284 KINETICS OF OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT OF PARTHENOGENETIC PORCINE EMBRYOS AFTER MEIOTIC INHIBITION WITH ROSCOVITINE

Kim, D.H.,Kim, S.W.,Im, G.S.,Yang, B.C.,Lee, D.R.,Park, H.S.,Hwang, I.S.,S Seo, J.,Yang, B.S.,Chang, W.K. CSIRO Publishing 2005 Reproduction, fertility, and development Vol.17 No.1-2

<P> Maturation of mammalian oocytes is a very important process for subsequent embryo development after fertilization. Prolonged maturation time by meiotic inhibitors could be an effective method for improvement in the meiotic and developmental competence of mammalian oocytes. Roscovitine, a cyclin dependent kinase inhibitor, is known to specifically inhibit M-phase promoting factor (MPF) kinase activity and prevent the resumption of meiosis. The aim of this study was to examine the effect of roscovitine on the maturation and subsequent development of porcine oocytes. Ovaries were collected from slaughtered prepubertal gilts and COCs were aspirated from 2- to 5-mm antral follicles. In control, porcine cumulus oocyte complexes (COCs) were cultured in the maturation medium (TCM-199 supplemented with 0.3% BSA, 1 μg/mL FSH, 1 μg/mL LH, and 10 ng/mL EGF) for 44 h. In the experimental group, COCs were cultured in the inhibition medium (TCM-199 supplemented with 0.3% BSA and roscovitine) for 24 h, and then further cultured in the maturation medium for 44 h. Matured oocytes from both groups were activated by electrical pulse (1.2 kV/cm for 30 μs), and then cultured in PZM-3 medium for 6 days. Apoptotic cells in blastocysts were detected by TUNEL assay and total cell number was examined by propidium iodide (PI) counterstaining. Data were analyzed by chi-square and Student's t-test. The first experiment was conducted to determine the effect of roscovitine (0, 12.5, 25, 50, and 100 μM) on meiotic inhibition of GV oocytes. This effect was dose-dependent, and a concentration of 50 μM was sufficient to prevent meiotic resumption in 79.2% (76/96, 5 replicates) of the porcine oocytes after 24 h of culture when compared to 0 (15.4%, 15/97), 12.5 (32.1%, 36/112), 25 (57.4%, 54/94), and 100 μM (77.8%, 77/99). The second experiment was carried out to examine the kinetics of maturation of roscovitine-treated porcine oocytes. The concentration of roscovitine used was 50 μM. A total of 75.8% (50/66, 3 replicates) of roscovitine-treated oocytes reached metaphase II stage compared with 70.8% (46/65) of control. The third experiment was performed to compare embryo development between control and treated group after parthenogenetic activation. No differences (P > 0.05) were found between the control and the treated group in cleavage rate (77.2%, 132/171 vs. 68.0%, 115/169), blastocyst rate (26.9%, 46/171 vs. 17.8%, 30/169), and total (33.7 ± 12.4 vs. 35.1 ± 12.6) and apoptotic (2.2 ± 2.4 vs. 2.2 ± 1.2) cell number per blastocyst (4 replicates). The results suggest that roscovitine can be used to prolong maturation time of porcine oocytes without reducing meiotic maturation but also without significantly decreasing their subsequent developmental competence. Further studies are necessary to improve the developmental competence of porcine oocytes treated with roscovitine.</P>

298 DIFFERENTIATION OF CANINE AMNIOTIC FLUID MESENCHYMAL STEM CELLS INTO NEURONAL PRECURSORS

Kim, E. Y.,Choi, S. A.,Lee, J. H.,Kim, K. J.,Park, K. S.,Park, Y. B.,Ha, Y. N.,Li, X.,Park, J. Y.,Kim, M. K. CSIRO Publishing 2011 Reproduction, fertility, and development Vol.23 No.1

