Impact of Adipose Tissue Depot Harvesting Site on the Multilineage Induction Capacity of Male Rat Adipose-Derived Mesenchymal Stem Cells: An In Vitro Study.

Recently, substantial attention has been paid toward adipose-derived mesenchymal stem cells (AdMSCs) as a potential therapy in tissue engineering and regenerative medicine applications. Rat AdMSCs (r-AdMSCs) are frequently utilized. However, the influence of the adipose depot site on the multilineage differentiation potential of the r-AdMSCs is still ambiguous. Hence, the main objective of this study was to explore the influence of the adipose tissue harvesting location on the ability of r-AdMSCs to express the stem-cell-related markers and pluripotency genes, as well as their differentiation capacity, for the first time. Herein, we have isolated r-AdMSCs from the inguinal, epididymal, peri-renal, and back subcutaneous fats. Cells were compared in terms of their phenotype, immunophenotype, and expression of pluripotency genes using RT-PCR. Additionally, we investigated their potential for multilineage (adipogenic, osteogenic, and chondrogenic) induction using special stains confirmed by the expression of the related genes using RT-qPCR. All cells could positively express stem cell marker CD 90 and CD 105 with no significant in-between differences. However, they did not express the hematopoietic markers as CD 34 and CD 45. All cells could be induced successfully. However, epididymal and inguinal cells presented the highest capacity for adipogenic and osteogenic differentiation (21.36-fold and 11.63-fold for OPN, 29.69-fold and 26.68-fold for BMP2, and 37.67-fold and 22.35-fold for BSP, respectively, in epididymal and inguinal cells (p < 0.0001)). On the contrary, the subcutaneous cells exhibited a superior potential for chondrogenesis over the other sites (8.9-fold for CHM1 and 5.93-fold for ACAN, (p < 0.0001)). In conclusion, the adipose tissue harvesting site could influence the differentiation capacity of the isolated AdMSCs. To enhance the results of their employment in various regenerative cell-based therapies, it is thus vital to take the collection site selection into consideration.

Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer.

Mammalian oocytes can reprogram differentiated somatic cells into a totipotent state through somatic cell nuclear transfer (SCNT), which is known as cloning. Although many mammalian species have been successfully cloned, the majority of cloned embryos failed to develop to term, resulting in the overall cloning efficiency being still low. There are many factors contributing to the cloning success. Aberrant epigenetic reprogramming is a major cause for the developmental failure of cloned embryos and abnormalities in the cloned offspring. Numerous research groups attempted multiple strategies to technically improve each step of the SCNT procedure and rescue abnormal epigenetic reprogramming by modulating DNA methylation and histone modifications, overexpression or repression of embryonic-related genes, etc. Here, we review the recent approaches for technical SCNT improvement and ameliorating epigenetic modifications in donor cells, oocytes, and cloned embryos in order to enhance cloning efficiency.

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate.

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture.

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle-invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient-derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA-sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28, CTSE, CNN3, TFPI2, COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two-dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle-invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Age-related changes in DNA methylation levels at CpG sites in bull spermatozoa and in vitro fertilization-derived blastocyst-stage embryos revealed by combined bisulfite restriction analysis.

Age-associated methylation changes in genomic DNA have been recently reported in spermatozoa, and these changes can contribute to decline in fertility. In a previous study, we analyzed the genome-wide DNA methylation profiles of bull spermatozoa using a human DNA methylation microarray and identified one CpG site (CpG-1) that potentially reflects age-related methylation changes. In the present study, cryopreserved semen samples from a Japanese Black bull were collected at five different ages, which were referred to as JD1-5: 14, 19, 28, 54, and 162 months, respectively, and were used for genome-wide DNA methylation analysis and in vitro fertilization (IVF). Distinct age-related changes in methylation profiles were observed, and 77 CpG sites were found to be differently methylated between young and adult samples (JD1-2 vs. JD4-5). Using combined bisulfite restriction analysis (COBRA), nine CpG sites (including CpG-1) were confirmed to exhibit significant differences in their age-dependent methylation levels. Eight CpG sites showed an age-dependent increase in their methylation levels, whereas only one site showed age-dependent hypomethylation; in particular, these changes in methylation levels occurred rapidly at a young age. COBRA revealed low methylation levels in some CpG regions in the majority of the IVF blastocyst-stage embryos derived from spermatozoa at JD2-5. Interestingly, bulls with different ages did not show differences in their methylation levels. In conclusion, our findings indicated that methylation levels at nine CpG sites in spermatozoa changed with increasing age and that some CpG regions were demethylated after fertilization. Further studies are required to determine whether age-dependent different methylation levels in bull spermatozoa can affect fertility.

