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Tetsuma MURASE, Ismail EL-KON, Hiroshi HARAYAMA, Koushi MUKOUJIMA, Mas ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
36-40
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: October 08, 2009
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This study investigated whether a cyclic adenosine 3',5'-monophosphate (cAMP) analogue, cBiMPS, could induce hyperactivated motility in frozen-thawed Japanese Black bull spermatozoa and compared the ability of spermatozoa to undergo hyperactivation between fertile and subfertile bulls. Frozen-thawed spermatozoa from 3 fertile and 2 subfertile bulls were washed, suspended in BO-Hepes medium and incubated in the presence of 0.1 mM cBiMPS for up to 4 h. At 1-h intervals, the spermatozoa were examined for hyperactivated motility. The proportions of spermatozoa showing a circular swimming pattern with asymmetrical flagellar beating and those showing whiplash beating of flagella to the total number of motile spermatozoa were expressed as C% and W%, respectively. The motile spermatozoa % was barely affected by treatment with cBiMPS or the fertility status of the sperm donor, although it gradually decreased in all sperm samples during the 4-h incubation. In the fertile bulls, the C% was 0% at 0 h of incubation but rapidly increased during the 1-h incubation with cBiMPS. It then decreased slightly towards 4 h concomitantly with a gradual increase in W% towards 4 h. In the subfertile bulls, however, the cBiMPS-induced increase of C% was delayed for 1-3 h, although the incubation time-related changes in mean W% were similar between the fertile and subfertile bulls. In the vehicle controls for cBiMPS, the C% and W% were 0% throughout incubation for all the samples examined. The results suggest that hyperactivation of the flagellum can be induced by the cAMP analogue, cBiMPS, in frozen-thawed Japanese Black bull spermatozoa and that induction of hyperactivation may serve as a useful tool for detection of functional abnormality of spermatozoa from subfertile Japanese Black bulls.
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Laura Francesca PISANI, Paola RAMELLI, Barbara LAZZARI, Silvia BRAGLIA ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
41-48
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: October 08, 2009
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Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis and control the developmental program until embryonic genome activation takes place. NLRP5 (NLR family, Pyrin domain containing 5), also called MATER (Maternal Antigen That Embryos Require) is one of the genes required for normal early embryonic development, although its precise function remains to be elucidated. The aim of the present study was to analyze the NLRP5 gene expression pattern and protein distribution in somatic tissues and germ cells in the pig. Reverse transcription was performed on mRNA from germinal vescicle (GV) oocytes and total RNA from spermatozoa and tissues from different organs. The transcript for NLRP5 gene was identified only in ovaries and oocytes. The presence of NLRP5 protein was detected only in ovaries by western blot analysis and immunohistochemistry.
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Kanokwan SRIRATTANA, Chanchao LORTHONGPANICH, Chuti LAOWTAMMATHRON, Su ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
49-54
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: October 08, 2009
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This study investigated the effect of donor cell types on the developmental potential and quality of cloned swamp buffalo embryos in comparison with cloned cattle embryos. Fetal fibroblasts (FFs), ear fibroblasts (EFs), granulosa cells (GCs) and cumulus cells (CCs) were used as the donor cells in both buffalo and cattle. The cloned cattle or buffalo embryos were produced by fusion of the individual donor cells with enucleated cattle or buffalo oocytes, respectively. The reconstructed (cloned) embryos and
in vitro matured oocytes without enucleation were parthenogenetically activated (PA) and cultured for 7 days. Their developmental ability to the blastocyst stage was evaluated. The total number of trophectoderm (TE) and inner cell mass (ICM) cells and the ICM ratio in each blastocyst was determined by differential staining as an indicator of embryo quality. The fusion rate of CCs with enucleated oocytes was significantly lower than for those of other donor cell types both in cattle and buffalo. The rates of cleavage and development to the 8-cell, morula and blastocyst stages of cloned embryos derived from all donor cell types did not significantly differ within the same species. However, the cleavage rate of cloned cattle embryos derived from FFs was significantly higher than those of cattle PA and cloned buffalo embryos. The blastocyst rates of cloned cattle embryos, except for the ones derived from CCs, were significantly higher than those of cloned buffalo embryos. In buffalo, only cloned embryos derived from CCs showed a significantly higher blastocyst rate than that of PA embryos. In contrast, all the cloned cattle embryos showed significantly higher blastocyst rates than that of PA embryos. There was no difference in ICM ratio among any of the blastocysts derived from any of the donor cell types and PA embryos in both species. FFs, EFs, GCs and CCs had similar potentials to support development of cloned cattle and buffalo embryos to the blastocyst stage with the same quality.
