Changing trends in gastric and colorectal cancer among surgical patients over 85 years old: A multicenter retrospective study, 2001-2021.

BACKGROUND: Whether patients over 85 years old with gastrointestinal cancer should undergo surgery remains controversial. We aimed to describe the changing trends of characteristics to provide more information to decision makers, and strive to find appropriate surgical plan. AIM: To describe the changing trends of characteristics to provide more information to decision makers, and strive to find appropriate surgical plan. METHODS: A total of 218 gastric cancer (GC) patients and 563 colorectal cancer (CRC) patients who underwent surgery between 2001 and 2021 were enrolled in this retrospective analysis. Changes in clinicopathological features, surgical treatments, and survival status were analyzed longitudinally at 5-year intervals. RESULTS: Only 14 GC patients underwent laparoscopic surgery where 219 CRC patients had this procedure. Cardia and esophagogastric junction cancer increased in GC patients, and the proportion of sigmoid colon cancer decreased in CRC patients. Pulmonary infection gradually became the most common postoperative complication, its incidence in period 4 reached 48.79%. However, the incidence of anastomotic leakage decreased from 26.79% to 9.38% (P < 0.01). Additionally, 30-d mortality significantly decreased from 32.14% to 9.01%. Increases were observed in 5-year overall survival (OS) in GC patients from period 1 to period 4 (18.18% vs 33.32%, respectively) and CRC patients (0 vs 36.32%, respectively). Disease-free survival (DFS) also increased in GC and CRC patients (7.14% vs 27.74% and 0 to 36.03%, respectively). The average survival time of GC patients following radial lymphadenectomy was higher than in patients that underwent limited lymphadenectomy (26 vs 22 mo, respectively), the same was seen in CRC patients (44 vs 33 mo, respectively). This advantage was particularly evident in patients with TNM I, but not in patients with TNM II/III period cancer. CONCLUSION: The safety as well as effectiveness of surgery in ultra-elderly patients is increasing. Radical lymphadenectomy has advantages in patients with TNM I gastrointestinal cancer, but not TNM II/III.

Cloning, expression, and characterization of two pectate lyases isolated from the sheep rumen microbiome.

Pectate lyases (Pels) have a vital function in degradation of the primary plant cell wall and the middle lamella and have been widely used in the industry. In this study, two pectate lyase genes, IDSPel16 and IDSPel17, were cloned from a sheep rumen microbiome. The recombinant enzymes were expressed in Escherichia coli and functionally characterized. Both IDSPel16 and IDSPel17 proteins had an optimal temperature of 60 ℃, and an optimal pH of 10.0. IDSPel16 was relatively stable below 60 °C, maintaining 77.51% residual activity after preincubation at 60 °C for 1 h, whereas IDSPel17 denatured rapidly at 60 °C. IDSPel16 was relatively stable between pH 6.0 and 12.0, after pretreatment for 1 h, retaining over 60% residual activity. IDSPel16 had high activity towards polygalacturonic acid, with a Vmax of 942.90 ± 68.11, whereas IDSPel17 had a Vmax of only 28.19 ± 2.23 µmol/min/mg. Reaction product analyses revealed that IDSPel17 liberated unsaturated digalacturonate (uG2) and unsaturated trigalacturonate (uG3) from the substrate, indicating a typical endo-acting pectate lyase (EC 4.2.2.2). In contrast, IDSPel16 initially generated unsaturated oligogalacturonic acids, then converted these intermediates into uG2 and unsaturated galacturonic acid (uG1) as end products, a unique depolymerization profile among Pels. To the best of our knowledge, the IDSPel16 discovered with both endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities. These two pectate lyases, particularly the relatively thermo- and pH-stable IDSPel16, will be of interest for potential application in the textile, food, and feed industries. KEY POINTS: • Two novel pectate lyase genes, IDSPel16 and IDSPel17, were isolated and characterized from the sheep rumen microbiome. • Both IDSPel16 and IDSPel17 are alkaline pectate lyases, releasing unsaturated digalacturonate and unsaturated trigalacturonate from polygalacturonic acid. • IDSPel16, a bifunctional pectate lyase with endo-Pel (EC 4.2.2.2) and exo-Pel (EC 4.2.2.9) activities, could be a potential candidate for industrial application.

