In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)- related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: 32.5±10.1% vs control: 77.8±5.3%). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, 200 μM) and melatonin (0.1 μM), in BIP/ GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, p-eIF2α, eIF2α, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

This study was performed to analyze genetic relationship of the four major Cucurbitaceae crop. We used 120 Expressed Sequence Tag(EST)-Simple Sequence Repeat(SSR) primer sets of developed from watermelon and published in International Cucurbit Genomics Initiative (ICuGI) database. Among 120 EST-SSR primer, 51(49.17%) EST-SSR primer set successfully amplified and 49(40.8%) EST-SSR primer set showed polymorphisms among eight cultivars of Cucurbitaceae. In the first instance, amplified PCR products analysis was conducted by the agarose-gel electrophoresis then further analyzed by using Fragment Analyzer. A total 382 PCR band were producted by 49 EST-SSR primers in 24 plant panels, used the analysis of pairwise similarity and dendrogram construction. Assessment of the genetic relationships resulted in similarity index with range of 0.0103 to 0.8452. In dendrogram, 24 plant panels were formed three major groups (A, B, C) and 7 subgroups (A-1, A-2, B-1, B-2, B-3, C-1, C-2). Major group A was comprised of 2 subgroups, subgroup A-1 (6 watermelon cultivars, Citrullus lanatus var. vulgaris Schrad.) and subgroup A-2 (3 wild type watermelon, Citrullus lanatus var. citroides Mats. & Nakai). Major group B was comprised of 3 subgroups, subgroup B-1 (4 melon cultivars, Cucumis melo var. cantalupensis Naudin.), subgroup B-2 (2 oriental melon cultivars, Cucumis melo var. conomon Makino.) and subgroup B-3 (5 cucumber cultivars, Cucumis sativus L.). Major group C was comprised of 2 subgroups, subgroup C-1 [2 squash/ pumpkin cultivars, Cucurbita moschata (Duch. ex Lam.)/Duch. & Poir. and Cucurbita maxima Duch.] and subgroup C-2(2 squash/pumpkin cultivars, Cucurbita pepo L./Cucurbita ficifolia Bouche.)