<P> The amniotic fluids contain mesenchymal stem cells and can be readily available for tissue engineering. Recently, regenerative treatments such as tissue engineering, cell therapy, and transplantation have shown potential in clinical trials of degenerative diseases. Physiologically, disease presentation and clinical responses in the dog are much more similar to that in the human compared with other traditional mammalian models. In addition, several researchers have demonstrated Canis familiaris is a suitable model for human diseases. The aim of the present study was to investigate whether canine amniotic fluid (cAF)-derived mesenchymal stem cells (MSC) can differentiate into neural precursor cells in vitro by neural induction reagent. The conditions of differentiation of MSC into neural cells were DMEM and N2-supplement, dibutyryl cyclic adenosine monophosphate, and butylated hydroxyanisole. During neural precursor differentiation, cAF-MSC can progressively acquire neuron-like morphology. Expressions of neuron cell-specific markers were examined before and after in vitro induction of differentiation. Changes in mRNA levels of specific genes were quantified by RT-PCR. The mRNA levels of NEFL (730%), GFAP (350%), β-tubuline 3 (2900%), and NSE (960%) were significantly increased after induction. The value of change in mRNA levels before and after induction was evaluated with the Image J program. In addition, the nestin, β-tubuline 3, and tyrosine hydroxylase protein expressions were confirmed by immunocytochemistry assay following the induction of differentiation, compared with the noninduction. In conclusion, this study demonstrated that cAF-MSC have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegeneration diseases including Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. </P>

Inactivated Sendai-virus-mediated fusion improves early development of cloned bovine embryos by avoiding endoplasmic-reticulum-stress-associated apoptosis

Song, Bong-Seok,Kim, Ji-Su,Yoon, Seung-Bin,Lee, Kyu-Sun,Koo, Deog-Bon,Lee, Dong-Seok,Choo, Young-Kug,Huh, Jae-Won,Lee, Sang-Rae,Kim, Sun-Uk,Kim, Sang-Hyun,Kim, Hwan Mook,Chang, Kyu-Tae CSIRO Publishing 2011 Reproduction, fertility, and development Vol.23 No.6

<P> Somatic cell nuclear transfer (SCNT) is a powerful tool, not only for producing cloned animals, but also in revealing various early developmental events. However, relatively little is known regarding the biological events and underlying mechanism(s) directly associated with early development of SCNT embryos. Here, we show that production of high-quality bovine SCNT blastocysts is dependent on the method used for fusion and the associated reduction in endoplasmic reticulum (ER) stress. During fusion between the donor cell and the enucleated oocyte, electrofusion triggers spontaneous oocyte activation, accompanied by an increase in intracellular Ca2+ and improper nuclear remodelling. These events can be greatly reduced by the use of Sendai virus (SV)-mediated fusion. Moreover, SV-SCNT improves the blastulation rate and blastocyst quality, defined by the number and ratio of inner cell mass and trophectoderm cells in each blastocyst, in comparison with electrofusion-mediated SCNT (E-SCNT). Interestingly, expression of ER-stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos. These increases could be reversed by inhibition of ER stress or by using the SV-mediated fusion method. Collectively, these results indicate that SV-mediated fusion improves the developmental competence and quality of SCNT blastocysts, by reducing ER-stress-associated apoptosis. </P>

Regulation of XFGF8 gene expression through SRY (sex-determining region Y)-box 2 in developing Xenopus embryos

Kim, Yong Hwan,Shin, Jee Yoon,Na, Wonho,Kim, Jungho,Ju, Bong-Gun,Kim, Won-Sun CSIRO Publishing 2012 Reproduction, fertility, and development Vol.24 No.6

<P> Fibroblast growth factors (FGFs) function as mitogens and morphogens during vertebrate development. In the present study, to characterise the regulatory mechanism of FGF8 gene expression in developing Xenopus embryos the upstream region of the Xenopus FGF8 (XFGF8) gene was isolated. The upstream region of the XFGF8 gene contains two putative binding sites for the SRY (sex-determining region Y)-box 2 (SOX2) transcription factor. A reporter assay with serially deleted constructs revealed that the putative SOX2-binding motif may be a critical cis-element for XFGF8 gene activation in developing Xenopus embryos. Furthermore, Xenopus SOX2 (XSOX2) physically interacted with the SOX2-binding motif within the upstream region of the XFGF8 gene in vitro and in vivo. Depletion of endogenous XSOX2 resulted in loss of XFGF8 gene expression in midbrain-hindbrain junction, auditory placode, lens placode and forebrain in developing Xenopus embryos. Collectively, our results suggest that XSOX2 directly upregulates XFGF8 gene expression in the early embryonic development of Xenopus. </P>

Effects of aroclor 1254 on the expression of the KAP3 gene and reproductive function in rats