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Tetsuya TAKUMA, Sayoko SAKAI, Daisuke EZOE, Hitoshi ICHIMARU, Takaomi ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
55-59
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: October 08, 2009
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The aim of the present study was to determine whether the season (hot and cool) and reproductive phase (pregnant and non-pregnant) of the cow affect follicular recruitment and oocyte development. Follicular oocytes were aspirated from Japanese black cows by the ovum pick-up (OPU) method, which was performed 2 to 6 times within 1.5 months in pregnant cows and 2 to 4 times within 2 months in non-pregnant cows, during the hot (July to September) and cool (October to November) seasons. After follicular aspiration, the number and morphology of cumulus-oocyte complexes (COCs) and the developmental competence of oocytes after
in vitro maturation (IVM),
in vitro fertilization (IVF) and
in vitro culture were evaluated. The quality of aspirated COCs did not differ between the hot and cool seasons, irrespective of the reproductive phase of the donor cows. In the pregnant cows, the season did not affect follicular recruitment, early embryonic development or the quality of embryos. In the non-pregnant cows, however, the mean number of aspirated follicles and collected oocytes decreased during the hot season as compared with the cool season. When the data for the 2 seasons were combined to assess the effects of reproductive phase on oocyte development, the total proportions of cleavage, development into blastocysts and freezable embryos were higher for embryos obtained from pregnant cows (P<0.05) than those obtained from non-pregnant cows. In conclusion, the season did not have any apparent effects on the quality of aspirated COCs and the developmental competence of oocytes after IVM-IVF, but it may affect follicular recruitment in non-pregnant cows. Moreover, the reproductive phase may influence the developmental competence of the recovered oocytes.
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Ken-ichi YAMANAKA, Ahmed Zaky BALBOULA, Miki SAKATANI, Masashi TAKAHAS ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
60-67
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: October 08, 2009
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A highly methylated genome, like a somatic donor cell, is observed in somatic cell nuclear transfer (SCNT) embryos. The aberrant DNA methylation status causes global gene expression failure, resulting in low developmental competence of SCNT embryos. In addition, recent studies have uncovered the relationship between DNA methylation status and reprogramming efficiency. Because DNA methylation is performed by DNA methyltransferases (DNMTs), developing a technique which specifically inhibits DNMTs is necessary for further SCNT studies. In the present study, we examined the potential use of RNA interference for knockdown of
DNMT mRNA in bovine fibroblast cells that were commonly used as karyoplast donors in SCNT studies. We designed three siRNAs corresponding to
DNMT1,
DNMT2 and
DNMT3a mRNA. In Experiment 1, to optimize transfection conditions, fluorescence and cell viability after transfection were evaluated at different concentrations of transfection reagent using a FITC-labeled nonsilencing control siRNA. Although fluorescence was observed in all groups transfected except for the negative control group, transfection with a higher concentration of transfection reagent significantly decreased in cell viability (P<0.05). In Experiment 2, the amount of
DNMT mRNA was measured by real-time PCR at 0, 48 and 96 h after siRNA transfection into the cells. The levels of each
DNMT mRNA were significantly decreased at 48 and 96 h after transfection (P<0.01). Furthermore, decreased expression of DNMT1 protein was confirmed by western blotting. In Experiment 3, the DNA methylation statuses were analyzed in each of the siRNA-transfected groups. The
DNMT1 siRNA-transfected group had a significantly decreased level of DNA methylation (P<0.05), but the other groups did not. Our data demonstrate that RNA interference with siRNA can be analyzed the function and expression of
DNMT genes in bovine fibroblast cells. The present study provides useful information for further SCNT studies.