Exosomal miR-181a-5p derived from SAOS-2 cells promotes macrophages M2 polarization by targeting RORA.

Although the interaction between tumor cells and tumor-associated macrophages (TAMs) has been widely studied; however, the mechanism of osteosarcoma cells in regulating the polarization of TAMs remains unclear. Exosomes from SAOS-2 cells were isolated and validated by electron microscopy and Western blot. Transfection of indicated plasmids was applied to modify the expressions of miR-181a-5p and RAR-related orphan receptor alpha (RORA). Flow cytometric analysis was carried out to analyze M1/M2 macrophage polarization. Quantitative real-time PCR was performed to determine the levels of miR-181a-5p and RORA. Protein levels of CD63, CD81, RORA, CD163, CD206, IL-10, CXCL10, and IL-1ß were evaluated by Western blot. The direct interaction of miR-181a-5p and RORA was validated by dual-luciferase activity assay. The expression of miR-181a-5p was upregulated in osteosarcoma tissues and presented in SAOS-2-derived exosomes. SAOS-2-derived exosomes promoted the polarization of M2 macrophages by transferring miR-181a-5p. In addition, RORA was downregulated in osteosarcoma tissues and showed a negative correlation with miR-181a-5p. RORA was found to be the downstream target of miR-181a-5p in SAOS-2 cells. Inhibition of RORA reversed the effects of miR-181a-5p knockdown on the polarization of M2 macrophages. The results showed that exosomal miR-181a-5p derived from osteosarcoma cells induced polarization of M2 macrophages via targeting RORA.

Risk and Prognostic Factors for Small Bowel Adenocarcinoma: A Multicenter Retrospective Observational Study in China.

Background: Small bowel adenocarcinoma (SBA) is a rare malignancy that accounts for 3% of all gastrointestinal tumors. We evaluated the clinical characteristics, outcomes, and prognostic factors of primary SBAs. Methods: We retrospectively analyzed the clinicopathological features and clinical outcomes of 300 patients with SBA from three institutions in China between January 2003 and July 2020. Overall survival (OS) was analyzed using the Kaplan-Meier method and it was statistically compared using the log-rank test. Single-variable and multivariate analyses were used to identify the significant correlates of OS. Results: The primary tumor was on the duodenal papilla in 156 patients (52%), in the duodenum in 60 patients (20%), and in the jejunum-ileum in 84 patients (28%). The median OS of the entire cohort was 32.5 months (range, 0-213 months), with a 1-year OS rate of 78.0%. For jejunoileal adenocarcinoma, advanced age, advanced T stage, advanced N stage, more positive lymph nodes, distant metastasis, high carcinoembryonic antigen (CEA), and lymphocyte-to-monocyte ratio < 2.32 predicted worse survival on single-variable analysis. Multivariate analysis showed that advanced age, advanced tumor node metastases (TNM) stage, high CEA level, high alpha fetoprotein (AFP) level, and low prealbumin level were independent prognostic factors for non-ampullary SBA. The independent prognostic factors for duodenal papilla adenocarcinoma included TNM Stage III, nerve invasion, low platelet/lymphocyte ratio, and high CA19-9. Conclusion: We found different independent prognostic factors for tumors at different locations. This finding warrants further investigation to ensure more effective management strategies for SBA.

Deficiency of Omentin-1 leads to delayed fracture healing through excessive inflammation and reduced CD31hiEmcnhi vessels.

Fracture healing is a complicated process affected by many factors, such as inflammatory responses and angiogenesis. Omentin-1 is an adipokine with anti-inflammatory properties, but whether omentin-1 affects the fracture healing process is still unknown. Here, by using global omentin-1 knockout (omentin-1-/-) mice, we demonstrated that omentin-1 deficiency resulted in delayed fracture healing in mice, accompanied by increased inflammation and osteoclast formation, and decreased production of platelet-derived growth factor-BB (PDGF-BB) and osteogenesis-promoting vessels that are strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in the fracture area. In vitro, omentin-1 treatment suppressed the ability of the tumor necrosis factor-α (TNF-α)-activated macrophages to stimulate multi-nuclear osteoclast formation, resulting in a significant increase in the generation of mono-nuclear preosteoclasts and PDGF-BB, a pro-angiogenic protein that is abundantly secreted by preosteoclasts. PDGF-BB significantly augmented endothelial cell proliferation, tube formation and migration, whereas direct treatment with omentin-1 did not induce obvious effects on angiogenesis activities of endothelial cells. Our study suggests a positive role of omentin-1 in fracture healing, which may be associated with the inhibition of inflammation and stimulation of preosteoclast PDGF-BB-mediated promotion of CD31hiEmcnhi vessel formation.

