The aim of the present study was to investigate the effects of centrifugation pretreatment on the viability and nuclear status of porcine in vitro matured (IVM) oocytes and on the developmental competence of in vitro fertilized (IVF) oocytes (zygotes) after cryopreservation by vitrification (Solid Surface Vitrification; SSV). Mature oocytes having the first polar body after IVM and zygotes having the second polar body at 10 h after IVF were centrifuged at 10,000 × g at 37 C for 20 min and then subjected to SSV. Their viability was evaluated by morphological appearance and fluorescein diacetate staining. The nuclear status of oocytes was evaluated 6 h after vitrification. The developmental ability to the blastocyst stage of vitrified zygotes was evaluated after 6 days of in vitro culture. Although centrifugation did not damage the oocytes directly, it drastically reduced the rate of live oocytes after SSV. The rates of vitrification-induced parthenogenetic activation were similar in both centrifuged and non-centrifuged oocytes (42.4 and 47.4%, respectively). Centrifugation had no significant effects on the viability of pronuclear oocytes. The development of vitrified zygotes to the blastocyst stage was significantly lower than that of the control irrespective of centrifugation pretreatment. There was no difference in the cleavage and blastocyst rates between the control and centrifuged zygotes after vitrification. There was also no difference in the total cell numbers of blastocysts between the control and centrifuged zygotes irrespective of vitrification. These results reveal that, in IVM porcine oocytes, centrifugation pretreatment is highly detrimental to cryotolerance; however, in zygotes, it has only a slight effect on viability and does not alter the developmental competence of surviving zygotes.
Miniature pigs have been recognized as valuable experimental animals in various fields such as medical and pharmaceutical research. However, the amount of information on somatic cell cloning in miniature pigs, as well as genetically modified miniature pigs, is much less than that available for common domestic pigs. The objective of the present study was to establish an efficient technique of cloning miniature pigs by somatic cell nuclear transfer. A high pregnancy rate was achieved following transfer of parthenogenetic (3/3) and cloned (5/6) embryos using female miniature pigs in the early pregnancy period as recipients after estrus synchronization with prostaglandin F2 alpha analog and gonadotrophins. The production efficiency of the cloned miniature pigs using male and female fetal fibroblasts as nucleus donors was 0.9% (2/215 and 3/331, respectively). Cloned miniature pigs were also produced efficiently (7.8%, 5/64) by transferring reconstructed embryos into the uteri of common domestic pigs. When donor cells transfected with the green fluorescent protein (GFP) gene were used in nuclear transfer, the production efficiency of the reconstructed embryos and rate of blastocyst development were comparable to those obtained by non-transfected cells. When transfected cell-derived reconstructed embryos were transferred to three common domestic pig recipients, all became pregnant, and a total of ten transgenic cloned miniature pigs were obtained (piglet production efficiency: 2.7%, 10/365). Hence, we were able to establish a practical system for producing cloned and transgenic-cloned miniature pigs with a syngeneic background.
Ginseng has been extensively used around the world for several thousand years as a food or drug. However, recently, several reports have indicated that the organogenesis of cultured embryos is inhibited by treatment with ginsenoside, the principal component of ginseng. In this study, we evaluated the morphological changes of embryos and the gene expression patterns of antioxidant enzymes, 3 types of glutathione peroxidases [GPx; cytosolic (cGPx), plasma (pGPx) and phospholipid hydroperoxide (phGPx) forms], in cultured rat embryos (embryonic days 9.5-11.5) exposed to ginsenosides Rb1, Rg1, Re and Rc at levels of 5, 50 and 100 μg/ml. With regard to total morphological scores, no significant differences were noted in the embryos exposed to all doses of ginsenosides, with the exception of 50 μg/ml of Rc. In the cultured embryos exposed to Rg1, a majority of the developmental parameters were normal, but growth of the hind- and mid- brains and the caudal neural tube was significantly increased compared with that observed in the control group (P<0.05). Furthermore, Rc significantly enhanced the growth of a variety of developmental parameters in the cultured embryos, with the exception of the hindlimbs. According to the results of our semiquantitative RT-PCR analysis, the levels of cGPx and phGPx mRNA in the cultured embryos were unaffected by treatment with the ginsenosides. However, the levels of pGPx mRNA increased significantly in the embryos treated with ginsenosides Re, Rc and Rb1 compared with the control group (P<0.05). These findings indicate that ginsenosides may exert a stimulatory effect on the growth of embryos via differential expression of GPx genes.