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Margareta WALLGREN, Fernando SARAVIA, Heriberto RODRIGUEZ-MARTINEZ
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
68-72
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: October 27, 2009
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In boars, sperm cohorts are sequentially emitted in epididymal cauda fluid and resuspended in different mixtures of accessory sex gland secretions while ejaculated in various fractions. During natural mating, these ejaculate fractions sequentially enter the cervix-uterine lumen, are quickly transported towards the tips of the uterine horns and colonize the oviductal sperm reservoirs (SR). Using a simple experiment, we tested the hypothesis that the first ejaculated sperm subpopulation (fortuitously present in the peak portion of the sperm-rich fraction [SRF], the so-called Portion 1, P1) is, by reaching first the SR, overrepresented there. Spermatozoa from P1- and of P2- (last portion of the SRF and the Post-SRF) were collected from 3 fertile boars. P1-spermatozoa were fluorophore DNA-stained, while P2-spermatozoa were kept unstained. Weaned estrous sows were conventionally inseminated (12 h after onset of estrus) with similar sperm numbers (approx 10 × 10
9 spermatozoa) per portion but in different orders as follows: (i) a mix of P1 and P2 aliquots (control, P1+P2, n=5), or testing (ii) a sequential order (P1-P2, Treatment A, n=5) or (iii) an inverse order (P2-P1, Treatment B, n=5) of cohort AI. Sows were euthanized approx 3 h post-AI, and the SRs were flushed to recover the spermatozoa, which were accounted for as stained or unstained. The total number of spermatozoa flushed did not differ between groups or boars (NS, ranging 0.9 to 2.0 × 10
9). Sequential,
in vivo-like, sperm deposition (P1-P2, Treatment A) yielded the highest proportion of stained P1-spermatozoa in the SRs (59.8 ± 5.66%, means ± SEM) compared with when the order was reversed (P2-P1, Treatment B; 15.6 ± 2.1% P1-spermatozoa, P<0.05) or P1 and P2 sperm suspensions were mixed (control, 36.9 ± 2.70% P1-spermatozoa, P<0.05). The tested hypothesis proved valid; if inseminated in the same order as ejaculated
in vivo, P1-spermatozoa become overrepresented in the SR. The physiological consequences of this skewed SR-colonization are discussed in this paper alongside the advantageous use of P1-spermatozoa for handling, including cryopreservation.
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Hong-Fei XIA, Xiao-Hua JIN, Pei-Pei SONG, Yi CUI, Chun-Mei LIU, Xu MA
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
73-78
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 02, 2009
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Mammalian embryonic implantation requires reciprocal interactions between implantation-competent blastocysts and a receptive uterus. Some microRNAs might play a key role during embryo implantation in the mouse, but the
let-7a expression profiles in the rat uterus during peri-implantation are unknown. In the study, the expression of
let-7a in the uterus during early pregnancy, pseudopregnancy, artificial decidualization and activation of delayed implantation was detected by Northern blotting and
in situ hybridization. The effect of steroid hormones on
let-7a expression was also detected by Northern blotting and
in situ hybridization. Here, we found that the expression level of
let-7a was higher on gestation day 6-7 (g.d. 6-7) in rats than on g.d.4-5 and g.d.8-9.
