Effect of Procyanidin on Canine Sperm Quality during Chilled Storage.

Procyanidin (PC) is a polyphenolic compound with antioxidant activity. The purpose of this study was to determine the influence of PC on canine sperm quality after 72 h of storage at 4 °C. The collected ejaculates were separated into four equal aliquots and treated with various concentrations of PC (0, 10, 30, and 50 µg/mL) in Tris-citric-fructose-egg yolk (TCFE) extender and stored at 4 °C for 72 h. The findings revealed that 30 µg/mL PC was the optimum concentration for significantly improving sperm motility (p < 0.05). Sperm samples treated with 30 µg/mL PC had substantially greater plasma membrane integrity, acrosome integrity, and mitochondrial membrane potential than the control group (p < 0.05). Furthermore, T-AOC and the expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (GPx1) genes were significantly higher in sperm treated with 30 µg/mL PC than those in control (p < 0.05). In summary, this study discovered that adding PC to the TCFE extender enhanced sperm quality and that 30 µg/mL PC was the optimal concentration for canine sperm when stored at 4 °C.

Effects of 25-hydroxycholecalciferol supplementation in maternal diets on reproductive performance and the expression of genes that regulate lactation in sows.

One hundred Yorkshire × Landrace sows were randomly assigned to one of two dietary treatments (diet ND: 6,000 IU vitamin D3 /d feed; diet 25-D: 200 µg/day 25OHD3 feed). The experiment began on d 90 of gestation and continued until weaning on day 21 of lactation. In sows that received 25OHD3 , the growth rate of the piglets before weaning was significantly accelerated (0.266 kg/day, p < .05). Sow serum was collected after weaning, and those in the 25OHD3 group were found to have significantly higher serum calcium (CA) and phosphorus (PI) levels (p < .05). Interestingly, the oestrus cycle of sows fed 25OHD3 was significantly shortened (p < .05), the oestrus time was concentrated on the fifth day after weaning, and the piglets were born with a higher degree of uniformity (p < .05). Colostrum was collected on the day of delivery, and the colostrum of sows fed 25OHD3 contained higher milk fat content than the control group (p < .05). 25OHD3 supplementation increased the mRNA and protein expression of INSIG1 and SREBP1, which regulate milk fat synthesis, in the mammary gland of lactating sows (p < .05). In conclusion, 25OHD3 supplementation in maternal diets improved reproductive performance, milk fat content and the mRNA and protein levels of genes regulating milk fat synthesis in lactating sows.

The impact of the bone marrow microenvironment on multiple myeloma (Review).

Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells in the bone marrow (BM). The interaction between the BM microenvironment and MM plasma cells can influence cell proliferation, drug resistance and prognosis of the disease. The BM microenvironment (BMME) consists of a cellular and non­cellular compartment. The cellular compartment includes stromal cells, endothelial cells, osteoclasts and osteoblasts, and the non­cellular compartment includes the extracellular matrix (ECM) and the liquid milieu, which contains cytokines, growth factors and chemokines. The complex interaction between the BM microenvironment and MM plasma cells influences disease development and prognosis. The present review focuses on the interaction between malignant plasma cells and the BM microenvironment during MM progression. An improved understanding of the interaction between MM plasma cells and their microenvironment will enable the development of novel therapeutic tools that can be used in the treatment of MM, a currently incurable blood cancer.

Effect of Hyperin and Icariin on steroid hormone secretion in rat ovarian granulosa cells.

