Rong Xun Han

Effect of Endoplasmic Reticulum Stress on Porcine Oocyte Maturation and Parthenogenetic Embryonic Development In Vitro

ABSTRACT X-box-binding protein 1 (XBP1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP1 was weak in mature oocytes and at the 1-, 2-, and 8-cell stages of embryos but abundant at the germinal vesicle (GV), 4-cell, morula, and blastocyst stages. In addition, RT-PCR revealed that both spliced XBP1 (XBP1-s) and unspliced XBP1 (XBP1-u) were expressed at the GV, 4-cell, morula, and blastocyst stages. Tunicamycin, an ER stress inducer, induced active XBP1 protein in nuclei of 4-cell embryos. Next, porcine embryos cultured in the presence of tauroursodeoxycholate, an ER stress inhibitor, were studied. Total cell numbers and the extent of the inner cell mass increased (P < 0.05), whereas the rate of nuclear apoptosis decreased (P < 0.05). Moreover, expression of the antiapoptotic gene BCL2 increased, whereas expression of the proapoptotic genes BCL2L1 (Bcl-xl) and TP53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress-mediated apoptosis in vitro.

Cardiovascular risk assessment of atypical antipsychotic drugs in a zebrafish model

The zebrafish model has been developed and evaluated for its ability to predict the toxicity of chemicals. Zebrafish additionally serve as an excellent model for assessing drug‐induced cardiotoxicity, although zebrafish and mammalian hearts differ in structure. Recently, regulatory authorities have expressed concerns about a possible relationship between antipsychotics and risk of QTc interval prolongation, serious arrhythmia and sudden cardiac death. In the current study, we performed a cardiovascular risk assessment of six atypical antipsychotic drugs in zebrafish, specifically, aripiprazole, clozapine, olanzapine, quetiapine, risperidone and ziprasidone. Visual endpoints, such as lethality, edema (the presence of heart and trunk edema), hemorrhage (clustering of a pool of blood in an area outside the normal circulation), abnormal body shape (including bent or misshapen caudal region of the larvae) and motility, were evaluated as general toxicity endpoints, and the heart beat rate calculated as the cardiovascular toxicity endpoint. The zebrafish model facilitates determination of the heart beat rate, and may thus be an attractive screening tool for cardiovascular risk assessment of atypical antipsychotic drugs to understand the variations in response to QT‐prolonging drugs. Copyright

A two-dimensional electrophoresis reference map for the bovine placenta during late pregnancy

An understanding of bovine placental gene expression is essential for the study of animal reproductive physiology. Recent reports have found that placental abnormalities occur frequently in cloned bovines and mice. However, the molecular mechanisms underlying bovine placenta function remain unclear. Here, we present a preliminary description of the bovine placenta proteome. Proteins within the isoelectric point ranging from 4.0 to 7.0 and 6.0 to 9.0 were analyzed separately using 2‐DE, using three replicates of bovine placenta. Approximately 2000 spots were detected in a placental 2‐D gel stained with Coomassie blue. Subsequent excision of 380 spots from gels and MALDI‐TOF MS analysis allowed the identification of 273 proteins. Our results revealed the composite profiles of key proteins in the bovine placenta during late pregnancy. These protein profiles will shed light on placental function during pregnancy and assist with functional analysis of the proteins.

Detection of early pregnancy-specific proteins in Holstein milk.

Bovine pregnancy is commonly diagnosed by rectal palpation or ultrasonography and changes in progesterone concentration. To determine a simpler and less expensive diagnostic method, we sought to identify early pregnancy-specific proteins in bovine milk by comparing samples collected from pregnant and non-pregnant Holstein cattle. Of the 600-700 protein spots visible on 2-DE gel images, 39 were differentially expressed in milk from pregnant and non-pregnant cattle. Antibodies generated against synthetic peptides of milk whey proteins expressed specifically during pregnancy were used to confirm protein expression patterns. Western blot analysis showed that the levels of expression of lactoferrin (lactotransferrin) and alpha1G T-type calcium channel subunit (alpha-1G) were higher in samples from pregnant than non-pregnant cattle. These findings suggest that assays for pregnancy-specific milk proteins may be used to diagnose pregnancy in cattle.

Aberrant protein expression in the placenta of cloned mouse derived from embryonic stem cell

Placentomegaly is a common phenotype in cloned mice. To assess differences in protein expression between placentae of cloned and uncloned mice, we used a proteomic approach involving 2-dimensional electrophoresis (DE) and MALDI-TOF MS. Proteins within isoelectric point range of 4-11 separately were analyzed in 2-DE with 3 replications of each sample. A total of approximately 3500 spots were detected in placental 2-DE stained with Coomassie blue. In the comparison of normal and cloned samples, a total of 41 spots were identified as differentially expressed proteins, of which 25 spots were up-regulated proteins such as TIMP-2, glutamate-ammonia, and esterase 10, while 16 spots were down-regulated proteins such as PBEF and annexin A1. The TIMP-2, which is related to extracellular matrix degradation and tissue remodeling processes, is an inhibitor of MMP-2. The PBEF is related to inhibition of apoptosis and induction of spontaneous labor. Western blot analysis confirmed increased TIMP-2 expression and decreased PBEF expression in cloned placentae compared with normal controls. Our results demonstrated composite profiles of key proteins involved in abnormal hypertrophic placenta derived from cloned mice and suggested that the reason for the placentomegaly may be due to abnormal gene expression in cloned mice.

Halogenated nucleotide labeling of nascent RNAs reveals dynamic transcription in growing pig oocytes.

Background: Germ cells differentiate into oocytes in females and are arrested at the first meiotic prophase. However, during arrest, oocytes undergo a growth phase leading to a dramatic increase in size, which is under control of transcription events. In the current study, we examined the transcriptional activity of growing pig oocytes using an immunocytochemical approach. Our data showed that fluorouridine (FU), a halogenated nucleotide, can be successfully incorporated into synthesizing RNAs and detected using a specific monoclonal antibody. Results: Using this method, we identified dynamic changes in transcriptional activity patterns in growing pig oocytes. Oocytes obtained from small follicles exhibited the highest level of transcription, while at the final phase of growth, transcription was no longer detected. These transcriptional changes were concomitant with chromatin compaction resulting in a tightly packed ring‐like chromatin conformation surrounding the nucleolar structure. Also, FU incorporation appeared sensitive to the biochemical manipulation of transcription, because transcriptional inhibitors induced a decrease in signal intensity from FU labeling and transcriptional activation caused an increase in FU signal intensity. Conclusions: Our data collectively support that a direct link exists between chromatin configuration and transcriptional activity in pig oocytes, and support the suitability of FU for studies on transcription‐related events in mammalian oocytes. Developmental Dynamics 242:16–22, 2013.