Relative Transcript Abundance Research Articles

GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process.

Simple SummaryThe knowledge of physiological events associated with canine reproduction involving oocyte developmental potential is essential to increase the success of reproductive biotechnologies in this species. In mammals, the oocytes are closely surrounded by a group of cells known as the cumulus cells. Although it is not well-known how these cells interact with the oocyte to promote maturation, they may provide important answers concerning oocyte development. The competence to undergo expansion is a unique characteristic of cumulus cells which is critical for normal oocyte maturation, however, the complete expansion of these cells takes longer in canines, which has been associated with the lengthy maturation process of the oocyte. Growth Differentiation Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are described as relevant players in the oocyte–cumulus cells’ regulatory mechanisms. Cumulus cells express many important genes from a very early stage, therefore, we proposed to study the gene expression of GDF-9 and BMP-15 in canine cumulus cells in relation to cumulus expansion and the maturation process. We demonstrate, for the first time, that these genes are differentially expressed in canine cumulus cells throughout the estrous cycle and that this expression is related to cumulus expansion and maturity status, suggesting specific regulation.The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.

Effects of Prostaglandins E2 and F2α on the in vitro maturation of bovine oocytes

We aimed to elucidate the effects of PGE2 and PGF2α on the in vitro maturation (IVM) of bovine oocytes. First, cumulus-oocyte complexes were matured in the media supplemented with or without PGE2, PGF2α, or PGE2 plus PGF2α for the final 24, 12, or 6 h of culture. Then, the cumulus-oocyte complexes were matured in the absence or presence of a PG endoperoxide synthase 2 (PTGS2) enzyme inhibitor (NS398) supplemented with PGE2, PGF2α, or PGE2 plus PGF2α. Finally, the expression of genes associated with PGs activity in cumulus cells (PTGS2, PG E-synthase-1 [PTGES1], and aldo-keto reductase 1 [AKR1B1]) or oocytes (receptors for PGE2 [PTGER2] and PGF2α [PTGFR]) of different competencies was quantified. Supplementation of the IVM medium with PGs did not improve in vitro embryo production or embryo quality (P > 0.05). During maturation, the relative abundance of PTGS2 transcripts increased (P < 0.05) only in the less-competent group, whereas those of PTGES1 increased in the less-competent and in the more-competent groups. Conversely, AKR1B1 expression decreased only in the less-competent group (P < 0.05). Receptors for the PGE2 and PGF2α genes were very low or undetectable in oocytes. In conclusion, PGE2 and PGF2α are not recommended for media supplementation during maturation because they have no effect on embryo development. Although genes related to PGs activity are differentially expressed in cumulus cells of cumulus-oocyte complexes of different competence during maturation, the expression of PGE2 and PGF2α receptor genes was either not detectable or was detected at low levels in oocytes.

Cortisol inhibits the Escherichia coli-induced endometrial inflammatory response through NF-κB and MAPK pathways in postpartum goats

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Molecular characteristics and abundance of insulin-like androgenic gland hormone and effects of RNA interference in Eriocheir sinensis

Insulin-like androgenic gland hormone (IAG) has an important function in sexual differentiation and somatic growth in crustaceans. In this study, there was cloning of the full-length sequence of IAG from Eriocheir sinensis (Es-IAG). The full-length Es-IAG gene was 1392 base pairs long and encoded a protein of 151 amino acid residues. The precursor peptide included a signal peptide, and the protein was a protein that is secreted from the cell in which it is produced with no transmembrane domain. Amino acid sequence alignment indicated there was the greatest homology between E. sinensis and Chaceon quinquedens (47 %), followed by Callinectes sapidus (44 %). Results from analysis of the relative abundances of Es-IAG mRNA transcript at different developmental stages indicated that Es-IAG may have an important endocrine function in early embryonic development, and that Stages I through Ⅲ may be an important period for sexual differentiation in juvenile E. sinensis. In vivo treatment with siRNA-391 resulted in a 66.7 % lesser relative abundance of the Es-IAG mRNA transcript. Three treatments with siRNA-391 to inhibit Es-IAG production during Stages Ⅲ to Ⅴ period resulted in about 10 % of male crabs being transformed into “neo-females.” These results provide the basis for further research into the sexual differentiation mechanism and monosex breeding of E. sinensis.

