increased SMAD5 phosphorylation. Moreover, miR-130a enhanced TGF-β signaling by activating SMAD5 protein, and TGF-β signaling further activated the PI3K/AKT signaling pathway to promote cell proliferation and inhibit cell apoptosis in porcine immature Sertoli cells. Collectively, miR-130a promoted immature porcine Sertoli cell growth by activating SMAD5 through the TGF-β-PI3K/AKT signaling pathway. This study, therefore, provides novel insights into the effects of miR-130a on porcine spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

Vitrification of Porcine Oocytes and Zygotes in Microdrops on a Solid Metal Surface or Liquid Nitrogen.

Oocyte cryopreservation is a potent approach to keep female germplasm safe from epidemic diseases. In the last decade, we developed simple, cheap, and robust vitrification protocols which enable quick cryopreservation of immature porcine oocytes and zygotes in large numbers. In this chapter, we describe vitrification procedures for porcine oocytes and zygotes where they are vitrified in 1-2μL aliquots of a defined (protein-free) vitrification medium and dropped either on a metal surface pre-cooled from the bottom with liquid nitrogen (solid surface vitrification) or directly into liquid nitrogen. Vitrified microdrops can be stored in cryo-vials in liquid nitrogen. Low concentrations of permeating cryoprotectants during equilibration and proper temperatures during equilibration and warming are crucial for achieving high survival rates. The device used for cooling does not seem to affect system efficacy as vitrification of oocytes or zygotes either on Cryotop® sheets or in microdrops were equally effective.

Vitrification of porcine cumulus-oocyte complexes at the germinal vesicle stage does not trigger apoptosis in oocytes and early embryos, but activates anti-apoptotic Bcl-XL gene expression beyond the 4-cell stage.

The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4–8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

Percutaneous epicardial placement of a prototype miniature pacemaker under direct visualization: An infant porcine chronic survival study.

Pacemaker implantation in infants typically consists of surgical epicardial lead placement with an abdominal generator. Here, we describe the chronic performance of our minimally invasive prototype miniature pacemaker implanted under direct visualization in an immature porcine model. Twelve piglets underwent miniature pacemaker implantation. A self-anchoring two-channel access port was inserted into a 1cm incision in the subxiphoid space, and a thoracoscope was inserted into the main channel to visualize the thoracic cavity under insufflation. The pacemaker leadlet was inserted through a sheath via secondary channel and affixed against the epicardium using a helical side-biting electrode. The miniature pacemaker was tucked into the incision, which was sutured closed. Ventricular sensing, leadlet impedance, and capture thresholds were measured biweekly. A limited necropsy was performed after euthanasia. Nine piglets were followed for a median of 78 (IQR 52-82) days and gained 6.6 ± 3.2kg. Three animals were censored from the analysis due to complications unrelated to the procedure. Capture thresholds rose above maximal output after a median of 67 (IQR 40-69) days. At termination, there was a significant decrease in R-wave amplitude (P=.03) and rise in capture thresholds at 0.4ms (P=.01) and 1.0ms pulse widths (P=.02). There was no significant change in leadlet impedance (P=.74). There were no wound infections. There were no infections following minimally invasive implantation of our prototype miniature pacemaker. Improvements to epicardial fixation are necessary to address diminished leadlet efficacy over time.