A microwell culture system that allows group culture and is compatible with human single media.

PURPOSE: A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. METHODS: Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. RESULTS: WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. CONCLUSIONS: In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies.

Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture.

Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air-liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer.

Differentially methylated CpG sites in bull spermatozoa revealed by human DNA methylation arrays and bisulfite analysis.

The methylation status of sperm DNA differs between individual bulls. However, the relationship between methylation status and bull sperm parameters is not well elucidated. The present study investigated genome-wide methylation profiles at 450,000 CpG sites in bull spermatozoa by using a human DNA methylation microarray. Semen samples from three adult Japanese Black bulls with different in vitro fertilization (IVF) results and from a young Holstein bull through sexual maturation (at ages 10, 10.5, 15, and 25 months) were used for the analysis. The heatmap displaying the results of microarray analysis shows inter- and intra-individual differences in methylation profiles. After setting a cut-off of 0.2 for differences between ages (10, 10.5 vs. 15, 25 months) or between IVF results (developed to the blastocyst-stage, > 20% vs. < 10%), different methylation levels were detected at approximately 100 CpGs. We confirmed the different DNA methylation levels of CpG sites by using combined bisulfite restriction analysis (COBRA); five of the CpG sites reflected methylation levels similar to those detected by the microarray. One of the CpG sites was thought to reflect an age-related increase in methylation levels, which was confirmed by COBRA and bisulfite sequencing. However, the relationship between methylation status and IVF results could not be shown here. In conclusion, methylation profiles of individual and age-related alterations in bull spermatozoa can be revealed using a human microarray, and methylation changes in some CpG sites can be easily visualized using COBRA. Combined analysis of DNA methylation levels and sperm parameters could be considered an effective approach for assessing bull fertility in the future.

Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2, PEG3, and PEG10, were obtained. These results illustrate the epigenetic characteristic of bovine parthenogenetic embryos and contribute to the identification of novel imprinted genes in cattle.

Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality.

Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was determined. Individual differences in semen were detected using the sperm TUNEL index in these bulls (P < 0.01). The sperm TUNEL index of cryopreserved semen obtained from test-mated Japanese Black (n = 30, including two bulls with a conception rate lower than 10%) and Holstein (n = 34) bulls were analyzed. The average sperm TUNEL index and conception rate resulting from artificial insemination (AI) were 4.7% and 55.7% for Japanese Black, and 4.9% and 39.5% for Holstein, respectively. A weak negative correlation between sperm TUNEL index and conception rate was observed in Holstein bulls (P < 0.05). Semen samples from six bulls with more than 10% sperm TUNEL index were studied, and these samples showed low sperm viability. However, semen resulting in a very low conception rate did not have a high sperm TUNEL index. Although it would be difficult to predict a low conception rate resulting from AI using the sperm TUNEL index alone, the index can be used as an additional parameter to provide a more comprehensive description of semen quality.

Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

Effects of trichostatin A on In vitro development and DNA methylation level of the satellite I region of swamp buffalo (Bubalus bubalis) cloned embryos.

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82-85%), cleavage (79-84%) and development to the 8-cell stage (59-65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.

Myogenic Differentiation and Immunomodulatory Properties of Rat Adipose-Derived Mesenchymal Stem/Stromal Cells.