Effects of selenium on the proliferation and apoptosis of sheep spermatogonial stem cells in vitro

The objective of this study was to investigate effects of selenium (Se) on proliferation and apoptosis of sheep spermatogonial stem cells (SSC) in vitro. The SSC were assigned to five treatment groups (0, 2.0, 4.0, 8.0 and 16.0 μmol/L Se). After treatment with Se for 96 h, cell proliferation and apoptosis were evaluated. The relative abundance of P53 mRNA transcript and protein, cell cycle and apoptosis-related genes were detected using real-time PCR and Western blot quantifications, respectively. The results indicate there were the least cell proliferation rates in the Se16.0 group. Treatments with relatively greater Se concentrations (8.0 and 16.0 μmol/L) resulted in a greater percentage of apoptotic cells, which was consistent with the relative abundances of P53, P21, P27 and pro-apoptosis mRNA transcripts. There were relatively greater ROS concentrations in the control, Se8.0 and Se16.0 groups. Compared with the control group, treatment with the Se concentration of 16.0 μmol/L resulted in an increased abundance of P53, P21, P27 and BAX proteins. Treatment with Pifithrin-α suppressed the increase in abundance of P53 and P21 proteins induced by the relatively greater concentration of Se (16.0 μmol/L), however, did not result in a change in abundances of P27 and BAX proteins. These results indicate the regulatory functions of Se on proliferation and apoptosis of sheep SSC is associated with the P21-mediated P53 signalling pathway. The P27 and BAX proteins have limited functions during the apoptotic process of SSC induced by the relatively greater concentrations of Se.

Mechanism of Ca2+-mediated NOX modulated in ROS metabolism induced by T-2 toxin in potato tuber

T-2 toxin at low concentrations can induce ROS accumulation and modulate host resistance in plants. NOX plays crucial roles in ROS production and is regulated by Ca2+via direct binding to EF-hand motifs. In this study, the effect of EGTA (Ca2+ chelating agent) on the expression and enzymatic activity of NOX, as well as the activities and corresponding gene expressions involved in ROS metabolism and cell membrane integrity, were investigated in treated slices. Results indicated that EGTA treatment significantly affected gene expression and activity of NOX, and reduced ROS accumulation and cell membrane integrity and the enzymatic activities and gene expression involved in ROS metabolism when exposed to treatment. The addition of exogenous Ca2+ restored the initial relative transcript abundance, ROS accumulation and their activities. Results suggest that Ca2+ affected by EGTA plays a crucial role in NOX activity regulation, ultimately affecting ROS metabolism in slices induced by T-2 toxin.

Effects of abundances of OCT-4 mRNA transcript on goat pre-implantation embryonic development

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8–16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed.

Nano-ZnO-Induced Drought Tolerance Is Associated with Melatonin Synthesis and Metabolism in Maize.

The applications of ZnO nanoparticles in agriculture have largely contributed to crop growth regulation, quality enhancement, and induction of stress tolerance, while the underlying mechanisms remain elusive. Herein, the involvement of melatonin synthesis and metabolism in the process of nano-ZnO induced drought tolerance was investigated in maize. Drought stress resulted in the changes of subcellular ultrastructure, the accumulation of malondialdehyde and osmolytes in leaf. The nano-ZnO (100 mg L−1) application promoted the melatonin synthesis and activated the antioxidant enzyme system, which alleviated drought-induced damage to mitochondria and chloroplast. These changes were associated with upregulation of the relative transcript abundance of Fe/Mn SOD, Cu/Zn SOD, APX, CAT, TDC, SNAT, COMT, and ASMT induced by nano-ZnO application. It was suggested that modifications in endogenous melatonin synthesis were involved in the nano-ZnO induced drought tolerance in maize.

Effect of kisspeptin and RFamide-related peptide-3 on the synthesis and secretion of LH by pituitary cells of pigs during the estrous cycle