Let-7a was specifically localized in glandular and luminal epithelia and decidua. The expression of
let-7a was not significantly different in the pseudopregnant uterus and increased significantly in the uteri of rats subjected to artificial decidualization and activation of delayed implantation. Treatment with estradiol-17
β or progesterone significantly increased
let-7a expression. Thus,
let-7a expression was significantly induced by the process of embryo invasion, and this increased expression level was mainly induced by active blastocysts and decidualization during the window of implantation, implying that
let-7a may participate in endometrial decidualization. Steroid hormones, estradiol-17
β or progesterone stimulated
let-7a expression.
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Manabu KAWAHARA, Qiong WU, Tomohiro KONO
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
79-85
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 02, 2009
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Imprinted genes in which only one of the two parental chromosome copies is expressed have a substantial effect on mammalian ontogenesis. On mouse distal chromosome 7, the paternally expressed gene insulin-like growth factor 2 (
Igf2) is separated by approximate 100 kb from the maternally expressed non-coding gene
H19. However, there is limited knowledge of the manner in which
Igf2 transcription affects the other genes involved in embryonic development. To clarify this, we performed quantitative gene expression analysis for representative angiogenic factors-
Vegf, Flt1, Flt4, Flk1, Ang1, Ang2,
Tie1, and
Tie2-for 3 types of bi-maternal conceptuses containing genomes with non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the ng oocytes were 1) the wild type (ng
wt), 2) mutant mice carrying a 3-kb deletion of the
H19 transcription unit (ng
H19Δ3-KO/fg) and 3) mutant mice carrying a 13-kb deletion in the
H19 transcription unit, including the germline-derived differentially methylated region on chromosome 7 (ng
H19Δ13-KO/fg). In the ng
wt/fg and ng
H19Δ3-KO/fg placentae,
Vegf and
Flt1 were upregulated compared with the mean value for the wt placenta, whereas in the ng
H19Δ13-KO/fg placenta, these transcriptional levels were restored. In the fetus, however, only 2 genes among the 8 genes analyzed were significantly changed in the bi-maternal fetuses, indicating that the effects of the
Igf2 mRNA level on angiogenic factor transcription in the fetus differed from those in the placenta. Our results indicated that the
Igf2 mRNA level affects transcription of angiogenic factors in both bi-maternal placentae and fetuses.
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Hui Wen LIM, Misa IWATANI, Naoko HATTORI, Satoshi TANAKA, Shintaro YAG ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
86-93
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: December 09, 2009
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The DNA methyltransferase (Dnmt) inhibitor and demethylating agent 5-aza-2'-deoxycytidine (5azadC) has been used to induce cellular differentiation and gene activation. It has been approved for treating several kinds of malignancies due to its ability to reactivate silenced tumor suppressor genes. Considering the potential effect of 5azadC on non-targeted genomic regions in normal cells, we investigated its effect on repetitive sequences and selected gene loci,
Oct-4,
Sall3,
Per1,
Clu,
Dpep1 and
Igf2r, including tissue-dependent and differentially methylated regions, by treating mouse NIH/3T3 fibroblast cells with concentrations of 5azadC ranging from 0.001 to 5
μM. Demethylation of minor satellite repeats and endogenous viruses was concentration dependent, and the demethylation was strong at 1 and 5
μM. In genic regions, the methylation level decreased only at 0.1
μM, but was minimally altered at concentrations lower or higher, regardless of the abundance of CpG sites. Thus, repeats are strongly demethylated, but genic regions are only demethylated at effective doses. Genes were activated by 5azadC treatment and were accompanied by a unique combination of histone modifications in genic regions, including an increased level of H3K9me3 and a decreased level of AcH3. Increase of H3K9me3 in genic regions was not observed in Dnmt knock out cells. We identified differential effects of 5azadC on repetitive sequences and genic regions and revealed the importance of choosing appropriate 5azadC doses to achieve targeted gene recovery.