Harmine targets inhibitor of DNA binding-2 and activator protein-1 to promote preosteoclast PDGF-BB production.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet-derived growth factor-BB (PDGF-BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF-BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding-2 (Id2) and activator protein-1 (AP-1) were important factors implicated in harmine-enhanced preosteoclast PDGF-BB production. Exposure of RANKL-induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP-1. Knockdown of Id2 by Id2-siRNA reduced the number of preosteoclasts as well as secretion of PDGF-BB in RANKL-stimulated BMMs administrated with harmine. Inhibition of c-Fos or c-Jun (components of AP-1) both reversed the stimulatory effect of harmine on preosteoclast PDGF-BB production. Dual-luciferase reporter assay analyses determined that PDGF-BB was the direct target of AP-1 which was up-regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF-BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

Akkermansia muciniphila promotes type H vessel formation and bone fracture healing by reducing gut permeability and inflammation.

Improving revascularization is one of the major measures in fracture treatment. Moderate local inflammation triggers angiogenesis, whereas systemic inflammation hampers angiogenesis. Previous studies showed that Akkermansia muciniphila, a gut probiotic, ameliorates systemic inflammation by tightening the intestinal barrier. In this study, fractured mice intragastrically administrated with A. muciniphila were found to display better fracture healing than mice treated with vehicle. Notably, more preosteclasts positive for platelet-derived growth factor-BB (PDGF-BB) were induced by A. muciniphila at 2 weeks post fracture, coinciding with increased formation of type H vessels, a specific vessel subtype that couples angiogenesis and osteogenesis, and can be stimulated by PDGF-BB. Moreover, A. muciniphila treatment significantly reduced gut permeability and inflammation at the early stage. Dextran sulfate sodium (DSS) was used to disrupt the gut barrier to determine its role in fracture healing and whether A. muciniphila still can stimulate bone fracture healing. As expected, A. muciniphila evidently improved gut barrier, reduced inflammation and restored the impaired bone healing and angiogenesis in DSS-treated mice. Our results suggest that A. muciniphila reduces intestinal permeability and alleviates inflammation, which probably induces more PDGF-BB+ preosteoclasts and type H vessel formation in callus, thereby promoting fracture healing. This study provides the evidence for the involvement of type H vessels in fracture healing and suggests the potential of A. muciniphila as a promising strategy for bone healing.This article has an associated First Person interview with the first author of the paper.

H-type blood vessels participate in alveolar bone remodeling during murine tooth extraction healing.

OBJECTIVES: We aimed to investigate whether skeletal-specific H-type blood vessels exist in alveolar bone and how they function in alveolar bone remodeling. MATERIALS AND METHODS: H-type vessels with high expression of CD31 and Endomucin (CD31hi Emcnhi ) were immunostained in alveolar bone. Abundance and age-related changes in CD31hi Emcnhi endothelial cells (H-ECs) were detected by flow cytometry. Osteoprogenitors association with H-type vessels and bone mass were detected in tooth extraction model of alveolar bone remodeling by immunohistofluorescence and micro-CT, respectively. Transcription and expression of H-EC feature genes during in vitro Notch inhibition were measured by RT-qPCR and immunocytofluorescence. RESULTS: We verified that H-type vessels existed in alveolar bone, the abundance of which was highest at infancy age, then decreased but maintained a constant level during aging. In tooth extraction model, H-ECs significantly increased with concomitant perivascular accumulation of Runx2+ osteoprogenitors and gradually augmentation of bone mass. Notch inhibition of in vitro cultured H-ECs resulted in decreased expression levels of Emcn and hes1, but not Pecam1 or Kdr genes, with decreased expression levels of H-EC numbers, accordingly. CONCLUSIONS: The present study suggests that H-type vessels promote osteogenesis during alveolar bone remodeling. Notch signaling pathway regulates expression of Emcn and possibly determines fate and functions of alveolar H-ECs.

