Xiang Shun Cui

Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts

Evaluation of apoptosis and expression level of apoptosis-related genes is useful for examining the variation in embryo quality according to environmental change. The objective of this study was to investigate DNA fragmentation and apoptosis-related gene expression patterns in frozen-thawed bovine blastocysts. In vitro produced day 7 blastocysts were frozen by two different vitrification methods (conventional 0.25 ml straw or MVC straw). After thawing, DNA fragmentation of surviving embryos was examined by TUNEL assay, and the expression patterns of their apoptotic genes (survivin, Fas, Hsp 70 and caspase-3) were evaluated using real-time quantitative reverse transcriptase polymerase chain reaction. In vitro survival rates of frozen-thawed embryos were higher following the MVC vitrification method (88.2% re-expanded at 24 h, 77.1% hatching at 48 h) than the conventional (C) vitrification method (77.0% re-expanded at 24 h, 66.7% hatching at 48 h). However, both vitrified methods resulted in a significantly higher apoptotic index (C vitrification method 11.9%, MVC vitrification method 11.0%) than in non-frozen embryos (3.0%). Expression levels of survivin, Fas, caspase-3, and Hsp 70 were also increased in the frozen-thawed embryos compared with non-frozen embryos. These results indicate that the cryopreservation procedure might cause damage that results in an increase in DNA fragmentation and apoptosis-related gene transcription, reducing developmental capacity of frozen-thawed embryos.

Aberrant epigenetic reprogramming of imprinted microRNA-127 and Rtl1 in cloned mouse embryos

The microRNA (miRNA) genes mir-127 and mir-136 are located near two CpG islands in the imprinted mouse retrotransposon-like gene Rtl1, a key gene involved in placenta formation. These miRNAs appear to be involved in regulating the imprinting of Rtl1. To obtain insights into the epigenetic reprogramming of cloned embryos, we compared the expression levels of mir-127 and mir-136 in fertilized mouse embryos, parthenotes, androgenotes and cloned embryos developing in vitro. We also examined the DNA methylation status of the promoter regions of Rtl1 and mir-127 in these embryos. Our data showed that mir-127 and mir-136 were highly expressed in parthenotes, but rarely expressed in androgenotes. Interestingly, the expression levels of mir-127 and mir-136 in parthenotes were almost twice that seen in the fertilized embryos, but were much lower in the cloned embryos. The Rtl1 promoter region was hyper-methylated in blastocyst stage parthenotes (75.0%), moderately methylated (32.4%) in the fertilized embryos and methylated to a much lower extent (approximately 10%) in the cloned embryos. Conversely, the promoter region of mir-127 was hypo-methylated in parthenogenetically activated embryos (0.4%), moderately methylated (30.0%) in fertilized embryos and heavily methylated in cloned blastocysts (63-70%). These data support a role for mir-127 and mir-136 in the epigenetic reprogramming of the Rtl1 imprinting process. Analysis of the aberrant epigenetic reprogramming of mir-127 and Rtl1 in cloned embryos may help to explain the nuclear reprogramming procedures that occur in donor cells following somatic cell nuclear transfer (SCNT).

A link between the interleukin-6/Stat3 anti-apoptotic pathway and microRNA-21 in preimplantation mouse embryos.

Signal transducers and activators of transcription‐3 (Stat3) plays a central role in interleukin‐6 (IL‐6)‐mediated cell proliferation by inhibiting apoptosis in a variety of cell types. MicroRNA‐21 (miRNA‐21), a ubiquitous miRNA, acts as an anti‐apoptotic factor that seems to be indirectly but strictly linked to Stat3. In order to determine whether the IL‐6 induced Stat3 anti‐apoptosis pathway is linked with miRNA‐21, we first determined the effects of recombinant mouse IL‐6 on Stat3 expression, mouse embryo viability, and the mRNA levels of apoptosis related genes and miRNA‐21 during mouse embryo development in vitro. Addition of 10 or 100 ng/ml of recombinant IL‐6 to the culture medium did not affect the developmental ability of 2‐cell stage embryos into blastocysts. However, total cell number was significantly increased and apoptosis was reduced in blastocyst stage embryos cultured in the presence of 100 ng/ml of recombinant IL‐6. Furthermore, addition of recombinant IL‐6 to the culture medium significantly increased the copy numbers of anti‐apoptotic miRNA‐21, up‐regulated Bcl2l1, and down‐regulated casp3. Similarly, the injection of mature miRNA‐21 into cells up‐regulated Bcl2l1 and down‐regulated casp3. These results suggest that the induction of the Stat3 anti‐apoptotic pathway by IL‐6 is linked to miRNA‐21 expression, which possibly results in the regulation of cell apoptosis in early mouse embryo development. Mol. Reprod. Dev. 76: 854–862, 2009.