Lee, Chae Kwan,Kang, Han Seung,Kim, Ju Ran,Lee, Byung Ju,Lee, Jong Tae,Kim, Jeong Ho,Kim, Dae Hwan,Lee, Chang Hee,Ahn, Jin Hong,Lee, Chae Un,Yu, Seong Jin,Kang, Sung Goo CSIRO Publishing 2007 Reproduction, fertility, and development Vol.19 No.4

<P> The present study investigated the effects of aroclor 1254 (A1254) on the expression of the kinesin superfamily associated protein 3 (KAP3) gene in F1 rat brain during brain sexual differentiation and puberty. In addition, the effects of A1254 on reproductive function were examined. The KAP3 gene is involved in the neurogenesis and synaptogenesis of sexual differentiation in rats and also during puberty. In the present study, pregnant Sprague-Dawley rats each received a daily dose of A1254 (0, 10, 50 mg kg-1) dissolved in 1.0 mL corn oil by gavage, from gestational Day (GD) 8 to postnatal Day (PD) 21. The mRNA levels of the KAP3 gene in hypothalamic tissues were analysed by northern blot hybridisation during the critical periods of brain sexual differentiation (GD18 and PD5) and puberty (PD28). Variables affecting reproduction in F1 female rats, such as vaginal opening (VO), vaginal oestrus (VE) and oestrous cyclicity, were recorded. Depending on the sex and A1254 exposure (control or 50 mg kg-1 day-1), F1 rats were divided into three mating groups, namely control male-control female, control male-A1254-treated female and A1254-treated male-control female. During the critical periods of brain sexual differentiation (GD18, PD5) and puberty (PD28), KAP3 mRNA levels were significantly reduced in A1254-treated fetal and pubertal rat brains relative to those of control groups. In A1254-treated F1 female rats, VO and VE were delayed, the percentage of irregular oestrous cycles was increased and the duration of the oestrous cycle was extended in a dose-dependent manner compared with control groups. Treatment with a high dose of A1254 significantly impaired the reproductive function of both male and female F1 rats, including mating and pregnancy indices and the number of live fetuses. These data suggest that A1254 disrupts transcriptional regulation of the KAP3 gene in fetal and pubertal rat brains and that these effects may be related to A1254-induced abnormal brain sexual differentiation and lowered reproductive function in F1 rats. </P>

Maternally derived transcripts: identification and characterisation during oocyte maturation and early cleavage

Cui, Xiang-Shun,Kim, Nam-Hyung CSIRO Publishing 2007 Reproduction, fertility, and development Vol.19 No.1

<P> The identification and characterisation of differentially regulated genes in oocytes and early embryos are required to understand the mechanisms involved in maturation, fertilisation, early cleavage and even long-term development. Several methods, including reverse transcription-polymerase chain reaction-based suppression subtractive hybridisation, differential display and cDNA microarray, have been applied to identify maternally derived genes in mammalian oocytes. However, conventional gene-knockout experiments to determine specific gene functions are labour intensive and inefficient. Recent developments include the use of RNA interference techniques to establish specific gene functions in mammalian oocytes and early embryos. Regulation of the poly(A) tail length is a major factor in controlling the activities of maternal transcripts in mammals. Further studies are required to clarify the mechanisms by which expression levels of maternally derived transcripts are regulated. In the present review, we focus on the identification and functions of the differentially expressed transcripts during oocyte maturation, fertilisation and early cleavage. </P>

390 THE EFFECT OF REDUCED FSH DOSE AND NUMBER OF TREATMENTS ON SUPEROVULATORY RESPONSE IN CIDR-TREATED KOREAN NATIVE COWS

Son, D. S.,Choe, C. Y.,Cho, S. R.,Choi, S. H.,Kim, H. J.,Kim, I. H. CSIRO Publishing 2007 Reproduction, fertility, and development Vol.19 No.1