27 Vitrification at the germinal vesicle stage does not trigger apoptosis in porcine oocytes and early embryos

Porcine immature oocytes can survive vitrification at high rates and retain their ability to undergo maturation and fertilization; however, the procedure reduces their competence for subsequent embryo development via unknown mechanisms (Somfai et al. 2014 Plos One 9, e97731). The aim of the present study was to clarify whether our vitrification procedure at the germinal vesicle stage triggers apoptosis in oocytes and subsequent developing embryos. Immature porcine cumulus-oocyte complexes obtained from slaughterhouse-derived ovaries were vitrified and warmed by our method (Appeltant et al. 2018 Cryobiology 85, 87-94) immediately after collection (vitrified group). The oocytes were equilibrated in 2% (vol/vol) ethylene glycol and 2% (vol/vol) propylene glycol for 13-15min. Then, they were vitrified by dropping them into liquid nitrogen in 2-μL microdrops of a medium composed of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3M sucrose, and 50mgmL−1 polyvinylpyrrolidone. After warming, they were subjected to IVM, fertilization (IVF), and embryo culture using chemically defined media (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208-213). From each collected batch, a group of oocytes was processed without vitrification (control group). Apoptosis was assayed in membrane-intact oocytes at the end of IVM and in cleavage-stage embryos on Day 2 after IVF (Day 0) by the CaspACE FITC-VAD-FMK In Situ Caspase Marker (Promega; Experiment 1), deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL; Experiment 2), and analysis of mRNA levels by RT-qPCR for the pro-apoptotic Bax and CASP3 genes (Experiment 3). Each experiment was replicated three times. Data were analysed by Kruskal-Wallis test followed by Dunn's multiple comparisons test. The mean survival rate of vitrified oocytes was 89.2%. There was no significant difference between the control and vitrified groups in relative caspase levels in IVM oocytes and in 2- to 4-cell embryos after IVF; however, significantly increased caspase activity (P&amp;lt;0.05) was detected in oocytes and embryos after treatment with 10 μM staurosporine (positive control). There was no significant difference between the control and vitrified groups in the proportion of TUNEL-positive oocytes (4.1 and 0.8%, respectively) and embryos (0 and 0%, respectively), whereas 96.6% of oocytes and 100% of cleavage stage embryos treated with 1000IUmL−1 deoxyribonuclease I (positive control) were proven to be TUNEL positive (P&amp;lt;0.05). Similar mRNA levels for Bax and CASP3 genes were detected in oocytes at the end of IVM and subsequent developing 4- to 8-cell embryos between the control and vitrified groups. In conclusion, vitrification of porcine oocytes at the germinal vesicle stage by our method did not trigger apoptosis in oocytes and subsequent developing embryos. This work was supported by the Japan Science and Technology Agency (JST)/Japan International Cooperation Agency (JICA) Science and Technology Research Partnership for Sustainable Development (SATREPS).

MiR-499 promotes immature porcine Sertoli cell growth through the PI3K/AKT pathway by targeting the PTEN gene.

Sertoli cells are indispensable for normal spermatogenesis, and increasing evidence has shown that miRNAs participate in the regulation of Sertoli cell growth. However, the functions and regulatory mechanisms of miRNAs in Sertoli cells of domestic animals have not been fully investigated. In the present study, we mainly investigated the regulatory roles of miR-499 in immature porcine Sertoli cells. The results showed that miR-499 was mainly located in the basement section of seminiferous tubules of prepubertal porcine testicular tissue. Overexpression of miR-499 promoted cell proliferation and inhibited apoptosis, whereas miR-499 inhibition resulted in the opposite effect. The PTEN gene was directly targeted by miR-499, and the expression of mRNA and protein was also negatively regulated by miR-499 in immature porcine Sertoli cells. siRNA-induced PTEN knockdown resulted in a similar effect as an overexpression of miR-499 and abolished the effects of miR-499 inhibition on immature porcine Sertoli cells. Moreover, both miR-499 overexpression and the PTEN knockdown activated the PI3K/AKT signaling pathway, whereas inhibition of the PI3K/AKT signaling pathway caused immature porcine Sertoli cell apoptosis and inhibited cell proliferation. Overall, miR-499 promotes proliferation and inhibits apoptosis in immature porcine Sertoli cells through the PI3K/AKT pathway by targeting the PTEN gene. This study provides novel insights into the effects of miR-499 in spermatogenesis through the regulation of immature Sertoli cell proliferation and apoptosis.

