Journal of Reproduction and Development
Online ISSN : 1348-4400
Print ISSN : 0916-8818
ISSN-L : 0916-8818
Volume 56, Issue 5
October
Displaying 1-11 of 11 articles from this issue
Review
  • Seung Pyo GONG, Jae Hee LEE, Jeong Mook LIM
    Article type: -Review-
    2010 Volume 56 Issue 5 Pages 481-494
    Published: 2010
    Released on J-STAGE: November 10, 2010
    JOURNAL FREE ACCESS
    Somatic cell nuclear transfer, the first established technique for producing patient-specific autologous stem cells, inevitably requires the sacrifice of viable embryos. To circumvent the serious ethical issues associated with this use of embryos, researchers have developed several alternative methods for the production of histocompatible stem cells. In our research, we have used two methods to derive histocompatible stem cells from murine ovarian tissue. First, we have established autologous stem cells by culturing degeneration-fated preantral follicles to produce developmentally competent, mature oocytes and then parthenogenetically activating these mature oocytes to acquire genetic homogeneity. Second, we have used cell-to-cell interactions to derive stem cells from ovarian stromal cells without undertaking genetic modification. We have successfully derived autologous murine stem cells by manipulating primary and early secondary follicles in vitro, and this method has proved successful even for follicles retrieved from aged ovaries. Furthermore, we believe that it will be possible to isolate stem cells directly from non-germline ovarian tissue or to derive stem cells by culturing the ovarian cells with other somatic cells. If achieved, these aims will greatly advance the development of induced pluripotent stem cell technology, as well as tissue-specific stem cell research. In this review, we introduce the relevant technologies for establishing histocompatible stem cells from ovarian tissue cells without undertaking genetic manipulation and review the current limitations of, and future research directions in, stem cell biology.
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Original Article
  • Sugako OGUSHI, Mitinori SAITOU
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 495-501
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 01, 2010
    JOURNAL FREE ACCESS
    During oocyte growth in the ovary, the nucleolus is mainly responsible for ribosome biogenesis. However, in the fully-grown oocyte, all transcription ceases, including ribosomal RNA synthesis, and the nucleolus adopts a specific monotonous fibrillar morphology without chromatin. The function of this inactive nucleolus in oocytes and embryos is still unknown. We previously reported that the embryo lacking an inactive nucleolus failed to develop past the first few cleavages, indicating the requirement of a nucleolus for preimplantation development. Here, we reinjected the nucleolus into oocytes and zygotes without nucleoli at various time points to examine the timing of the nucleolus requirement during meiosis and early embryonic development. When we put the nucleolus back into oocytes lacking a nucleolus at the germinal vesicle (GV) stage and at second metaphase (MII), these oocytes were fertilized, formed pronuclei with nucleoli and developed to full term. When the nucleolus was reinjected at the pronucleus (PN) stage, most of the reconstructed zygotes cleaved and formed nuclei with nucleoli at the 2-cell stage, but the rate of blastocyst formation and the numbers of surviving pups were profoundly reduced. Moreover, the zygotes without nucleoli showed a disorder of higher chromatin organization not only in the female pronucleus but also, interestingly, in the male pronucleus. Thus, the critical time point when the nucleolus is required for progression of early embryonic development appears to be at the point of the early step of pronucleus organization.
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  • Ken-ichiro ABE, Azusa INOUE, Masataka G. SUZUKI, Fugaku AOKI
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 502-507
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 16, 2010
    JOURNAL FREE ACCESS
    As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 μm). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP II from the DNA. We also observed dissociation of RPB1 from the DNA in full-grown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.