<P> Reducing the total dose and numbers of treatments with FSH for superstimulation without decreasing embryo yield may be less stressful and more economical for bovine embryo transfer. The objective of this study was to investigate the effect of dose and the number of days of FSH treatment on superovulatory responses in CIDR-treated Korean native cows. Forty-two cows, at random stages of the estrous cycle, received a CIDR device (CIDRTM; InterAg, Hamilton, New Zealand), 1 mg estradiol benzoate (SY Esrone; Samyang, Seoul, Korea) and 50 mg progesterone (SY Ovaron; Samyang); gonadotropin treatment began 4 days later. Cows were divided into 2 groups based on the dose and numbers of days of treatment with porcine FSH (pFSH): T1 group (n = 20): a total of 28 mg pFSH (recommended dose of Antorin�; Kawasaki Pharmaceutical, Tokyo, Japan) was given in twice daily IM injections in decreasing doses over 4 days (5, 5, 4, 4, 3, 3, 2, and 2 mg); and T2 group (n = 22): a total of 24 mg pFSH given in twice daily decreasing doses over 3 days (5, 5, 4, 4, 3, and 3 mg). Otherwise, all cows received the same treatments. Twenty-five and 15 mg dinoprost (PGF2α; Lutalyse; Pharmacia & Upjohn, Puurs, Belgium) were given with the 5th and 6th injections of pFSH, respectively. CIDR devices were withdrawn with the 6th pFSH injection, and the cows received 100 �g Gonadorelin (GnRH; Fertagyl; Intervet, Boxmeer, The Netherlands) 36 h after CIDR device removal. Cows were artificially inseminated using commercial semen from 4 Korean native bulls twice, at 48 and 60 h after CIDR device removal, and embryos were recovered 6 or 7 days after the 2nd insemination. The number of CL was counted on the day of embryo recovery by transrectal ultrasonography (Sonovet 600 with 5.0 MHz linear-array transducer; Medison Co., Ltd., Seoul, Korea). The recovered embryos were evaluated according to the IETS Manual for stage of development and quality. All data between groups were compared using Student&apos;s t-test. The numbers of CL (9.7 � 1.1 vs. 9.4 � 1.3), total ova/embryos (7.2 � 1.1 vs. 6.3 � 1.4), transferable embryos (4.4 � 1.0 vs. 3.6 � 0.9), degenerate embryos (0.9 � 0.3 vs. 1.3 � 0.4), and unfertilized ova (2.0 � 0.6 vs. 1.5 � 0.5) did not differ between groups (T1 vs. T2), respectively (P &gt; 0.05). Data indicate that the reduced dose (24 vs. 28 mg) and numbers of treatments (6 vs. 8) of pFSH for superstimulation of Korean native cows does not affect the embryo yield. </P>

165 EXPRESSION OF CALCIUM TRANSPORTER 1 GENE IN THE UTERUS OF RATS DURING PREGNANCY

Lee, B.-M.,Lee, G.-S.,Jeung, E.-B. CSIRO Publishing 2007 Reproduction, fertility, and development Vol.19 No.1

<P>Calcium Transporter 1 (CaT1), which is highly expressed in calcium-absorption organs, i.e. duodenum and kidney, is a critical mediator for calcium uptake during transcellular calcium transport. It has been shown that CaT1 gene is also expressed in the placenta and uterine smooth muscle in female reproductive organs. We previously demonstrated that uterine CaT1 mRNA is highly expressed at diestrus and tightly regulated by progesterone (P4) related to calcium homeostasis for uterine functions during the estrous cycle in rats. Thus, in the recent study, we further examined the expression of CaT1 mRNA in the uterus and placenta of rats to elucidate its role during pregnancy in these tissues. Female Sprague Dawley rats (n = 2) were employed and their pregnancy days (PD) were determined by a vaginal plug every morning; the rats were euthanized daily (PD 0 to 21). The expression of CaT1 mRNA decreased from PD 0 to 4 and highly increased on PD 5 to 10. Its increased transcripts gradually decreased at the end of pregnancy. Taken together, these results indicate that the expression level of CaT1 mRNA is regulated in the uterus of rats during pregnancy, probably via sex steroid hormones and their receptors through a complex pathway. A further study is warranted to verify relevant factors which regulate CaT1 gene and to provide further insight into its role(s) in the female reproductive tissues.</P>

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

Lee, Gi-Ho,Sohn, Seong-Han,Park, Eun-Young,Park, Young-Doo CSIRO Publishing 2012 Functional plant biology Vol.39 No.9

<P> The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3-9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene. </P>

Effects of increasing supplementation levels of rice bran on milk production and fatty acid composition of milk in Saanen dairy goats

Park, J.K.,Kwon, E.G.,Kim, C.-H. CSIRO PUBLISHING 2013 Animal production science Vol.53 No.5