The myogenic differentiation potential of MSCs is a key factor in their potential use as a cell source for muscle tissue repair and regeneration. Additionally, evaluating the immunomodulatory properties of MSCs is important to highlight their potential for regulating inflammation and supporting tissue regeneration. Given the limited literature on muscle differentiation potential and immunomodulatory properties, this study aims to characterize rat ADP MSCs for treating muscle disease. We isolated MSCs from adipose tissues around the periscapular region of the rats. We used a monoculture method for the myogenic differentiation and modified the myogenic induction medium by supplementing it with the growth factors FGF, HGF, and IGF. In rat ADP MSCs, expression of the MSC-specific marker, CD90, was 87.7%, while CD44 was 42.8%. For genes involved in immunomodulation, IGF1 and TGFB1 were highly expressed, while IL6 was poorly expressed. In addition to their trilineage differentiation potential, ADP MSCs exhibited the capacity to differentiate into myogenic cell lines, as evidenced by changes in cell morphology, leading to elongated and aligned structures and the expression of the MyoD and MYOG antibodies. The study found that ADP MSCs show great clinical promise for muscle regeneration.

Potential protective effects of L-carnitine against myocardial ischemia/reperfusion injury in a rat model.

Myocardial ischemia/reperfusion (I/R) injury is a growing concern for global public health. This study seeks to explore the potential protective effects of L-carnitine (LC) against heart ischemia-reperfusion injury in rats. To induce I/R injury, the rat hearts underwent a 30-min ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. We evaluated cardiac function through electrocardiography and heart rate variability (HRV) and conducted pathological examinations of myocardial structure. Additionally, the study investigated the influence of LC on myocardial apoptosis, inflammation, and oxidative stress in the context of I/R injury. The results show that pretreatment with LC led to improvements in the observed alterations in ECG waveforms and HRV parameters in the nontreated ischemic reperfusion model group, although most of these changes did not reach statistical significance. Similarly, although without a significant difference, LC reduced the levels of proinflammatory cytokines when compared to the values in the nontreated ischemic rat group. Furthermore, LC restored the reduced expressions of SOD1, SOD2, and SOD3. Additionally, LC significantly reduced the elevated Bax expressions and showed a nonsignificant increase in Bcl-2 expression, resulting in a favorable adjustment of the Bcl-2/Bax ratio. We also observed a significant enhancement in the histological appearance of cardiac muscles, a substantial reduction in myocardial fibrosis, and suppressed CD3 + cell proliferation in the ischemic myocardium. This small-scale, experimental, in vivo study indicates that LC was associated with enhancements in the pathological findings in the ischemic myocardium in the context of ischemia/reperfusion injury in this rat model. Although statistical significance was not achieved, LC exhibits potential and beneficial protective effects against I/R injury. It does so by modulating the expression of antioxidative and antiapoptotic genes, inhibiting the inflammatory response, and enhancing autonomic balance, particularly by increasing vagal tone in the heart. Further studies are necessary to confirm and elaborate on these findings.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Production of fertile offspring from oocytes grown in vitro by nuclear transfer in cattle.

Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte-granulosa cell complexes with a mean oocyte diameter of approximately 100 µm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360,000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

Supplementation of culture medium with L-carnitine improves development and cryotolerance of bovine embryos produced in vitro.