Actions of kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) at the hypothalamus modulate female reproduction. The action of KISS and RFRP-3 in the pituitary gland of pigs during the estrous cycle has not yet been delineated. The present study was conducted to assess the effect of KISS and RFRP-3 on relative abundance of αGSU and βLH mRNA transcript and LH secretion in vitro by pituitary cells of gilts during the estrous cycle. The cells were isolated from gilts on Days 2–3, 10–12, 15–16 and 19–20 of the estrous cycle and cultured in vitro without inclusion of GnRH (control) or with GnRH (100 ng/ml), KISS (10−6 M, 10−7 M) and RFRP-3 (10−6 M, 10−7 M) alone or in combination. The relative abundance of α-GSU and β-LH mRNAs was examined. Treatment with KISS increased the synthesis and/or secretion of LH by pituitary cells and RFRP-3 inhibited the synthesis and secretion of LH in the presence of GnRH on Days 10–12 and 15–16 of the estrous cycle. The synthesis and secretion of LH was greater when there was treatment with KISS and GnRH during the late follicular phase. Treatments with KISS and RFRP-3 affected the synthesis and/or secretion of LH during the luteal phase and luteolysis. In conclusion, KISS and RFRP-3 apparently affects the synthesis and secretion of LH by pituitary cells of estrous cyclic pigs. There appears to be a greater effect of KISS in modulation of LH secretion than RFRP-3 in pigs.

Epigenetic changes associated with increased estrogen receptor alpha mRNA transcript abundance during reproductive maturation in chicken ovaries

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that regulates cellular responses to estrogens and transcription processes of target genes. In this study, changes in DNA methylation and histone modifications in the promoter region and Exon 1 of the ERα gene were analyzed to ascertain epigenetic changes associated with increased ERα mRNA abundance during reproductive maturation from 90 (egg production not yet initiated) to 160 (after egg production was initiated) d of age (d post-hatching) in chicken ovaries. The results indicate there was no difference in CpG methylation at the promoter and Exon 1 except at the region analyzed with primer pairs F2 and R2, where percentage of methylated CpG of Sites 2 and 8 after reproductive maturation was greater compared with before reproductive maturation. By using the chromatin immunuoprecipitation (ChIP) assay combined with SYBR green quantitative PCR, effects of histone modifications were evaluated, including histone H3K4 di + tri methylation, H3K9 phosphorylation and trimethylation, H3K36 methylation and H3K27 acetylation on chicken ERα mRNA transcript abundance. The results indicated that there was a greater histone H3K27 acetylation and lesser H3K36 trimethylation associated with increased abundance of ERα mRNA transcript in chicken ovaries after reproductive maturation (90 compared with 160 d of age). In consistent with this finding, the relative abundance of transcriptional coactivator p300 mRNA transcript and protein in the ovaries was markedly greater in reproductively mature than immature chickens. Findings provide insights into the epigenetic regulations of the chicken ERα gene expression that is required for chicken ovarian development.

Relative abundance of interferon-stimulated genes STAT1, OAS1, CXCL10 and MX1 in ovine lymph nodes during early pregnancy

Lymph nodes have functions in the adaptive immune response, and interferon-tau (IFNT), a primary pregnancy recognition signal in domestic ruminants has effects on immune regulation. It, however, is unclear whether early pregnancy induces an increase in the abundance of interferon-stimulated gene (ISG) mRNA transcripts and proteins in lymph nodes of sheep. In this study, lymph nodes were obtained on day 16 of the estrous cycle from non-pregnant ewes and days 13, 16 and 25 of gestation from pregnant ewes, and the abundance of ISG mRNA transcripts, including signal transducer and activator of transcription 1 (STAT1), phosphorylated STAT1 (p-STAT1), 2′,5′-oligoadenylate synthetase (OAS1), myxovirus resistance protein 1 (MX1) and C-X-C motif chemokine 10 (CXCL10), was analyzed using real-time quantitative PCR. Furthermore, Western blot and immunohistochemistry analysis was conducted to assess relative abundance of proteins encoded by these genes. The results indicated that there was a larger abundance of STAT1 mRNA transcript and protein, and p-STAT1 protein in the maternal lymph node at days 16 and 25 of gestation, and that abundances of OAS1, MX1 and CXCL10 mRNA transcripts and protein were greatest on day 16 of gestations. In addition, STAT1 protein was located in the subcapsular sinus, lymph sinuses, B cells and T cells. The larger relative abundances of STAT1, p-STAT1, OAS1, MX1 and CXCL10 mRNA transcripts and/or protein in the lymph nodes of ewes may be associated with maternal immunoregulation through blood circulation and lymph circulation during early pregnancy.