IL-1ß secreted by macrophage M2 promotes metastasis of osteosarcoma via NF-κB/miR-181α-5p/RASSF1A/Wnt pathway.

BACKGROUND: Ras-associated domain family protein1 isoform A (RASSF1A) was significantly absent in clinical samples and many osteosarcoma (OS) cell lines. Overexpression of RASSF1A could suppress OS metastasis, which may be mediated by tumor-associated macrophages polarized M2 (M2-TAMs). However, the relationship between IL-1ß secreted by M2-TAMs and RASSF1A remains unknown. METHODS: The expression levels of M2-TAMs markers CD68 and CD204 were measured by flow cytometry, and arginase-1 (Arg-1) and interleukin-1ß (IL-1ß) secreted by M2-TAMs were examined by real-time quantitative PCR (RT-qPCR). MTT assay was employed to determine the proliferation of OS cells, while scratch wound healing assay and Transwell assay were used to evaluate their migration and invasion, respectively. The level of miR-181α-5p was measured by RT-qPCR, while the levels of RASSF1A, GSK-3ß, p-GSK-3ß, ß-catenin, MMP-2 and MMP-9 were evaluated by Western blot. The direct binding of miR-181α-5p and RASSF1A was identified using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: The levels of CD68, CD204, Arg-1 and IL-1ß were elevated in M2-TAMs compared with control group. Overexpression of RASSF1A and knockdown of miR-181α-5p could both suppress invasion and migration of OS cells through Wnt pathway. IL-1ß secreted by M2-TAMs facilitated the OS metastasis via RASSF1A/Wnt pathway, which could be targeted by miR-181α-5p and affected by nuclear factor-kappa B (NF-κB). CONCLUSIONS: IL-1ß secreted by M2-TAMs contributed to OS metastasis, which could be suppressed by knockdown of miR-181α-5p or overexpression of RASSF1A through NF-κB/miR-181α-5p/RASSF1A/Wnt pathway. These findings can guide new target discovery for drug development in OS treatment.

[Relationship between variations of severe isthmic spondylolisthesis spino-pelvic parameters and clinical symptoms].

OBJECTIVE: To explore the relationship between variations of severe isthmic spondylolisthesis spino-pelvic parameters and clinical symptoms. METHODS: A retrospective study of spino-pelvic parameters was conducted for 45 patients with L5 severe isthmic spondylolisthesis. Their spino-pelvic parameters were analyzed on preoperative full spinal radiography: grade of spondylolisthesis, pelvic incidence (PI), sacral slope (SS), pelvic tilt (PT), lumbar lordosis (LL), thoracic kyphosis (TK), SFHD, SFVD, SC7D and T9 tilting angle. According to the Oswestry score, the patients with low back pain were divided into light and severe groups. T-tests were used to compare their parameters. The analyses of multiple factor correlation, linear regression and stepwise regression were used to examine the associations between all parameters and Oswestry score. RESULTS: Grade of spondylolisthesis, PT, LL, T9 tilting angle, SFHD, SC7D, PT/SS, SFHD/SFVD, LL/TK were significantly greater and SS, SFVD were significantly smaller for patients with severe low back pain versus light low back pain (P < 0.05). PI or TK had no statistically significant difference between two groups. Multiple factor correlation, linear regression and stepwise regression: Grade of spondylolisthesis, PT, SC7D, LL and SFHD had significant positive correlations with Oswestry score. Degree of positive correlation: Grade of spondylolisthesis > SC7D > PT > LL > SFHD. And PI, TK or T9 tilting angle had no significant correlation with Oswestry score. SS and SFVD had significant negative correlations with Oswestry score. Degree of negative correlation: SS > SFVD. PT/SS, SFHD/SFVD and LL/TK had significant positive correlation with Oswestry score. Degree of positive correlation: PT/SS> SFHD/SFVD > LL/TK. CONCLUSION: Clinical symptoms of severe isthmic spondylolisthesis have significant positive correlations with grade of spondylolisthesis, PT, SC7D, LL, SFHD, PT/SS, SFHD/SFVD, LL/TK and significant negative correlations with SS and SFVD. Low back pain symptom has no significant correlation with PI, TK or T9 tilting angle.