Genes regulating programmed cell death are significantly upregulated in porcine immature oocytes

Abstract Correct maturation of the oocyte is crucial for further fertilization and embryogenesis. It comprises of both nuclear and cytoplasmic maturation, during which the proteins, nutrients and mRNAs are assembled. Cumulus cells are connected with the oocyte via gap-junctions, which enable bi-directional transfer of molecules, forming cumulus-oocyte complex (COC). The expression pattern in CCs is thought to resemble the genes expressed in the oocyte. The CCs surrounding the gamete of high developmental competence have an increased expression of apoptotic markers. Therefore, our aim in this study was to determine whether any apoptosis-related genes are upregulated in porcine oocytes before or after IVM. We isolated COCs from 45 pubertal crossbred gilts, performed brilliant cresyl blue (BCB) staining and analyzed the gene expression pattern in oocytes before and after IVM with the use of microarray analysis. The results include 419 differentially expressed transcripts, 25 of which belong to „regulation of apoptosis” and „regulation of cell death” GO BP terms. This set of genes includes BCLAF1, EIF2AK3, KLF10, MIF, MAP3K1, NOTCH2, TXNIP and APP, all of which have been upregulated in immature porcine oocytes. Our results suggest that they play part in porcine oocyte maturation and could be used as potential markers of female gamete’s developmental competence. This knowledge could serve as a basis to improve ART in pigs.

MiR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene

MicroRNAs (miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34c is indispensable in the late steps of spermatogenesis. However, whether miR-34c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34c. The results from experiment I demonstrated that the relative expression level of miR-34c in swine testicular tissues increased (P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7 (SMAD7) decreased (P=0.0009) with curve. Furthermore, miR-34c expression levels showed a significant negative correlation (P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses (P<0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34c inhibited (P<0.05) proliferation and enhanced (P<0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8 (CCK-8) assay, the 5-Ethynyl-2'-deoxyuridine (EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA (siRNA) gave a similar result. It is concluded that miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.

Creation of a Porcine Kyphotic Model

Study DesignLarge animal study. ObjectiveCreate a thoracic hyperkyphotic deformity in an immature porcine spine, so that future researchers may use this model to validate spinal instrumentation and other therapies used in the treatment of hyperkyphosis. Summary of Background DataAlthough several scoliotic animal models have been developed, there have been no reports of a thoracic hyperkyphotic animal model creation in an immature animal. The present study was designed to produce a porcine hyperkyphotic model by the time the pig weighed 25 kg, which corresponds to the approximate weight of a child undergoing surgery for early-onset scoliosis (EOS). MethodsSuccessful surgical procedures were performed in 6 consecutive 10-kg (male, 5-week-old) immature Yorkshire pigs. Procedure protocol consisted of 1) a left thoracotomy at T10–T11, 2) screw placement at T9 and T11, 3) partial vertebrectomy at T10, 4) posterior interspinous ligament transection, and 5) placement of wire loop around screws and tightening. Weekly x-ray imaging was performed preoperatively and postoperatively, documenting progressively increasing kyphosis as the pig grew. Necropsy was performed 5–6 weeks after surgery, with CT, slab section, and histologic analysis. ResultsAverage T9–T11 kyphosis (measured by sagittal Cobb angle) was 6.1° ± 1.4° (mean ± SD) preoperatively, 30.5° ± 1.0° immediately postoperation, and significantly increased to 50.3° ± 7.2° (p < .0001) over 5–6 weeks in 6 consecutive pigs at time of necropsy. ConclusionsAn animal model of relatively more rigid-appearing thoracic hyperkyphotic deformities in immature pigs has been created. Subsequent studies addressing management of early-onset kyphosis with spinal instrumentation are now possible. Level of EvidenceLevel V

Increased Hip Intracapsular Pressure Decreases Perfusion of the Capital Femoral Epiphysis in a Skeletally Immature Porcine Model.