Use of a prediction method for early pregnancy status utilizing receiver operating characteristic curve analysis of peripheral blood leukocyte interferon-stimulated genes in Japanese-Black cattle

A prediction method for early pregnancy status (pregnant or non-pregnant) in cattle that can be used within 3 weeks after insemination is desired. Interferon-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) have been examined as prediction molecules for determination of pregnancy status. Relative abundances of ISG15 and MX2 gene transcripts in PBLs were suitable biomarkers for the prediction of pregnancy status when there were assessments of Holstein cattle. In the present study, it was determined whether ISG biomarkers are applicable for predicting gestation in Japanese-Black (JB) cattle and evaluation of the applicability of receiver operating characteristic (ROC) analysis procedures for this purpose. There was assessment of the reliability of using average ISG values in PBLs collected during the estrous cycle (AVE) as a cutoff compared to the Youden index cutoff values. Application of AVE to assessment of pregnancy status in JB cattle indicated there was reliable predictions for pregnancy status when using ISG15 and MX2 values on day 21 after insemination, which coincided with the time of assessment in the previous study with Holstein cattle. The area under the curve values of the ROC curves confirmed the reliability of using ISGs to predict pregnancy from days 18 to 21 after insemination. Comparing AVE with Youden index values, there was confirmation of the accuracy of AVE for predicting gestation. The average mRNA transcript abundance values of ISG15 and MX2 may serve as excellent pregnancy biomarkers for cattle within 3 weeks of insemination.

Effect of prepartum energy intake and supplementation with ruminally protected choline on innate and adaptive immunity of multiparous Holstein cows

Objectives were to evaluate the effect of prepartum energy intake and peripartal supplementation of ruminally protected choline (RPC) on select indicators of immune status in blood plasma and on lipopolysaccharide-stimulated blood cells ex vivo. At 47 ± 6 d before the expected calving date, 93 multiparous Holstein cows were assigned randomly to 1 of 4 dietary treatments in a 2 × 2 factorial arrangement. Cows were fed energy to excess [EXE; 1.63 Mcal of net energy for lactation (NEL)/kg of dietary dry matter (DM)] or to maintenance (MNE; 1.40 Mcal of NEL/kg of dietary DM) ad libitum throughout the nonlactating period. The RPC was fed at 0 or 60 g/d to supply 0 or 12.9 g/d of choline ions top-dressed for 17 ± 4.6 d prepartum through 21 d postpartum. After calving, cows were fed the same methionine-supplemented diet, apart from RPC supplementation. During the last 2 wk before calving and during the first 5 wk postpartum, blood was sampled repeatedly and analyzed for cell types, acute-phase proteins, tumor necrosis factor-α (TNFα), and neutrophil function. Samples of whole blood were collected at 3 and 14 DIM and stimulated with 1 μg/mL lipopolysaccharide (LPS) in vitro for 6 and 24 h. After 6 h of LPS exposure, peripheral blood leucocytes (PBL) were harvested, and relative transcript abundance for select cytokines were measured. Supernatant was analyzed for TNFα after 24 h of LPS exposure. The PBL from cows fed EXE diets during the whole dry period had increased transcripts for the proinflammatory cytokines CXCL8 and TNF, although the plasma concentrations of the acute-phase proteins haptoglobin and fibrinogen, and the killing activity of the blood neutrophils in the postpartum period, were not affected by feeding different energy levels prepartum. Feeding RPC to cows overfed energy prepartum modulated their inflammatory state, as evidenced by decreased IL6 in PBL and reduced mean fluorescence intensity of CD14 during the postpartum period, compared with cows not fed RPC. Feeding RPC also decreased TNFα protein production, abundances of IL1B, CXCL8, and TNF transcripts, and mean fluorescence intensity of CD80 of PBL stimulated by LPS, regardless of prepartum energy intake. In contrast, proportions of blood neutrophils undergoing phagocytosis and oxidative burst were increased at 17 d postpartum in cows supplemented with RPC. Collectively, these data indicate that transition cows supplemented with RPC experienced less inflammation, which may partially explain increased milk production in cows supplemented with RPC.

Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification.

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3' untranslated regions is associated with decreased relative transcript abundance and defective RNA 3' end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

Oxygen tension during in vitro oocyte maturation and fertilization affects embryo quality in sheep and deer