AIM OF THE STUDY: This study was designed to investigate the effect of different concentrations of Hyperin and Icariin (ICA)on proliferation and the secretion of estrogen (E2), and progesterone (P) in granulosa cells, and to explore the effect of Hyperin and Icariin on the expression of CYP17 and CYP19. MATERIALS AND METHODS: Rat ovary granulosa cells were cultured in vitro and treated with different concentrations of Hyperin and Icariin. The proliferation of ovarian granulosa cells was measured with the MTT assay. The concentration of estradiol was measured with a magnetic particle-based enzyme-linked immunosorbent assay (ELISA) kit. The CYP17 and CYP19 mRNA expression was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The CYP17 and CYP19 protein expression was determined with Western blotting. RESULTS: Hyperin (50 µg/l) and Icariin (10 µg/l) significantly increased proliferation of ovarian granulosa cells and secretion of estrogen and progesterone. Hyperin and Icariin stimulated the mRNA and protein expression of CYP17 and CYP19. CONCLUSIONS: These results showed that Hyperin and Icariin can promote the secretion of E2 and P through up-regulation of CYP17 and CYP19. Frequently used Chinese herbs like Cuscuta Chinensis Lam and Epimedium Brevicornu maxim, which contain Hyperin and Icariin, could improve the ovarian endocrine function through these effects.

Antigenicity of tissues and organs from GGTA1/CMAH/ß4GalNT2 triple gene knockout pigs.

Clinical xenotransplantations have been hampered by human preformed antibody-mediated damage of the xenografts. To overcome biological incompatibility between pigs and humans, one strategy is to remove the major antigens [Gal, Neu5Gc, and Sd(a)] present on pig cells and tissues. Triple gene (GGTA1, CMAH, and ß 4GalNT2) knockout (TKO) pigs were produced in our laboratory by CRISPR-Cas9 targeting. To investigate the antigenicity reduction in the TKO pigs, the expression levels of these three xenoantigens in the cornea, heart, liver, spleen, lung, kidney, and pancreas tissues were examined. The level of human IgG/IgM binding to those tissues was also investigated, with wildtype pig tissues as control. The results showed that αGal, Neu5Gc, and Sd(a) were markedly positive in all the examined tissues in wildtype pigs but barely detected in TKO pigs. Compared to wildtype pigs, the liver, spleen, and pancreas of TKO pigs showed comparable levels of human IgG and IgM binding, whereas corneas, heart, lung, and kidney of TKO pigs exhibited significantly reduced human IgG and IgM binding. These results indicate that the antigenicity of TKO pig is significantly reduced and the remaining xenoantigens on porcine tissues can be eliminated via a gene targeting approach.

Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/ß4GalNT2/CMAH.

Bioprosthetic heart valves (BHVs) originating from pigs are extensively used for heart valve replacement in clinics. However, recipient immune responses associated with chronic calcification lead to structural valve deterioration (SVD) of BHVs. Two well-characterized epitopes on porcine BHVs have been implicated in SVD, including galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) whose synthesis are catalyzed by α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Ac hydroxylase (encoded by the CMAH gene), respectively. It has been reported that BHV from αGal-knockout pigs are associated with a significantly reduced immune response by human serum. Moreover, valves from αGal/Neu5Gc-deficient pigs could further reduce human IgM/IgG binding when compared to BHV from αGal-knockout pigs. Recently, another swine xenoantigen, Sd(a), produced by ß-1,4-N-acetyl-galactosaminyl transferase 2 (ß4GalNT2), has been identified. To explore whether tissue from GGTA1, CMAH, and ß4GalNT2 triple gene-knockout (TKO) pigs would further minimize human antibody binding to porcine pericardium, TKO pigs were successfully produced by CRISPR/Cas9 mediated gene targeting. Our results showed that the expression of αGal, Neu5G and Sd(a) on TKO pigs was negative, and that human IgG/IgM binding to pericardium was minimal. Moreover, the analysis of collagen composition and physical characteristics of porcine pericardium from the TKO pigs indicated that elimination of the three xenoantigens had no significant impact on the physical proprieties of porcine pericardium. Our results demonstrated that TKO pigs would be an ideal source of BHVs. STATEMENT OF SIGNIFICANCE: Surgical heart valve replacement is an established lifesaving treatment for diseased heart valve. Bioprosthetic heart valves (BHVs) made from glutaraldehyde-fixed porcine or bovine tissues are widely used in clinics but exhibit age-dependent structural valve degeneration (SVD) which is associated with the immune response against BHVs. Three major xenoantigens present on commercial BHVs, Galactosea α1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc) and glycan products of ß-1,4-N-acetyl-galactosaminyl transferase 2 (ß4GalNT2) are eliminated through CRISPR/Cas9 mediated gene targeting in the present study. The genetically modified porcine pericardium showed reduced immunogenicity but comparable collagen composition and physical characteristics of the pericardium from wild-type pigs. Our data suggested that BHVs from TKO pigs is a promising alternative for currently available BHVs from wild-type pigs.