Increased intracapsular hip pressure is thought to be one of the possible etiologies of femoral head avascular necrosis after intra-articular proximal femoral fractures or acute slipped capital femoral epiphysis. The purpose of this study was to evaluate the relationship between intra-articular hip pressure (IAP) and epiphyseal perfusion pressure (EPP), and its dependency on skeletal maturity using a porcine model. Seven female Yorkshire-hybrid pigs were used to study the direct relationship between IAP and EPP. A needle inserted into the capsule provided both IAP monitoring and saline infiltration until IAP was above mean arterial pressure (MAP). Video simultaneously documented IAP, EPP, MAP. Parameters for all trials in each hip were averaged and compared between the 2 age groups. Significance was P<0.05. Four young hips (in pigs 10.3±1.0 wk, 27.4±2.0 kg) and 5 older hips (21.1±0.1 wk, 89.4±7.1 kg) were studied. There was no significant difference in the MAP (50.0±11.8 and 55.5±7.0 mm Hg respectively, P=0.411) between the 2 age groups. In the older hips, biphasic EPP persisted despite increasing IAP to an average of 177 mm Hg over MAP. In the young pigs, the biphasic EPP waveform ceased with increased IAP to an average of 28 mm Hg over MAP. Biphasic waveforms returned once IAP fell to an average of 5 mm Hg over MAP. Increased IAP resulted in tamponade of epiphyseal perfusion in the young, but not in the older hips. An intact physis may preclude intraosseous metaphyseal vessels from penetrating the epiphysis, leaving it vulnerable to retinacular artery tamponade. The IAP and EPP relationship has direct clinical practice implications. Hip capsulotomy and decompression in young patients with intra-articular proximal femoral fractures and increased intracapsular pressure may decrease avascular necrosis risk.

MiR-196a Promotes Proliferation and Inhibits Apoptosis of Immature Porcine Sertoli Cells.

Our previous study showed that the expression of miR-196a was significantly higher in immature porcine testes than in mature porcine testes. However, the role of miR-196a in immature Sertoli cells remains unclear. In this study, miR-196a mimics, miR-196a inhibitor, and microRNA-small hairpin negative control (miRNA-ShNC) were transfected into immature Sertoli cells, respectively. Subsequently, the cell cycle and apoptosis rate of the immature Sertoli cells were measured by flow cytometry, and the viability of the Sertoli cells was measured by the MTS assay. Furthermore, the candidate target genes of miR-196a were analyzed by bioinformatics, and the target genes were validated by dual luciferase reporter assays, then the expression of target genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assays. The results showed that miR-196a promotes the proliferation and inhibits the apoptosis of immature Sertoli cells. miR-196a directly binds the 3' untranslated region (3' UTR) of RCC2 and ABCB9. The expression of miR-196a was shown to be negatively correlated with the messenger RNA and protein levels of the RCC2 and ABCB9 genes. The study demonstrates that miR-196a regulates immature Sertoli cell proliferation and apoptosis and inhibits the expression of RCC2 and ABCB9.

Increase Functional β-Cell Mass in Subcutaneous Alginate Capsules With Porcine Prenatal Islet Cells but Loss With Human Adult Islet Cells.

Alginate (Alg)-encapsulated porcine islet cell grafts are developed to overcome limitations of human islet transplantation. They can generate functional implants in animals when prepared from fetal, perinatal, and adult pancreases. Implants have not yet been examined for efficacy to establish sustained, metabolically adequate functional β-cell mass (FBM) in comparison with human islet cells. This study in immune-compromised mice demonstrates that subcutaneous implants of Alg-encapsulated porcine prenatal islet cells with 4 × 105 β-cells form, over 10 weeks, a FBM that results in glucose-induced plasma C-peptide >2 ng/mL and metabolic control over the following 10 weeks, with higher efficiency than nonencapsulated, while failing in peritoneum. This intracapsular FBM formation involves β-cell replication, increasing number fourfold, and maturation toward human adult β-cells. Subcutaneous Alg-encapsulated human islet cells with similar β-cell number establish implants with plasma C-peptide >2 ng/mL for the first 10 weeks, with nonencapsulated cells failing; their β-cells do not replicate but progressively die (>70%), explaining C-peptide decline and insufficient metabolic control. An Alg matrix thus helps establish β-cell functions in subcutis. It allows formation of sustained metabolically adequate FBM by immature porcine β-cells with proliferative activity but not by human adult islet cells. These findings define conditions for evaluating its immune-protecting properties.