Incubation gas atmosphere affects the development of in vitro produced embryos. In this study, there was examination of effects of two different oxygen (O2) tensions (5 % and 21 %) during in vitro maturation (M5 and M21) and/or fertilization (F5 and F21) on embryo production and quality in deer and sheep. There was assessment of the percentage of embryos with cell cleavage occurring, percentage that developed to the blastocyst stage, and analysis of the relative abundance of mRNA transcript for genes important for development to the blastocyst stage. The O2 tension treatment did not affect (P > 0.05) percentage cleavage or blastocyst development in either species. In sheep, there was a greater abundance of SHC1, GPX1, TP53, BAX and NRF1 mRNA transcript (P < 0.05) in M21 F5-derived embryos. In deer, there was a greater abundance of SOD2 mRNA transcript (P < 0.05) when oocytes had been matured under relatively lesser O2, regardless of the tension used during fertilization. There was a lesser abundance of SOX2 mRNA transcript (P < 0.05) in the M5F21 compared to the other three treatment groups. The AKR1B1 mRNA transcript was in greater abundance (P < 0.05) in M21 F21 as compared to M21 F5 and M5F21 group, and there was a greater abundance PLAC8 mRNA transcript (P < 0.05) in M21 F21, as compared to all other treatment groups. In conclusion, while O2 tension had no effect on developmental rates it did affect the relative abundance of mRNA transcript of multiple genes related to important cell functions during development.

Heat stress impairs in vitro development of preantral follicles of cattle

Although detrimental effects of heat stress on antral follicle development have been well studied, long-term effects - affecting the preantral follicle pool – are still largely unknown. The goal of this study was to evaluate effects of heat stress on growth, viability, gene expression and ATP production of preantral follicles of cattle. Follicles at the primary, early secondary and secondary stages were isolated from cattle ovaries and individually cultured while imposing physiological (CON; 38.5 °C) or intermittent heat stress (HS; 38.5 °C for 16 h and 41 °C for 8 h daily) conditions for 7 days. Individual follicles were subjected to real-time qPCR for determination of relative abundance of BAX, HSPA1A and SOD1 mRNA transcripts and evaluated for ATP production. Treatment for 7 days with intermittent HS decreased viability (P = 0.01) and diameter (P = 0.03) of preantral follicles. Relative abundances of BAX and HSPA1A mRNA transcripts were greater in follicles of the CON and HS groups that became non-viable during culture (P < 0.05); relative abundance of SOD1 mRNA transcript, however, was only greater in non-viable follicles of the HS group (P < 0.05), but not non-viable follicles of the CON group (P = 0.3). The ATP production was not different between viable follicles of the CON and HS group (P = 0.86). In conclusion, all stages of growing preantral follicles of cattle were susceptible to negative effects of heat stress. Follicles at the secondary stage of development were most sensitive, followed by early secondary and primary follicles.

Expression analysis of CD24 and CD44 transcripts in Iranian breast cancer patients.

The importance of cancer stem cells (CSCs) in initiation and progression of breast cancer has been well established. This population of cells is characterized by high expression of CD44 and low expression of CD24. However, the relative abundance of CD24 and CD44 transcripts in breast cancer tissues and adjacent non-cancerous tissues (ANCTs) has not been quantified yet. In the present investigation, we assessed expression of CD24 and CD44 at transcript level in breast cancer tissues and ANCTs in association with clinical determinants of patients' outcome and parameters that predict response to therapeutic options. There was no significant difference in expression of CD24 and CD44 in breast cancer tissues compared with ANCTs (Expression ratios: 1.03 and 0.84, P values: 0.92 and 0.61, respectively). However, CD44 expression was associated with tumor size in a way this gene was up-regulated in all of small sized (≤2cm) tumors compared with the corresponding ANCTs (P value = 0.04). Besides, CD44 expression was significantly higher in tumors with extracapsular nodal extension compared with those without extension (P =0.04). Expression of CD24 was higher in grade3 tumors compared with grade 2 tumors (P =0.04). Expression levels of CD24 and CD44 were correlated with each other in ANCTs but not in tumoral tissues. The current study shows another aspect of CSC markers in the development of breast cancer.

Developmental and mRNA transcript relative abundance pattern of vitellogenin receptors, LR8-/Lrp13, during ovarian development in the large yellow croaker (Larimichthys crocea)