In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.
Mouse bi-maternal embryos (BMEs) that contain two haploid sets of genomes from non-growing (ng) and fully-grown (fg) oocytes develop to embryonic day (E) 13.5. However, the ng/fg BMEs never develop beyond E13.5 because of repression of the paternally expressed imprinted genes, Igf2 and Dlk1. The present study was conducted to address the issue of whether fetal hematopoietic disorder is involved in the restricted development of BMEs. FACS analysis revealed that the livers of ngwt/fg BMEs contained increased numbers of immature c-kit+/ter119- hematopoietic cells, were while the numbers of mature c-kit-/ter119+ hematopoietic cells were decreased. This finding was supported by histological observations. Quantitative gene expression analysis revealed that Igf2 and Dlk1 expression was repressed in the liver. To understand the role of paternally-methylated imprinted genes on chromosomes 7 and 12, particularly Igf2 and Dlk1, in fetal liver hematopoiesis, we constructed ngΔch7/fg, ngΔch12/fg and ngΔDouble/fg BMEs using ng oocytes harboring deletion of differentially methylated regions at distal chromosomes 7 and/or 12. The ngΔch7/fg, ngΔch12/fg and ngΔDouble/fg BMEs, respectively, express Igf2, Dlk1 and both, and these embryos developed to term with specific phenotypes; the ngΔch7/fg and ngΔch12/fg BMEs develop to term with severe growth retardation, and the ngΔDouble/fg BMEs can survive to become normal female adults. By inducing Igf2 and Dlk1 expression, the proportions of mature and immature hematopoietic cells in the livers of the ngΔch7/fg, ngΔch12/fg and ngΔDouble/fg BMEs were considerably restored, and particularly in the ngΔDouble/fg BMEs, hematopoiesis occurred normally with appropriate expressions of the related genes. These data suggest that inappropriate expression of Igf2 and Dlk1 is involved in impaired fetal hematopoiesis.
Endothelial cells derived from the corpus luteum (CLENDOs) exhibit diverse characteristics presumably serving their wide-ranging roles in luteal function and fate. Here, several attributes of CLENDOs derived from cows at midcycle (days 9-12 of the estrous cycle) were compared with CLENDOs from early pregnancy (day 60 of pregnancy). Flow cytometric analysis of cells fluorescently-tagged with the lectins Bandeiraea simplicifolia (BS-1) and Concanavalin A (ConA) indicated that CLENDOs of midcycle CL do not differ from those of pregnancy. Mean fluorescence intensity for BS-1 was 15 ± 1 and 23 ± 7 fluorescent units for midcycle CLENDOs and CLENDOs of pregnancy, respectively (P>0.05). For ConA, mean fluorescence was 25 ± 2 and 26 ± 1 fluorescent units, respectively (P>0.05). The CLENDOs were also exposed to cytokines to assess differences in activation of nuclear factor kappa B signaling (NF-κB), induction of the transcription factor interferon regulatory factor 1 (IRF1), cytokine production, and cytokine-induced cell death. In response to TNF, for instance, both types of CLENDOs exhibited a rapid, 5-fold decrease in NF-κB inhibitor alpha (NFKBIA) protein expression (P<0.05), and a 4-fold increase in IRF1 expression (P<0.05), that did not differ with phenotype (P>0.05). Similarly, both types of CLENDOs produced tumor necrosis factor alpha and chemokine ligand 2 in response to IFNG stimulation (P<0.05) that did not differ with phenotype (P>0.05). Lastly, extended exposure of CLENDOs of midcycle CL to cytokines induced cell death (~50% cell death vs. control) similar to the incidence of cell death seen previously in CLENDOs of early pregnancy. The results indicate that several physical and functional characteristics of CLENDOs of midcycle CL are retained through early pregnancy, including lectin-binding properties, sensitivity to cytokines, and the activation of cytokine-initiated intracellular signals.
We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.
