[Changes and clinical significance of erythrocyte lifespan in megaloblastic anemia].

Objective: To investigate the lifespan of erythrocytes in megaloblastic anemia (MA) patients. Methods: A prospective cohort study analysis. Clinical data from 42 MA patients who were newly diagnosed at the Department of Hematology, Lanzhou University Second Hospital from January 2021 to August 2021 were analyzed, as were control data from 24 healthy volunteers acquired during the same period. The carbon monoxide breath test was used to measure erythrocyte lifespan, and correlations between erythrocyte lifespan and laboratory test indexes before and after treatment were calculated. Statistical analysis included the t-test and Pearson correlation. Results: The mean erythrocyte lifespan in the 42 newly diagnosed MA patients was (49.05±41.60) d, which was significantly shorter than that in the healthy control group [(104.13±42.62) d; t=5.13,P=0.001]. In a vitamin B12-deficient subset of MA patients the mean erythrocyte lifespan was (30.09±15.14) d, and in a folic acid-deficient subgroup it was (72.00±51.44) d, and the difference between these two MA subsets was significant (t=3.73, P=0.001). The mean erythrocyte lifespan after MA treatment was (101.28±33.02) d, which differed significantly from that before MA treatment (t=4.72, P=0.001). In MA patients erythrocyte lifespan was positively correlated with hemoglobin concentration (r=0.373), and negatively correlated with total bilirubin level (r=-0.425), indirect bilirubin level (r=-0.431), and lactate dehydrogenase level (r=-0.504) (all P<0.05). Conclusions: Erythrocyte lifespan was shortened in MA patients, and there was a significant difference between a vitamin B12-deficient group and a folic acid-deficient group. After treatment the erythrocyte lifespan can return to normal. Erythrocyte lifespan is expected to become an informative index for the diagnosis and treatment of MA.

αv ß3 Integrin may participate in conceptus attachment by regulating morphologic changes in the endometrium during peri-implantation in ovine.

The objective of this study was to determine expression and potential functions of α(v) and ß(3) integrin subunits in ovine endometrium during the peri-implantation period (days 8-17 after fertilization). The morphologic changes in the endometrium were observed histochemically following haematoxylin/eosin (HE) staining, whereas the expressions of α(v) and ß(3) integrin subunits were analysed by RT-PCR, immunohistochemistry and Western blot. The filamentous conceptus attached to the luminal epithelium (LE) on day 17 of pregnancy, with no differences in endometrial morphology between days 8-12 of pregnancy. However, endometrial glands in the endometrial stroma (S) underwent extensive hyperplasia from day 14 to day 17, increased reductus of the LE with an obvious proliferation of caruncles, and an increased number and diameter of blood vessels (V) in the endometrium. The relative expression levels of α(v) and ß(3) integrin subunits mRNA gradually increased until day 16, but sharply declined on day 17. Western blot analysis revealed that the expression pattern of α(v) and ß(3) integrin subunit proteins paralleled that of the corresponding mRNA. In addition, immunohistochemical localization of α(v) and ß(3) integrin subunits confirmed their presence in the glandular epithelium (GE), LE and endometrial stroma. Immunostaining on LE and stroma varied with the increasing days of pregnancy, with the strongest immunostaining on days 16 and 17. In conclusion, expression of α(V) and ß(3) integrin subunits was closely related to the early progression of pregnancy and conceptus attachment; therefore, we inferred that α(v) ß(3) integrin may participate in conceptus attachment by the regulation of endometrial morphology during peri-implantation in ovine.

Expression of interferon-tau mRNA in bovine embryos derived from different procedures.

Interferon-tau (IFN-tau) is a secreted conceptus protein which plays a critical role in the establishment of ruminant pregnancy by its antiluteolytic and antiviral effects. In the present study, we hypothesized that IFN-tau expression was temporally and spatially regulated in different pre-implantation embryos and the levels of IFN-tau expression were different among bovine embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). By using in situ hybridization with Digoxingenin (DIG)-labelled IFN-tau cDNA as a probe, we detected IFN-tau mRNA in bovine embryos from days 3 to 9 in culture. However, the timing of the initiation of IFN-tau mRNA expression was different among PA, IVF and SCNT embryos. Interferon-tau mRNA was first expressed in 16-cell stage IVF embryos on day 4, in SCNT morula on day 5 and early PA blastocyst on day 6. Semi-quantitative RT-PCR analysis showed that the expression levels of IFN-tau mRNA did not differ significantly among IVF, SCNT and PA embryos on day 7. In addition, freezing and thawing did not have a major impact either on IFN-tau mRNA expression in IVF or in vivo-produced bovine blastocysts.