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  • Ai TAKESHITA, Tomohiro KONDO, Toshiya OKADA, Ken Takeshi KUSAKABE
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 508-514
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 23, 2010
    JOURNAL FREE ACCESS
    Supplementary material
    The complement system is thought to be precisely regulated during pregnancy. We have examined specific gene profiles in mouse placentas causing spontaneous abortion and found notable up-regulation of adipsin, a complement activating factor. The aim of the present study was to determine the basic kinetics and localization of adipsin in the placenta and the difference in complement activity between normal placentas and placentas of abortuses. Normal and spontaneously absorbed implantation sites obtained from naturally-mated mouse uteri on days 10.5 and 14.5 of pregnancy were processed for histologic studies and protein purification. Adipsin immunoreaction was detected at the decidua basalis in normal placentas and additionally at the placental labyrinth in the absorbed placentas. The quantity of adipsin was increased in the absorbed placentas compared with the normal placentas. In concert with the increase in adipsin, the amounts of complement component 3 and degradation products were elevated and complemental activity was up-regulated in the absorbed placenta. These findings suggest that local expression of adipsin has a reproductive effect at the feto-maternal interface and possibly plays a role in spontaneous abortion.
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  • Takuo HOJO, Akihiro ODA, Seung-Hyung LEE, Tomas J. ACOSTA, Kiyoshi OKU ...
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 515-519
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 16, 2010
    JOURNAL FREE ACCESS
    The corpus luteum (CL) is mainly composed of luteal steroidogenic cells (LSCs) and luteal endothelial cells (LECs). Cell death of LSCs and LECs is essential for structural luteolysis. Therefore, it is important to understand the mechanisms regulating cell death in both types of luteal cells. We previously reported that a treatment combining tumor necrosis factor α (TNF) and interferon γ (IFNG) induced cell death in LSCs and that LECs express TNF receptor type I (TNFRI). To investigate the mechanism of cell death in LECs, in the present study we determined the effects of the same cytokines on cell viability and TNFRI mRNA expression in cultured LECs. To induce cell death in LECs, LECs were treated with TNF or IFNG (0, 0.05, 0.5, 1.0, 2.5 nM) and a combination of TNF (0.5 nM) and IFNG (0.5 nM) for 24 h. The viability of LECs was reduced by a single treatment with TNF or IFNG dose-dependently (P<0.05). Cell viability was further decreased by treatment with a combination of TNF and IFNG (P<0.05). Cells with DNA fragmentation (TUNEL-positive cells) were observed after the treatment with TNF and IFNG. Semi-quantitative RT-PCR analysis revealed that treatment with IFNG alone or in combination with TNF increased the expression of TNFRI mRNA compared with the control (P<0.05). In summary, TNF and IFNG increased cell death in cultured bovine LECs. The upregulation of TNFRI mRNA expression by IFNG suggests that TNF and IFNG synergistically affect the death of LECs resulting in acute luteolysis.
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  • Mohamed M.M. KANDIEL, Gen WATANABE, Alaa E. ABDEL-GHAFFAR, Gamal A. SO ...
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 520-526
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: July 28, 2010
    JOURNAL FREE ACCESS
    The present study was conducted to characterize follicular development and its hormonal control during early pregnancy in goats. The ovaries of goats (n=8) were scanned daily for follicles (> or = 2 mm in diameter) and corpora lutea by transrectal ultrasound with blood sampling from the jugular vein for monitoring the hormonal changes during the first thirty-five days after mating. During early pregnancy, three (37.5%), four (50%) and one (12.5%) goat showed nine, eight and seven waves of follicular development, respectively. The corpora lutea were detected as early as Day 3.61 ± 0.45 (7.47 ± 0.43 mm) of pregnancy (Day 0=day of mating) and attained their maximal cross-sectional diameter (10.64 ± 0.37 mm) on Day 25.7 ± 0.8 of pregnancy, respectively. A transient rise in FSH levels was temporally associated with the day of follicular wave emergence (up to three days prior to wave emergence). The plasma LH and estradiol levels were negatively correlated with the progesterone concentration. The rise in plasma immunoreactive (ir) inhibin levels was negatively correlated with the FSH concentration and positively correlated with the number of large-sized follicles. Alternatively, the mean plasma ir-inhibin levels showed a noticeable decline with the progression of pregnancy. The present results demonstrated that follicular development during early pregnancy shows a wave-like pattern, with seven to nine waves developing until Day 35 after breeding, and that the number of follicular waves can be predicted by the number of FSH peaks. The current study also demonstrated that the role of inhibin as an FSH regulator is maintained throughout early pregnancy.