Wolbachia infection decreased the resistance of Drosophila to lead.

BACKGROUND: The heavy metal lead has been shown to be associated with a genotoxic risk. Drosophila melanogaster is a model organism commonly utilized in genetic toxicology testing. The endosymbionts--Wolbachia are now very common in both wild populations and laboratory stocks of Drosophila. Wolbachia may induce resistance to pathogenic viruses, filarial nematodes and Plasmodium in fruit fly and mosquito hosts. However the effect of Wolbachia infection on the resistance of their hosts to heavy metal is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Manipulating the lead content in the diet of Drosophila melanogaster, we found that lead consumption had no different effects on developmental time between Wolbachia-infected (Dmel wMel) and -uninfected (Dmel T) flies. While in Pb-contaminated medium, significantly reduced amount of pupae and adults of Dmel wMel were emerged, and Dmel wMel adults had significantly shorter longevity than that of Dmel T flies. Lead infusion in diet resulted in significantly decreased superoxide dismutase (SOD) activity in Dmel T flies (P<0.05), but not in Dmel wMel flies. Correspondingly, lead cultures induced a 10.8 fold increase in malonaldehyde (MDA) contents in Dmel T larvae (P<0.05). While in Dmel wMel larvae, it resulted in only a 1.3 fold increase. By quantitative RT-PCR, we showed that lead infused medium caused significantly increased expression level of relish and CecA2 genes in Dmel T flies (P<0.01). Lead cultures did not change dramatically the expression of these genes in Dmel wMel flies. CONCLUSIONS/SIGNIFICANCE: These results suggest that Wolbachia infection decreased the resistance of Drosophila to lead likely by limiting the production of peroxides resulted from lead, thus being unable to activate the immunological pathway in the host to prevent them from lead damage. This represents a novel Wolbachia-host interaction and provides information that researchers working on Drosophila toxicology should take in consideration the presence of Wolbachia in the stocks they are analyzing.

Taurine inhibits serum deprivation-induced osteoblast apoptosis via the taurine transporter/ERK signaling pathway

Taurine has positive effects on bone metabolism. However, the effects of taurine on osteoblast apoptosis in vitro have not been reported. The aim of this study was to investigate the activity of taurine on apoptosis of mouse osteoblastic MC3T3-E1 cells. The data showed that 1, 5, 10, or 20 mM taurine resulted in 16.7, 34.2, 66.9, or 63.75 percent reduction of MC3T3-E1 cell apoptosis induced by the serum deprivation (serum-free α-MEM), respectively. Taurine (1, 5, or 10 mM) also reduced cytochrome c release and inhibited activation of caspase-3 and -9, which were measured using fluorogenic substrates for caspase-3/caspase-9, in serum-deprived MC3T3-E1 cells. Furthermore, taurine (10 mM) induced extracellular signal-regulated kinase (ERK) phosphorylation in MC3T3-E1 cells. Knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 (10 μM) blocked the activation of ERK induced by taurine (10 mM) and abolished the anti-apoptotic effect of taurine (10 mM) in MC3T3-E1 cells. The present results demonstrate for the first time that taurine inhibits serum deprivation-induced osteoblast apoptosis via the TAUT/ERK signaling pathway.

Taurine inhibits serum deprivation-induced osteoblast apoptosis via the taurine transporter/ERK signaling pathway.

Taurine has positive effects on bone metabolism. However, the effects of taurine on osteoblast apoptosis in vitro have not been reported. The aim of this study was to investigate the activity of taurine on apoptosis of mouse osteoblastic MC3T3-E1 cells. The data showed that 1, 5, 10, or 20 mM taurine resulted in 16.7, 34.2, 66.9, or 63.75% reduction of MC3T3-E1 cell apoptosis induced by the serum deprivation (serum-free α-MEM), respectively. Taurine (1, 5, or 10 mM) also reduced cytochrome c release and inhibited activation of caspase-3 and -9, which were measured using fluorogenic substrates for caspase-3/caspase-9, in serum-deprived MC3T3-E1 cells. Furthermore, taurine (10 mM) induced extracellular signal-regulated kinase (ERK) phosphorylation in MC3T3-E1 cells. Knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 (10 µM) blocked the activation of ERK induced by taurine (10 mM) and abolished the anti-apoptotic effect of taurine (10 mM) in MC3T3-E1 cells. The present results demonstrate for the first time that taurine inhibits serum deprivation-induced osteoblast apoptosis via the TAUT/ERK signaling pathway.