Porcine cumulus cell influences ooplasmic mitochondria-lipid distributions, GSH-ATP contents and calcium release pattern after electro-activation.

The objective was to explore mechanisms of the influence of porcine cumulus cells (CC) on oocyte maturation. Immature porcine oocytes were matured in groups of denuded oocyte (DOs), cumulus-oocyte complexes (COCs), denuded oocytes co-cultured with CC (DoCC), or with cumulus-oocyte complexes (DoCOCs). Ooplasmic mitochondria-lipid distributions, glutathione (GSH)-adenosine triphosphate (ATP) contents, calcium release pattern, and developmental competence after parthenogenetic activation were assessed after IVM. The portion of matured oocytes after IVM and the developmental competence and GSH content in single oocytes were lower in DOs than in COCs (P<0.05). In contrast, the maturation rate and development in DoCOCs and COCs were higher than in DoCC and DOs (P<0.05). The blastocyst rate in DoCOCs was higher than in DOs (P<0.05), and ATP content in COCs was higher than in all other groups (P<0.01). In addition, the rate of oocytes with damaged oolemma in DOs (35%) was significantly higher than in COCs (3%), DoCOCs (7%), and DoCC (10%). The rate of oocytes with evenly distributed mitochondria was 70% in DOs, which was significantly lower than in COCs and DoCC (89 and 84%, respectively). The percentage of oocytes with normal lipid droplets distributions in COCs (70%) was significantly higher than in three other groups, whereas both percentages in DoCC and DoCOCs were higher than in DOs (P<0.05). The duration of [Ca(2+)] rise in DOs was longer than in three other groups, whereas the duration was shortest in COCs. The amplitude of the [Ca(2+)] rise in DOs was significantly lower than in other groups (P<0.05), but the amplitude did not differ significantly among DoCC, DoCOCs and COCs. In conclusion, the presence of porcine CC during IVM functionally affected ooplasmic mitochondria-lipid distributions and GSH-ATP contents, which may affect the calcium release pattern and developmental competence of oocytes after electro-activation.

Heat shock at the germinal vesicle breakdown stage induces apoptosis in surrounding cumulus cells and reduces maturation rates of porcine oocytes in vitro.

The objectives were to determine the effects of cumulus cells (CC) on porcine oocyte maturation in vitro (IVM) after heat shock (HS). Treated oocytes were cultured at 39 degrees C for 20h, followed by HS treatment (42 degrees C for 1h), and then matured in vitro for 23h. The CC were removed before maturation (H1), after HS (H2), or after maturation (H3). Control oocytes were continuously cultured under the same conditions and CC were similarly removed before maturation (C1), after 21h of IVM (C2), and after maturation (C3). Maturation rates were affected by HS (P<0.01) and by an interaction between HS and CC (P<0.01). A significant decrease in maturation rate only occurred when CC were not removed from cumulus oocyte complexes during IVM after HS (H3, 39.2+/-5.7% versus C3, 78.2+/-8.2%, P<0.01). Mature oocytes in all treatment groups were electrically activated and cultured for 8 d in NCSU23. Blastocyst rates in group H1 (7.2+/-3.5%) and C1 (6.3+/-3.1%) were lower than in other groups (H2, 21.4+/-4.4%, C2, 20.5+/-7.0%, H3, 23.1+/-2.0%, C3, 24.3+/-3.1%, P<0.05). Damaged DNA was detected in CC by a comet assay at 0h after HS (60.8+/-12.5% compared with 9.2+/-2.2% in control, P<0.05); in HS groups, both DNA damage (comet assay, 74.9+/-6.3% compared with 10.0+/-2.1% in control) and apoptosis (TUNEL assay, 21.6+/-1.6% compared with 5.6+/-0.6% in control) in CC were increased (P<0.05) at 44h of maturation. In conclusion, heat shock (42 degrees C for 1h) during IVM induced DNA damage and apoptosis of porcine CC; furthermore, apoptotic CC may contribute to maturation failure of oocytes in vitro.