For vitellogenin (Vtg) absorption to occur, there needs to binding of the glycolipophosphoproteins to the oocyte membrane in oviparous species, including teleosts. The cDNAs encoding homologous Vtg receptors (VgRs) LR8- and Lrp13 were cloned from ovaries of the large yellow croaker (Larimichthys crocea), an economically important species in Chinese aquaculture. The full-length Lc-lr8-/lrp13 cDNAs contained 4266/3760 base pairs (bp) and the deduced Lc-LR8-/Lrp13 proteins had 844/1218 amino acids, respectively. The VgRs comprised a ligand-binding domain, an epidermal growth factor precursor homology domain, YWXD motifs forming a β-propeller structure, and transmembrane and cytoplasmic domains. There was a marked relative abundance of Lc-lr8-/lrp13 transcripts in the tissues that were evaluated, with the largest abundance in the ovaries at Stage II of development. Furthermore, there was a lesser relative abundance of Lc-lr8-/lrp13 mRNA transcript during ovarian development (Stages II to IV). In situ hybridization technology was used to analyze decreasing relative abundance pattern of Lc-lr8-/lrp13 mRNA transcript during oogenesis in Stage II to IV of ovarian development. By combining mRNA relative abundance with morphological results, a model was developed to explain the reduction in Lc-lr8-/lrp13 mRNA transcript relative abundance during ovarian development. During the early developmental stages, transcription, translation, and differential accumulation of VgRs in previtellogenic and vitellogenic oocytes may occur and result in Vtg absorption in teleost oocytes. Overall, there is preliminary evidence indicating that at least two VgRs (Lc-LR8-/Lrp13) are present in the large yellow croaker and may be important for Vtg transport from the blood into the oocyte during ovarian development.

Characterization of splice-altering mutations in inherited predisposition to cancer

Mutations responsible for inherited disease may act by disrupting normal transcriptional splicing. Such mutations can be difficult to detect, and their effects difficult to characterize, because many lie deep within exons or introns where they may alter splice enhancers or silencers or introduce new splice acceptors or donors. Multiple mutation-specific and genome-wide approaches have been developed to evaluate these classes of mutations. We introduce a complementary experimental approach, cBROCA, which yields qualitative and quantitative assessments of the effects of genomic mutations on transcriptional splicing of tumor suppressor genes. cBROCA analysis is undertaken by deriving complementary DNA (cDNA) from puromycin-treated patient lymphoblasts, hybridizing the cDNA to the BROCA panel of tumor suppressor genes, and then multiplex sequencing to very high coverage. At each splice junction suggested by split sequencing reads, read depths of test and control samples are compared. Significant Z scores indicate altered transcripts, over and above naturally occurring minor transcripts, and comparisons of read depths indicate relative abundances of mutant and normal transcripts. BROCA analysis of genomic DNA suggested 120 rare mutations from 150 families with cancers of the breast, ovary, uterus, or colon, in >600 informative genotyped relatives. cBROCA analysis of their transcripts revealed a wide variety of consequences of abnormal splicing in tumor suppressor genes, including whole or partial exon skipping, exonification of intronic sequence, loss or gain of exonic and intronic splicing enhancers and silencers, complete intron retention, hypomorphic alleles, and combinations of these alterations. Combined with pedigree analysis, cBROCA sequencing contributes to understanding the clinical consequences of rare inherited mutations.

Maternal supplementation with fish oil modulates inflammation-related MicroRNAs and genes in suckling lambs

Dietary n-3 long-chain fatty acids (n-3 LCFA) have been shown to modify lipid metabolism and immune function. The objective of this study was to evaluate the effect of periparturient fish oil (FO) supplementation on the inflammation and metabolic health of ewes and their lambs at a molecular level. Prepartum ewes were fed control diet (CON, n = 12) or CON supplemented with 2% DM of calcium soap of FO (n = 12) from 28 days before until 21 days after parturition. The ewes were evaluated for plasma metabolites and milk composition. The experiment was followed by analyzing the relative transcript abundance of circulating microRNAs (miRNAs) in plasma and targeted miRNA/mRNA expression in peripheral blood mononuclear cells (PBMCs) in both ewes and lambs. FO treatment decreased prepartum feed intake (1812 ± 35 vs 1674 ± 33 g/day, P < 0.01), whereas the influence on plasma metabolites was negligible. Dietary FO supplementation decreased milk fat percentage (8.82 ± 0.49 vs 7.03 ± 0.45, P = 0.02) and reduced milk n-6/n-3 (P < 0.05). Also, it altered the expression of plasma-circulating miRNAs in both ewe and lamb (P < 0.05). Furthermore, maternal nutrition of FO downregulated the relative expression of miR-33a and miR-146b and transcript abundance of genes IL-1β (0.41-fold) and NF-κB (0.25-fold) in lambs' PBMC. In conclusion, results showed that FO supplementation starting antepartum affects milk composition and circulating miRNA in dams and the inflammatory markers in lambs delivered by the supplemented ewes. These may provide a strategy to maintain immune balance during gestation and develop the immune system in lambs.