Molecular Characterization and Polyadenylation-Regulated Expression of Cyclin B1 and Cdc2 in Porcine Oocytes and Early Parthenotes

Meiotic maturation of mammalian oocytes is controlled by the maturation/M‐phase promotion factor (MPF), a complex of Cdc2 kinase and cyclin B protein. To better understand the molecular mechanism of oocyte maturation, we characterized porcine cyclin B1 and Cdc2 genes, both of which are widely expressed in pig tissues. We further analyzed their expression profiles during in vitro maturation of pig oocyte and early embryonic development at both the mRNA and protein level. Two isoforms of cyclin B1, comprising the same open reading frame but differing in 3′‐UTR length, were identified. Cyclin B1 transcripts was up‐regulated after 30 hr of maturation, while Cdc2 mRNA levels were unchanged during maturation except for a sharp decline at 44 hr. Cyclin B1 protein synthesis increased with oocyte maturation. Cdc2 protein expression was relatively low during 0–18 hr, followed by a higher level of expression up to 44 hr of maturation. Poly(A)‐test PCR clearly revealed that both cyclin B1 isoforms underwent cytoplasmic polyadenylation starting around 18–24 hr during maturation, while a substantial de‐adenylation and degradation of Cdc2 isoforms were observed in metaphase II oocytes and during embryo development after parthenogenetic activation. Porcine MII oocytes derived from small follicles (≤3 mm) and bad quality 2‐cell parthenotes showed lower developmental competence and lower levels of cyclin B1 protein, and Cdc2 mRNA or both gene mRNAs, respectively, compared to their control counterparts. These results suggested that cyclin B1 was regulated posttranscriptionally by cytoplasmic polyadenylation during porcine oocyte maturation. Further, the decreased expression of maternal cyclin B1 and Cdc2 at the mRNA or protein level in developmentally incompetent oocytes and embryos was responsible for, at least in part, a profound defect in further embryonic development. Mol. Reprod. Dev. 77: 38–50, 2010.

Involvement of ER–calreticulin–Ca2+ signaling in the regulation of porcine oocyte meiotic maturation and maternal gene expression

Calcium is one of the most ubiquitous signaling molecules, and controls a wide variety of cellular processes. It is mainly stored in the endoplasmic reticulum (ER), bound to lumenal proteins. Calreticulin is the major Ca2+‐binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER– calreticulin–Ca2+ pathway in oocyte maturation and early embryogenesis, we characterized the porcine calreticulin gene and investigated its expression profile during oocyte maturation and early embryonic development. Calreticulin was widely expressed in pig tissues and its transcripts were downregulated during maturation, especially at 44 hr, and were undetectable at the blastocyst stage. We also investigated the effect of increased cytosolic Ca2+ induced by the Ca2+‐ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 µM did not inhibit germinal vesicle breakdown, but did result in the arrest of 38.6% oocytes at or before the MI stage. In addition, expression of the maternal genes C‐mos, BMP15, GDF9, and Cyclin B1 was significantly increased in CPA‐treated MII oocytes compared with control groups. These data were supported by the results of poly(A)‐test PCR, which revealed that the cyclin B1 short isoform (CB‐S), GDF9, and C‐mos underwent more intensive polyadenylation modification in CPA‐treated oocytes than control oocytes, suggesting that polyadenylation may influence Ca2+‐modulated changes in gene expression. Furthermore, CPA treatment decreased the percentage of four‐cell parthenotes that developed into blastocysts, suggesting the need for functional SR/ER Ca2+‐ATPase pumps or Ca2+ signals during early embryo development after zygotic genome activation. Together, these data indicate that ER–calreticulin‐associated Ca2+ homeostasis plays a role in oocyte and embryo development, and that alterations in maternal gene expression may contribute to the underlying molecular mechanism, at least partially, via polyadenylation. Mol. Reprod. Dev. 77: 462–471, 2010.

Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.

Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa.

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.

Male pronuclear formation and sperm mitochondria in porcine oocytes following intracytoplasmic injection of pig or mouse sperm

In the present study we determined the chromatin organization and fate of introduced mitochondria in porcine embryos following intracytoplasmic injection of pig or mouse sperm cells. At 3, 6, 9 and 12 h following injection of pig or mouse spermatozoa or isolated sperm heads, the oocytes were fixed and stained with propidium iodide. Between 3 and 6 h following injection, both porcine and murine sperm chromatin developed into pronuclei. The male and female pronuclei were apposed within 12 h in porcine oocytes following sperm injection from either source. We also introduced foreign mitochondria from either mouse or pig sperm midpiece into porcine oocytes following sperm injection. While porcine sperm mitochondria rapidly disappeared from the actively developing porcine oocytes, mouse sperm mitochondria remained in the embryos until the 8-cell stage. These results suggest that pronuclear formation and movement occur between 6 and 12 h following sperm incorporation into the cytoplasm, and that foreign mitochondria are selectively removed in a species-specific manner.

Effect of anti-PMSG on distribution of estrogen receptor alpha and progesterone receptor in mouse ovary, oviduct and uterus.

It is well established that estrogen and progesterone are critical endogenous hormones that are essential for implantation and pregnancy in females. However, the distribution of estrogen receptor α (ERα) and progesterone receptor (PR) in female reproductive tracts is elusive. Herein, we report that after serial treatments with pregnant mares serum gonadotrophin (PMSG) with or without anti-PMSG (AP), mice could regulate the distribution of ERα and PR in the murine ovary, oviduct and uterus and the level of estradiol in serum. ERα and PR regulation by PMSG and anti-PMSG was estrous cycle-dependent and critical for promoting the embryo-implantation period. Furthermore, our results suggested that AP-42 h treatment is more effective than the other treatments. In contrast, other treatment groups also affected the distribution of ERα and PR in mouse reproductive tracts. Thus, we found that anti-PMSG has the potential to restore the distribution of ERα and PR, which could effectively reduce the negative impact of residual estrogen caused by the normal superovulation effect of PMSG in mice.