Novel use of telescoping growth rods in treatment of early onset scoliosis: An in vivo and in vitro study in a porcine model.

IntroductionTreatment of early‐onset scoliosis (EOS) can be difficult. Various forms of growing rods exist to correct deformity while delaying definitive spinal fusion. The disadvantage of traditional growing rods is need for repeated surgical lengthening procedures. Telescoping growth rods (TelGR) are a prototype new, guided growth technology with a rod mechanism that allows spontaneous longitudinal growth over time without manual lengthening. We hypothesized that the TelGR system will permit unrestricted growth with limited complications through 12 weeks in vivo, and that the range of motion (RoM) in each of three directions and stiffness of the TelGR system would not be significantly different than the rigid rod system in vitro.Materials and Methods In vivo: Six immature pigs were surgically implanted with TelGR with cephalad fixation at T6‐7 and caudal fixation at T14‐L1. Radiographs of the involved vertebral segments were measured postoperatively and after 12 weeks. In vitro: A robotic testing system was utilized for flexibility tests in flexion‐extension (FE), lateral bending (LB), and axial rotation (AR) of eight immature porcine specimens (T3‐T15). Testing was performed on both dual rigid rods and bilateral TelGR with instrumentation at T4‐5 and T13‐14.Results In vivo: Over the 12‐week period, the rod length of the TelGR increased an average of 65 mm. In vitro: TelGR demonstrated significantly increased motion in LB and AR RoM compared with rigid rods. No difference was noted in FE RoM.DiscussionThe in vivo results in this study showed expected skeletal growth with spines instrumented with TelGR. In vitro findings of increased RoM in AR and LB suggest that the TelGR system may be less rigid than traditional growing rods. Treatment with TelGR might, if proven efficacious in the clinical setting, decrease the need for repeated surgical intervention compared with traditional growing rods. This study adds to the limited body of biomechanical evidence examining guided growth technology.

Effects of age and rate of twist on torsional fracture patterns in infant porcine femora

ObjectiveLong bone fractures are a common injury in the pediatric population. Differentiation between abusive, or non-accidental trauma, and accidental trauma in children remains challenging for forensic practitioners. A recent clinical-based study was able to separate pediatric abusive from accidental trauma based on femoral fracture pattern using the ratio of fracture length over bone diameter (fracture ratio), as determined from radiographic analysis of this fractured bone. The forensic literature indicates more cases of abuse in younger pediatric victims than accidental cases. While this was the case in the clinical study, the effect was not shown to be statistically significant. Furthermore, while speed of trauma was not considered in the clinical study, a laboratory study with an immature bovine model indicates rotational speed influences fracture pattern, but specimen age was not varied in that study. Therefore, the objective of the current study was to use immature porcine femora to investigate the effects of age and rate of twist on a modified version of this fracture ratio parameter. MethodsFifteen pairs of porcine femora with various ages were twisted until observable failure using a custom-built torsional fixture. The left femur of each pair was twisted to failure at a rate of 3 deg/s, while the right femur was twisted at a rate of 90 deg/s. The torque and angle of rotation were recorded at a sampling rate of 10,000 Hz. Fracture ratio was defined as total fracture length divided by bone diameter. ResultsFracture ratio increased with specimen age, with specimens under the low rate of twist yielding a consistently lower fracture ratio than those from specimens under the high rate of twist. The results showed that both specimen age and rate of twist were significant factors influencing fracture ratio. ConclusionThe determination of abusive from accidental trauma in criminal cases, based on the pattern of long bone fracture alone, may need to include additional data on the specific age of the pediatric victim and the potential speed of the traumatic event.