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Michela ISOLA, Margherita COSSU, Antonello DE LISA, Raffaella ISOLA, D ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
94-97
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 05, 2009
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Oxytocin is a cyclic nonapeptide whose best known effects are stimulation of uterine smooth muscle cells during labor and of milk ejection during lactation. Circulating oxytocin originates from the hypothalamus, but its production has also been documented in peripheral tissues. Furthermore, seminal plasma also contains oxytocin, but its functional role is still unknown, although its secretion is generally ascribed to the prostate. In this study, we investigated the possibility that seminal oxytocin is also secreted by other exocrine glands of the human male genital tract. Intramural (Littrè's) glands isolated from bioptic specimens of normal urethrae were processed for immunogold localization of oxytocin. Immunostaining was detected in principal cells, with gold particles specifically found on secretory granules. Basal and endocrine cells were unstained. The present findings suggest that urethral glands not only produce the mucinous layer that protects and lubricates the urethral wall, but also are potential sources of other seminal components, such as oxytocin, which probably play still unclear roles in reproductive physiology.
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Kazumasa HONDA, Takashi HIGUCHI
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
98-102
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 02, 2009
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The dorsomedial nucleus of the hypothalamus (DMH) has been suggested to be a final relay site for the afferent pathway of milk-ejection reflex (MER). The present experiments were undertaken to examine whether unilateral lesion of the DMH abolished MER induced by unilateral suckling by pups. The unilateral DMHs of urethane-anesthetized lactating rats were electrolytically lesioned. Then 5 pups were applied to nipples ipsilateral or contralateral to the lesioned DMH and the intramammary pressure was monitored to observe the occurrence of MER. The effects of bilateral suckling by 10 pups were also examined. Eighteen of the 29 rats displayed MER during ipsi- or bilateral suckling for about 1 h. Seventeen of these 18 rats did not show MER during contralateral suckling for about 1 h. The remaining rat that showed milk ejection during contralateral suckling had a lesion outside the DMH. In 2 additional rats, extracellular action potentials of single oxytocin (OT) cells located in the supraoptic nucleus ipsilateral to the lesioned DMH were recorded during suckling ipsi- and conralateral to the lesioned DMH. OT cells showed milk-ejection burst during ipsilateral suckling but not during contralateral suckling. The results suggest that the unilateral DMH receives information of suckling stimuli applied to contralateral nipples.
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Ryosuke SUZUKI, Sueo NIIMURA
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
103-109
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 02, 2009
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The role of actin filaments and contractions in hatching was determined in mouse blastocysts whose actin filament bundling abilities had been suppressed by H-89, an inhibitor of protein kinase A. The hatching rate of blastocysts developed from morulae in a medium containing H-89 at a concentration of 4.0 μM was 17.2%, which was significantly lower than the 76.7% of the control blastocysts developed from morulae in a medium without H-89. The rates of blastocysts starting hatching and forming a slit in the zona pellucida were significantly lower in H-89-treated blastocysts (84.4 and 21.9%) than in control blastocysts (100.0 and 90.6%). The lengths of time needed for slit formation in the zona pellucida and for completion of hatching were significantly longer in the H-89-treated blastocysts (27.4 and 43.3 h) than in the control blastocysts (6.5 and 18.8 h). Over the course of 32 h after blastocoel formation, the number of strong contractions was similar in the H-89-treated and control blastocysts, but the number of weak contractions was significantly fewer in the H-89-treated blastocysts (2.41 times) than in the control blastocysts (4.19 times). Although the distribution of actin filaments was similar in the H-89-treated and control blastocysts in the pre-hatching, hatching and post-hatching periods, the rate of H-89-treated blastocysts in which most trophectoderm cells possessed the fluorescence of actin filaments (12.7%) was significantly lower than the 95.0% of the control blastocysts in the pre-hatching period. These results suggest that actin filament-mediated movements of trophectoderm cells contribute to hatching by facilitating the protrusion of trophectoderm cells from a small hole in the zona pellucida and by enlarging the protrusion. We also suggest that the low hatching ability of the treated blastocysts is related to weak contractions with a low frequency and to strong contractions requiring a longer time for re-expansion.