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  • Luciano BONILLA, Daniel LUCHINI, Estelle DEVILLARD, Peter J. HANSEN
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 527-532
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 16, 2010
    JOURNAL FREE ACCESS
    The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium - bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 μmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 μmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 μmol/l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 μmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine. In conclusion, the methionine requirement for preimplantation development is between 14 and 21 μmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of embryonic mortality.
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  • Yong FAN, Yumei LUO, Xinjie CHEN, Xiaofang SUN
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 533-539
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: July 20, 2010
    JOURNAL FREE ACCESS
    Human embryonic stem cells (HESCs) are defined as self-renewing cells that retain their ability to differentiate into all cell types of the body. They have enormous potential in medical applications and as a model for early human development. There is a need for derivation of new HESC lines to meet emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. Here, we describe a modified culture medium containing human recombinant leukemia inhibitory factor and human basic fibroblast growth factor that significantly increases the number of human blastocysts formed and their quality, as well as the efficiency of HESC derivation from poor-quality embryos. Culturing poor-quality embryos in modified medium resulted in a two-fold increase in the blastocyst formation rate and a seven-fold increase over the derivation efficiency in conventional medium. We derived 15 HESC lines from poor-quality embryos cultured in modified culture medium and two HESC lines from quality embryos cultured in conventional culture medium. All cell lines shared typical human pluripotent stem cell features including similar morphology, normal karyotypes, expression of alkaline phosphatase, pluripotency genes, such as Oct4, and cell surface markers (SSEA-4, TRA-1-60, TRA-1-81), the ability to form teratomas in SCID mice, and the ability to differentiate into cells of three embryonic germ layers in vitro. Our data suggest that poor-quality embryos that have reached the blastocyst stage in our modified culture medium are a robust source for normal HESC line derivation.
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  • Kathryn L. GATFORD, Christopher G. GRUPEN, Roger G. CAMPBELL, Brian J. ...
    Article type: -Original Article-
    2010 Volume 56 Issue 5 Pages 540-545
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 23, 2010
    JOURNAL FREE ACCESS
    Litter size and progeny birth weights are lower in gilts than in sows. Somatotropin (ST) is an important regulator of ovulation, fetal growth and survival. We therefore investigated effects of pST treatment of gilts for two to four weeks before mating on ovulation rate, behavioural estrus, fetal growth and survival, litter size and birth weights. In Experiment One, gilts were injected with 0, 30, 60 or 90 μg pST/kg/day for 14 days commencing 7 days after first estrus. Reproductive tracts were collected and corpora lutea and follicle numbers counted 5.5 days after second estrus. Ovulation rate (P=0.031) and number of medium-sized follicles (P=0.059) correlated positively with pST dose. In Experiment Two, gilts were injected with 0, 12.5, 25 or 50 μg pST/kg/day for 21 days from first estrus, and mated at second estrus. Numbers of corpora lutea, follicles and fetuses were counted at day 31 of pregnancy. Numbers of medium follicles and ovary weights were positively related to pST dose. In Experiment Three, 31 week old (1st replicate) or 27 week old (2nd replicate) gilts were injected daily with 0 or 12.5 μg pST/kg/day until mating 25.9 ± 0.6 days later, and delivered at term. Pre-mating pST increased total litter size in younger gilts in the 2nd replicate only (P<0.05). In conclusion, injecting gilts with pST before mating does not consistently alter ovulation rate, increases the number of medium follicles available for recruitment at the second mating after treatment and increases subsequent litter size in younger gilts.
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Technology Report
  • Ivan VASSILIEV, Svetlana VASSILIEVA, Luke F.S. BEEBE, Stephen M. MCILF ...
    Article type: -Technology Report-
    2010 Volume 56 Issue 5 Pages 546-551
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 01, 2010
    JOURNAL FREE ACCESS
    In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.
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  • Takayuki KOIKE, Koji MATSUURA, Keiji NARUSE, Hiroaki FUNAHASHI
    Article type: -Technology Report-
    2010 Volume 56 Issue 5 Pages 552-557
    Published: 2010
    Released on J-STAGE: November 10, 2010
    Advance online publication: June 16, 2010
    JOURNAL FREE ACCESS
    Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence.
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