[The study on the value of using the chronic obstructive pulmonary disease and asthma physiology score to assess the severity of acute exacerbation in patients with chronic obstructive pulmonary disease complicated by type II respiratory failure].

OBJECTIVE: To explore the value of the chronic obstructive pulmonary disease (COPD) and asthma physiology score (CAPS) in evaluating the severity and prognosis of patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated by type II respiratory failure. METHODS: Eighty-two cases with AECOPD complicated by type II respiratory failure between January 2005 and March 2009 were retrospectively analyzed. The severity in survivors and non-survivors was evaluated by CAPS and acute physiology and chronic health evaluation system (APACHE II score, APACHE III score), and retrospective and statistical analyses of all data were performed. RESULTS: CAPS, APACHE II score, APACHE III score, duration of invasive positive pressure ventilation (IPPV) and days in intensive care unit of 19 cases in the death group were 34.21+/-9.89, 22.53+/-7.49, 75.11+/-18.07, (25.06+/-24.64) days, (32.42+/-25.49) days , respectively, while 63 cases of the survival group were 27.41+/-8.15, 18.65+/-5.34, 64.11+/-15.92, (5.23+/-5.50) days, (12.51+/-20.70) days, respectively, and there were significant differences between two groups (P<0.05 or P<0.01). The areas under receiver operating characteristic (ROC) curves of CAPS, APACHE II score and APACHE III score were 0.712 (P=0.005), 0.654 (P=0.043) and 0.655 (P=0.042), respectively. When CAPS score was 30.5, Youden index was the highest (0.435). The mortality rate had a positive correlation with CAPS. When the CAPS score was over 30, there was a tendency of increase in mortality rate. CONCLUSION: CAPS is very useful to evaluate the severity and prognosis of patients with AECOPD complicated by type II respiratory failure. It is easy to perform, and better than APACHE II and APACHE III.

Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization.

Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5mm) and small follicles (SF, 3-5mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.

Stage-specific expression of bone morphogenetic protein type I and type II receptor genes: Effects of follicle-stimulating hormone on ovine antral follicles.

We investigated the mRNA expression patterns of receptor genes for bone morphogenetic proteins-15 (BMP15) and growth differentiation factor-9 (GDF-9) in granulosa cells of sheep treated with FSH. The effects of FSH and estradiol (E2) on the regulation of BMPRII, BMPRIB and ALK-5 in ovine granulosa cells were also examined. Ovaries were collected on day 16 of the estrous cycle and granulose cells were harvested from follicles of two sizes (3-5 and >5mm in diameter). For in vitro studies, granulosa cells were obtained from follicles of 3-5mm in diameter and cultured in serum-free McCoy's 5A medium supplemented with different doses of FSH (0, 1, 5, 10ng/ml) or a combination of 5ng/ml FSH with 1ng/ml E2. Expression of BMPRII, BMPRIB and ALK-5 mRNA was estimated by quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than of small follicles. Treatment of granulose cells with FSH (1-10ng/ml) alone down-regulated the expression of BMPRIB (P<0.05). BMPRII and ALK-5 mRNA expression was not significantly different at an FSH concentration of 5ng/ml compared to control. A further increase in FSH (10ng/ml) down-regulated the expression of BMPRII and ALK-5 (P<0.05). The combination of FSH (5ng/ml) and E2 (1ng/ml) up-regulated the expression of BMPRII, BMPRIB and ALK-5 in granulose cells (P<0.05). Therefore, the present study establishes the expression levels of the receptor genes of BMP15 and GDF-9 and suggests that the expression of BMPRII, BMPRIB and ALK-5 may be regulated by FSH and E2 in ovine granulosa cells.