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Ryo NISHIMURA, Kiyoshi OKUDA
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
110-116
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 02, 2009
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Hypoxia-inducible factor 1 (HIF1) has been demonstrated to have critical roles in angiogenesis via transcriptional regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). In the ovary, angiogenesis is known to occur after ovulation in the developing corpus luteum (CL) in mammals. To determine whether HIF1 participates in angiogenesis in bovine CL, the present study investigated the mRNA and protein expressions of the HIF1 alpha subunit (HIF1A) and VEGF in bovine CL during the estrous cycle. The effects of hypoxia on the expressions of HIF1A protein,
VEGF mRNA and VEGF protein in bovine luteal cells were also examined by using a cell culture system.
HIF1A mRNA expression was less at the regressed stage than at the other stages, whereas protein expression of HIF1A was highest at the early luteal stage and decreased thereafter.
VEGF mRNA expression was highest at the developing luteal stage and decreased thereafter. VEGF protein expression was highest at the early luteal stage and decreased significantly at the regressed luteal stage. Hypoxia increased the amounts of HIF1A protein,
VEGF mRNA and VEGF protein in cultured bovine luteal cells. Furthermore, we found that hypoxia inhibited progesterone production in the mid luteal cells, but not in the early luteal cells. The overall findings indicate that HIF1 is one of the factors promoting VEGF-induced angiogenesis during luteal development, and suggest that the hypoxic conditions formed after follicle rupture contribute to establishing luteal vascularization in cattle.
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Daisuke HAYAKAWA, Motoki SASAKI, Masatsugu SUZUKI, Toshio TSUBOTA, Hir ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
117-123
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 19, 2009
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Testicular steroidogenesis and spermatogenesis during developmental and seasonal changes were investigated in male sika deer (
Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P450c17, 3βHSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3βHSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.
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Koumei SHIRASUNA, Takayuki ASAHI, Motoki SASAKI, Takashi SHIMIZU, Akio ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
124-130
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 05, 2009
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We have shown that luteal blood flow increases in the peripheral vasculature of the mature corpus luteum (CL) prior to the onset of luteolysis in response to prostaglandin F
2α (PGF
2α) in the cow, but this phenomenon does not occur in the early CL. We therefore hypothesize that this acute increase of luteal blood flow occurs by vasodilation of large blood vessels due to local release of nitric oxide (NO). In the present study, we characterized the CL vasculature together with endothelial NO synthase (eNOS) expression during the estrous cycle in the cow. Immunohistochemistry was used to quantify the number of arteriolovenous vessels (surrounded with smooth muscle actin-positive smooth muscle cells), capillaries (with von Willebrand Factor-positive endothelial cells) and eNOS protein. The arteriolovenous vessels existed more in the periphery of the matured CL (mid, late and regressing CL) than in the center region. In the early CL, there were as many arteriolovenous vessels in the periphery as in the center, while more capillaries existed in the center than in the periphery of the mid and late CL. Also, eNOS protein existed in the periphery more than in the center of the matured CL. These results indicate that the early CL has a homogeneous vascular and eNOS distribution. In contrast, the matured CL is a heterogeneous organ having a higher vascular and eNOS distribution in the periphery than in the center. In conclusion, the distribution of arteriolovenous vessels and eNOS in the matured CL was higher in the periphery than in the center of the CL. Thus, this suggests that this structural change from the early (homogeneous) to the mid (heterogeneous) luteal phase is related to the difference in the CL response of blood flow increase due to PGF
2α, which is only observed in the mature CL.