High lipid content in embryos is associated with low freezing tolerance. This study assessed the effects of exogenous L-carnitine, an enhancer of lipid metabolism, on the in vitro development and freezing survival of bovine embryos. Also, effects on metabolic activity, reactive oxygen species (ROS) and apoptosis were investigated. Supplementation of embryo culture medium with 1.518 mM or 3.030 mM L-carnitine significantly increased the rates of zygote development to the blastocyst stage and blastocyst cell numbers whereas 6.072 mM of this compound did not improve embryo development. Survival rates after slow freezing of blastocysts were significantly higher when embryos were cultured in the presence of 1.518 mM or 3.030 mM L-carnitine compared with the control. A lower density of lipid droplets was detected in L-carnitine-treated blastocysts compared with the control. L-carnitine significantly reduced ROS levels in 2-cell embryos but did not reduce ROS levels at later stages. The apoptotic cell rate was not different between control and L-carnitine-treated blastocysts. L-carnitine significantly increased ATP levels in 2-cell embryos but not at the 8-cell or blastocyst stages. L-carnitine increased the expression of metabolism-related ATP6 and COX1 genes in blastocysts. In conclusion, L-carnitine supplementation enhanced lipid metabolism in embryos resulting in improved development and cryotolerance of bovine blastocysts produced in vitro.

Downregulation of histone methyltransferase genes SUV39H1 and SUV39H2 increases telomere length in embryonic stem-like cells and embryonic fibroblasts in pigs.

Telomere is a nucleoprotein structure at the ends of chromosomes that helps to protect the ends of chromosomes from being fused with other chromosomes. Knockout of histone methyltransferases Suv39h1 and Suv39h2 increases the telomere length in murine cells, whereas downregulation of SUV39H1 and SUV39H2 genes decreases the telomere length in human cells, suggesting that telomere biology is different among mammalian species. However, epigenetic regulation of the telomere has not been studied in mammals other than the human and mouse. In the present study, the effect of knockdown of SUV39H1 and SUV39H2 genes on telomere length was examined in porcine embryonic stem-like cells (pESLCs) and porcine embryonic fibroblasts (PEFs). The telomeres in SUV39H1 and SUV39H2 knockdown (SUV39KD) pESLCs (37.1 ± 0.9 kb) were longer (P<0.05) compared with those of the control (33.0 ± 0.7 kb). Similarly, SUV39KD PEFs had longer telomeres (22.1 ± 0.4 kb; P<0.05) compared with the control (17.8 ± 1.1 kb). Telomerase activities were not different between SUV39KD pESLCs (10.4 ± 1.7) and the control (10.1 ± 1.7) or between SUV39KD PEFs (1.0 ± 0.3) and the control (1.0 ± 0.4), suggesting that telomerase activities did not contribute to the telomere elongation in SUV39KD pESLCs and SUV39KD PEFs. Relative levels of trimethylation of histone H3 lysine 9 and expressions of DNMT1, DNMT3A and DNMT3B were decreased in SUV39KD cells, suggesting that telomere lengthening in SUV39KD pESLCs and SUV39KD PEFs might be not only related to the loss of histone modification marks but also linked to the decrease in DNA methyltransferase in pigs.

Characterization of Rabbit Mesenchymal Stem/Stromal Cells after Cryopreservation.

Adipose tissues (ADPs) are an alternative source for mesenchymal stem/stromal cells (MSCs), given that conventional bone marrow (BM) collection is painful and yields limited cell numbers. As the need for easily accessible MSCs grows, cryopreservation's role in regenerative medicine is becoming increasingly vital. However, limited research exists on the characteristics and functional properties of rabbit-derived MSCs from various anatomical sources before and after cryopreservation. We examined the effects of cryopreservation using Bambanker. We found that cryopreservation did not adversely affect the morphology, viability, and adipogenic or chondrogenic differentiation abilities of ADP MSCs or BM MSCs. However, there was a notable drop in the proliferation rate and osteogenic differentiation capability of BM MSCs post-cryopreservation. Additionally, after cryopreservation, the surface marker gene expression of CD90 was not evident in ADP MSCs. As for markers, ADIPOQ can serve as an adipogenic marker for ADP MSCs. ACAN and CNMD can act as chondrogenic markers, but these two markers are not as effective post-cryopreservation on ADP MSCs, and osteogenic markers could not be validated. The study highlights that compared to BM MSCs, ADP MSCs retained a higher viability, proliferation rate, and differentiation potential after cryopreservation. As such, in clinical MSC use, we must consider changes in post-cryopreservation cell functions.