Overexpressing dominant-negative FGFR2-IIIb impedes lung branching morphogenesis in pigs.

Genetic studies with mouse models have shown that fibroblast growth factor receptor 2-IIIb (FGFR2-IIIb) plays crucial roles in lung development and differentiation. To evaluate the effect of FGFR2-IIIb in pig lung development, we employed somatic cell nuclear transfer (SCNT) technology to generate transgenic pig fetuses overexpressing the transmembrane (dnFGFR2-IIIb-Tm) and soluble (dnFGFR2-IIIb-HFc) forms of the dominant-negative human FGFR2-IIIb driven by the human surfactant protein C (SP-C) promoter, which was specifically expressed in lung epithelia. Eight dnFGFR2-IIIb-Tm transgenic and twelve dnFGFR2-IIIb-HFc transgenic pig fetuses were collected from three and two recipient sows, respectively. Repression of FGFR2-IIIb in lung epithelia resulted in smaller lobes and retardation of alveolarization in both forms of dnFGFR2-IIIb transgenic fetuses. Moreover, the dnFGFR2-IIIb-HFc transgenic ones showed more deterioration in lung development. Our results demonstrate that disruption of FGFR2-IIIb signaling in the epithelium impedes normal branching and alveolarization in pig lungs, which is less severe than the results observed in transgenic mice. The dnFGFR2-IIIb transgenic pig is a good model for the studies of blastocyst complementation as well as the mechanisms of lung development and organogenesis.

DNA demethylation pattern of in-vitro fertilized and cloned porcine pronuclear stage embryos.

Recent studies in mice showed that the Ten-eleven translocation Enzymes (TET) family is involved in the active DNA demethylation. The isotype TET-3 is responsible for the conversion of 5mc (5-methylcytosine) to 5hmc (5-hydroxymethylcytosine) at the pronuclear stages of mouse embryo. This study was performed to investigate the pattern of methylation change and the role of TET family in the demethylation process of porcine in-vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryo. Bisulfite-sequencing PCR (BSP) and DNA glucosylation and digestion before quantitative PCR (qGluMS-PCR) were done to evaluate the exact change of methylation during porcine pronuclear stages. The results showed that the amount of 5hmc detected increased whereas 5mc decreased in IVF embryo from pronuclear stage 2 (PN2) to pronuclear stage 5 (PN5). In addition, Immunofluorescent staining showed that the 5hmc signal, also detected in oocytes, significantly increased in both pronucleus from fertilization to PN2. The amount of 5hmc continued to rise in male pronucleus but decreased to a very low level in female pronucleus from PN2 to PN5. The above results indicate that female pronucleus might undergo active demethylation only at early pronuclear stages. On the other hand, male pronucleus might undergo active demethylation throughout all pronuclear stages. The expression of three TET isotypes (TET-1, TET-2, TET-3) were tested and TET-3 was found to be the highest expressed isotype. High TET-3 concentrations observed mainly in male pronucleus using immunofluorescent staining, implying that TET-3 might be the main enzyme which catalyzes the conversion of 5mc to 5hmc. In contrast, no TET-3 signal was detected in female pronucleus through the pronuclear stages. The demethylation pattern of SCNT embryos resembled that of the male pronucleus of IVF embryos, suggesting that active demethylation might happen in porcine cloned embryo.