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Mohammad Musharraf Uddin BHUIYAN, Yo SUZUKI, Hiroyuki WATANABE, Eunson ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
131-139
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 05, 2009
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The objectives of this study were to choose an effective embryo reconstruction method and an effective post-activation agent for
in vitro production of sei whale (
Balaenoptera borealis) interspecies somatic cell nuclear transfer (iSCNT) embryos. Moreover, trichostatin A (TSA) treatment of whale iSCNT embryos was performed to improve the
in vitro embryo development. In Experiment 1, the fusion rate was significantly higher (88.1%) in embryos reconstructed using the intracytoplasmic cell injection method (ICI) than that (48.7%) in the subzonal cell insertion (SUZI) counterpart. The rates of pseudopronucleus (PPN) formation (77.4
vs. 77.2%) and cleavage (24.5
vs. 37.0%) did not vary between ICI and SUZI. However, the PPN formation and cleavage rates were significantly (P<0.05) lower in the iSCNT embryos than in the parthenogenetic control (95.7% and 64.4%, respectively). Although 21.5% of the bovine parthenogenetic embryos developed to the blastocyst stage, no iSCNT embryo developed beyond the 6-cell stage. In Experiment 2, the cleavage rate did not vary between the TSA (50 nM)-treated and non-treated whale iSCNT embryos (30.5
vs. 32.3%, respectively). Moreover, it did not vary between the TSA-treated iSCNT and SCNT embryos (30.5
vs. 32.0%, respectively). Only one TSA non-treated iSCNT embryo developed to a compacted morula with 20 nuclei. One TSA-treated whale SCNT embryo developed to the 8-cell stage, and out of five whale iSCNT embryos, a 6-cell stage embryo was positive for whale DNA. In conclusion, bovine oocytes have the ability to support development of sei whale nuclei up to the 6-cell stage.
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Woojin KANG, Chong ZHOU, Yoshitaka KOGA, Tadashi BABA
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
140-144
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 19, 2009
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Sperm hyaluronidase has long been believed to participate in sperm penetration through the cumulus matrix. However, our previous works using male mice lacking either one of two sperm hyaluronidases, SPAM1 and HYAL5, conclusively showed that neither of these hyaluronidases is essential for fertilization. In this study, we examined whether the hyaluronan-degrading activity of mouse epididymal sperm is indeed required for the fertilization process. When the oocyte-cumulus complex was incubated with sperm protein extracts or capacitated epididymal sperm in the presence of the hyaluronidase inhibitor apigenin, dispersal of cumulus cells from the cumulus was effectively inhibited. Despite the presence of apigenin, capacitated epididymal sperm normally entered the oocyte-cumulus complex, traversed the cumulus matrix and reached the oocyte zona pellucida. Importantly, epididymal sperm were also capable of normally fertilizing the metaphase II-arrested oocytes in the presence of apigenin. These data suggest that the hyaluronan-degrading activity of sperm hyaluronidase may not be required for fertilization, at least in the mouse.
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Mito KANATSU-SHINOHARA, Narumi OGONUKI, Hiromi MIKI, Kimiko INOUE, Hir ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
145-153
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: November 19, 2009
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Spermatogonial stem cells (SSCs) are slowly dividing cells that undergo self-renewal division to support spermatogenesis. Although the effects of genetic background in stem cell self-renewal have been well studied in hematopoietic stem cells, little is known about its effect on stem cells in other self-renewing tissues, including SSCs. To examine whether genetic factors are involved in regulation of SSC self-renewal, we first studied spermatogenesis in different inbred mouse strains (C57BL/6, DBA/2, AKR, BALB/C and C3H) after chemical damage caused by busulfan. Spermatogenesis in the DBA/2 and AKR strains was relatively resistant to busulfan treatment, whereas spermatogenesis was diminished in C57BL/6 mice and nearly ablated in C3H and BALB/C mice. Serial germ cell transplantation experiments provided functional evidence that SSCs with the DBA/2 background expanded more rapidly than those with the B6 background. Finally, we also employed the Germline Stem (GS) cell culture technique to examine the self-renewal activity
in vitro. Although genetic manipulation of GS cells has been limited to those from the DBA/2 background, we produced transgenic offspring of the C3H background by electroporation of GS cells with a plasmid vector. Our results underscore the importance of genetic factors in SSC self-renewal. Furthermore, application of genetic modification techniques to GS cells with non-DBA/2 backgrounds extends the potential of a SSC-based approach in male germline modification.