Faster, cheaper, defined and efficient vitrification for immature porcine oocytes through modification of exposure time, macromolecule source and temperature

Vitrification reduces the developmental competence of porcine immature oocytes. We investigated the effects of modifying various factors on the viability and development of oocytes after vitrification. These factors included: 1) exposure to the vitrification solution, 2) macromolecule addition (bovine serum albumin (BSA) or polyvinyl pyrrolidone (PVP)), 3) treatment with cytochalasin B, 4) equilibration temperature, and 5) vitrification method (microdrop or Cryotop). Oocytes were equilibrated and vitrified using medium containing ethylene glycol and propylene glycol. After warming, oocytes were subjected to in vitro maturation, stimulated parthenogenetically, and cultured in vitro. Survival rate, nuclear maturation, cleavage, development to the blastocyst stage and their quality were compared between the vitrified groups and the non-vitrified control group. It was found that 1) exposure to the vitrification solution for longer than 30 s was detrimental to embryo development; 2) replacement of BSA with PVP improved embryo development; 3) cytochalasin B treatment reduced the survival rates, but did not affect the blastocyst development rates, 4) equilibration at room temperature (25 °C) was the most beneficial, and 5) the microdrop method improved survival rates. With these adjustments, we were able to establish a simplified and defined cryopreservation system for porcine immature oocytes with improved efficacy.

Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5′-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

Biomechanical properties and microstructure of neonatal porcine ventricles

Neonatal heart disorders represent a major clinical challenge, with congenital heart disease alone affecting 36,000 new-borns annually within the European Union. Surgical intervention to restore normal function includes the implantation of synthetic and biological materials; however, a lack of experimental data describing the mechanical behaviour of neonatal cardiac tissue is likely to contribute to the relatively poor short- and long-term outcome of these implants. This study focused on characterising the mechanical behaviour of neonatal cardiac tissue using a porcine model, to enhance the understanding of how this differs to the equivalent mature tissue. The biomechanical properties of neonatal porcine cardiac tissue were characterised by uniaxial tensile, biaxial tensile, and simple shear loading modes, using samples collected from the anterior and posterior walls of the right and left ventricles. Histological images were prepared using Masson’s trichrome staining, to enable assessment of the microstructure and correlation with tissue behaviour. The mechanical tests demonstrated that the neonatal cardiac tissue is non–linear, anisotropic, viscoelastic and heterogeneous. Our data provide a baseline describing the biomechanical behaviour of immature porcine cardiac tissue. Comparison with published data also indicated that the neonatal porcine cardiac tissue exhibits one-half the stiffness of mature porcine tissue in uniaxial extension testing, one-third in biaxial extension testing, and one-fourth stiffness in simple shear testing; hence, it provides an indication as to the relative change in characteristics associated with tissue maturation. These data may prove valuable to researchers investigating neonatal cardiac mechanics.

TALEN-mediated gene targeting in porcine spermatogonia.

Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.

Measurement and Finite Element Model Validation of Immature Porcine Brain-Skull Displacement during Rapid Sagittal Head Rotations.

Computational models are valuable tools for studying tissue-level mechanisms of traumatic brain injury, but to produce more accurate estimates of tissue deformation, these models must be validated against experimental data. In this study, we present in situ measurements of brain–skull displacement in the neonatal piglet head (n = 3) at the sagittal midline during six rapid non-impact rotations (two rotations per specimen) with peak angular velocities averaging 51.7 ± 1.4 rad/s. Marks on the sagittally cut brain and skull/rigid potting surfaces were tracked, and peak values of relative brain–skull displacement were extracted and found to be significantly less than values extracted from a previous axial plane model. In a finite element model of the sagittally transected neonatal porcine head, the brain–skull boundary condition was matched to the measured physical experiment data. Despite smaller sagittal plane displacements at the brain–skull boundary, the corresponding finite element boundary condition optimized for sagittal plane rotations is far less stiff than its axial counterpart, likely due to the prominent role of the boundary geometry in restricting interface movement. Finally, bridging veins were included in the finite element model. Varying the bridging vein mechanical behavior over a previously reported range had no influence on the brain–skull boundary displacements. This direction-specific sagittal plane boundary condition can be employed in finite element models of rapid sagittal head rotations.