Effects of different activation protocols on preimplantation development, apoptosis and ploidy of bovine parthenogenetic embryos.

The objective of this study was to optimize the protocols for bovine oocytes activation through comparing the effectiveness of different treatments on the activation and subsequent development of oocytes and examining the effects of two combined activation treatments on the blastocyst apoptosis and ploidy. Cumulus-oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation, cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage. In Experiment 1, the matured oocytes were treated with single activation agents, including ionomycin (5 microM for 5 min), ethanol (7% for 7 min), calcium ionophore A23187 (5 microM for 5 min) or strontium (10mM for 5h). The pronuclear formation and cleavage rate were higher significantly in ionomycin (39.0 and 30.7%) and ethanol (41.5 and 28.1%) treatment alone compared to other treatments (9.7-25.2 and 11.3-23.7%, respectively, P<0.05). Very low blastocyst rates (3.9-5.3%) resulted which were not significantly different among treatments (P>0.05). For the combined activation treatment (Experiment 2), the same concentrations of ionomycin and ethanol as in Experiment 1 were used in combination with either 6-dimethylaminopurine (6-DMAP, 2.0 mM for 3 h) or cycloheximide (CHX)+cytochalasin B (CB, 10 microg/ml for 3 h). The pronuclear formation, cleavage rate, blastocyst rate and cell number of blastocyst were higher significantly (P<0.05) in ionomycin+6-DMAP treatment (67.1, 69.2, 28.0 and 91.3%, respectively) and ethanol+CHX+CB treatment (68.9, 70.2, 25.5 and 89.3%, respectively) compared to other treatments (11.7-58.1, 10.2-47.1, 1.5-24.2 and 34.2-62.7%, respectively). In Experiment 3, the parthenogenetic blastocysts produced by activation with ionomycin+6-DAMP and ethanol+CHX+CB and in vitro fertilized blastocysts (control group) were examined for apoptosis using a terminal deoxynucleotidyl transferase mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The ethanol+CHX+CB treatment (7.0%) showed significantly lower blastocyst apoptosis index compared to ionomycin+6-DAMP treatment (9.1%, P<0.05). Furthermore, the chromosomal composition in the parthenotes embryos differed (P<0.05) among treatments. The percentage of haploid parthenotes was higher in ionomycin+6-DMAP treatment than ethanol+CHX+CB treatment. These results suggested that ethanol+CHX+CB treatment was more favorable protocol for parthenogenesis of bovine oocytes.

Improvement in bovine embryo production in vitro by treatment with green tea polyphenols during in vitro maturation of oocytes.

The present study examined the effect of green tea polyphenols (GTP) during in vitro maturation (IVM) of bovine oocytes on in vitro fertilization (IVF) parameters, intracellular glutathione (GSH) concentration and subsequent embryo development. Cumulus-oocyte complexes were aspirated from the ovaries derived from slaughterhouse and cultured in modified synthetic oviduct fluid (m-SOF) supplemented with 0-25 microM GTP for 24h. After IVM, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 15-18 h. Putative embryos were transferred to m-SOF and cultured for 8 days (Experiment 1). In comparison with the absence of GTP, treatment with GTP at a concentration of 15 microM showed a significant increase in the proportion of pronuclear (PN) formation after sperm penetration (65% versus 80%, P<0.05). No significant differences in the rates of sperm penetration and polyspermic fertilization were found among treatments. The cleavage rate at 48 h of in vitro insemination showed no difference in oocytes matured with or without GTP. However, compared to no addition (23.5%), the presence of 15 and 20 microM GTP during IVM significantly (P<0.05) increased the proportion of blastocysts (38.1% and 36.4%) on day 9 of in vitro insemination. A further increase from 20 to 25 microM GTP reduced (P<0.05) the proportion of blastocysts. In Experiment 2, after IVM, oocytes were fixed to analyze the GSH concentration. Compared to no addition, a higher (P<0.05) level of GSH was found in oocytes matured with 15 microM GTP and compared with 15 microM GTP, GSH was low (P<0.05) at 20 and 25 microM GTP. The results suggest that at certain concentrations of GTP (15 microM) in IVM medium has beneficial effects on subsequent embryo development, and is correlated with intracellular GSH level in bovine oocytes.