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Marcos B. VALDEZ Jr., Makoto MIZUTANI, Keiji KINOSHITA, Akira FUJIWARA ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
154-161
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: December 09, 2009
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To elucidate strain differences in the sex reversal of genetic females to phenotypic males, GSP and PNP/DO females were left ovariectomized (ovx) between one to three days after hatching, and the degree of masculinization based on sex-related characters, histological analysis of the right gonad and hormone assay were assessed at one year of age. The GSP and PNP/DO inbred lines were both derived from the Fayoumi breed and are only differentiated based on the red blood cell antigen type carried by each inbred line. Combs and wattles were found to be significantly bigger (P<0.05) in the GSP ovx compared with the PNP/DO ovx chickens, although male plumage patterns were more pronounced in the PNP/DO ovx. Spurs were observed both in the GSP and PNP/DO ovx chickens with no significant difference (P>0.05) in length compared with the respective male controls, and body weight was not significantly different (P>0.05) compared with the female controls. The weight of the right gonad was significantly heavier (P<0.05) in the GSP ovx than in the PNP/DO ovx. Positive correlations were found in the sex-related characters as well as the plasma testosterone level and right gonad weight in both the GSP and PNP/DO ovx chickens, but not in the spur length, which showed a negative correlation in the PNP/DO ovx chickens. Histological analysis revealed that the right gonads of the PNP/DO ovx chickens were morphologically developed compared with the GSP ovx chickens, which showed more advance stages of spermatogenesis. It could be inferred that PNP/DO females that exhibit a hereditary persistent right oviduct are more responsive to the masculinizing effect of ovariectomy compared with GSP females, suggesting that genetic background may have a possible contribution to the degree of masculinization and subsequent development of sex related characters.
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Wei DING, Wei WANG, Bo ZHOU, Wei ZHANG, Pan HUANG, Fangxiong SHI, Kazu ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
162-168
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: December 09, 2009
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The assembly of primordial follicles and subsequent development and transition of the primordial follicle to the primary follicle are critical processes in ovarian biology. In order to examine follicle formation and development in fetal and neonatal pigs, ovarian samples were obtained from a famous local breed of Chinese pigs, Erhualian pigs, ranging in age from 50 days postcoitum to 1 day postpartum in our current study. Morphological changes in the ovaries of the fetal and neonatal pigs indicated that egg nests were the earliest recognizable gamete cells. The proportion of egg nests decreased from 98.4 to 25.6% and the proportion of single follicles increased from 1.6 to 74.4% between 70 and 90 days postcoitum. The proportions of primordial follicles increased between 70 and 90 days postcoitum but decreased from 90 days postcoitum to 1 day postpartum. Our results suggested that the key stage of primordial follicle formation was between 70 and 90 days postcoitum and that the major stage of transition from primordial follicles into primary follicles was between 90 days postcoitum and 1 day postpartum. Experiments were also conducted to examine the localization of PTEN, PKB and FOXO3A proteins in the porcine ovaries by immunohistochemistry and immunoblotting. The results indicated that PTEN, PKB and FOXO3A were localized in the germ cells of egg nests, cytoplasm of oocytes and granulosa cells of follicles ranging from the primordial to secondary stages and that the staining intensity was weak in granulosa cells but strong in oocytes. The different staining patterns of PTEN, FOXO3A and PKB suggested that these proteins were expressed in a stage- and cell-specific manner during ovarian follicle formation and development in the fetal and neonatal pig.
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Katsuyoshi FUJIWARA, Daisuke SANO, Yasunari SEITA, Tomo INOMATA, Junya ...
Article type: Full Paper
2010 Volume 56 Issue 1 Pages
169-175
Published: 2010
Released on J-STAGE: March 05, 2010
Advance online publication: December 09, 2009
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Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after
in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after
in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.
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