Acute central lipoprivation suppresses pulsatile luteinizing hormone (LH) release and increases blood glucose levels through noradrenergic input to the hypothalamic paraventricular nucleus (PVN) in female rats. The present study was conducted to identify adrenergic receptor subtypes involved in central lipoprivation-induced suppression of pulsatile LH secretion and increases in plasma glucose levels in female rats. Acute hindbrain lipoprivation was produced by injection into the fourth cerebroventricle (4V) of 2-mercaptoacetate (MA), an inhibitor of fatty acid oxidation, in estradiol-implanted ovariectomized rats. Two min before MA injection, α1-, α2- or β-adrenergic receptor antagonist was injected into the PVN. Injection of MA into the 4V suppresses pulsatile LH release in PVN vehicle-treated rats, whereas pretreatment of animals with injection of α1- or α2-adrenergic antagonist into the PVN blocked the effect of the 4V MA injection on LH pulses. β-Adrenergic antagonist did not affect MA-induced suppression of LH pulses. The counter-regulatory increase in plasma glucose levels after 4V MA injection was also partially blocked by pretreatment with α1- and α2-adrenergic receptor antagonists. These results suggest that α1- and α2-adrenergic receptors in the PVN mediate hindbrain lipoprivation-induced suppression of LH release and counter-regulatory increases in plasma glucose levels in female rats.
A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of γH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of γH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of γH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.
We have previously indicated that porcine blastocysts can be produced by in vitro fertilization (IVF) and culture (IVC) in chemically defined porcine gamete medium (PGM) and porcine zygote medium (PZM)-5, respectively, In the present study, the effects of basic media and macromolecular components on in vitro maturation (IVM) were investigated to develop a defined system for in vitro embryo production using a single basic medium through IVM, IVF and IVC. Porcine immature oocytes were matured in porcine oocyte medium (POM) or modified North Carolina State University (mNCSU) 37, which were supplemented with either 10% (v/v) porcine follicular fluid (pFF) or 3 mg/ml polyvinyl alcohol (PVA) as a macromolecular component (designated POM+pFF, POM+PVA, mNCSU37+pFF and mNCSU37+PVA). In the maturation with mNCSU37+PVA, the percentages of oocytes that reached the metaphase II stages were significantly lower than those in the other treatments. Following IVM with the above media, oocytes were treated with an electrical stimulus and cycloheximide for parthenogenetic activation and were cultured in PZM-5 for 5 days. The rates of cleavage and blastocyst formation of parthenogenetic oocytes were significantly lowered for maturation with mNCSU37+PVA compared with the other treatments, while there were no significant differences in the total numbers of cells in blastocysts among the treatments. Following IVF and IVC, the rates of penetration, male pronucleus formation, cleavage and blastocyst formation were significantly lower when oocytes were matured in mNCSU37+PVA than in other maturation media. The normal fertilization rate was significantly higher in POM+PVA compared with the other treatments, although the total number of cells in blastocysts was reduced with the addition of PVA to both POM and mNCSU37 compared with pFF supplementation. These results demonstrate that porcine blastocysts can be produced by the defined system using a single basic medium.
The purpose of this study was to determine the relationships between conception rate and other parameters before estrus synchronization with a Controlled Internal Drug Release Device (CIDR) and estradiol benzoate (EB). In the estrus synchronization program, animals were injected with 2 mg EB and then received a CIDR. Seven days later, the CIDR was removed and the animals were given an injection of Prostaglandin F2α. Twenty-four hours later, they received an injection of 1 mg EB, and they were artificially inseminated 24 h after that. This program was applied to 258 Holstein cows in Tohoku-machi (Aomori, Japan). Blood was collected at the beginning of the program, and the conception rate was determined about 40 days after insemination. The relationships among conception rate, blood biochemical values, age, body condition score and days in milk were statistically analyzed to determine better conditions for cow conception. The conception rate of the cows in the high progesterone group (more than 1 ng/ml, P4+) was significantly higher than that of the low progesterone group (less than 1 ng/ml, P4-; 47.9% vs. 28.6% P<0.01). In the P4- groups, the serum phospholipid level was significantly higher in the conception group than in the non-conception group, and the same tendency was seen in the P4+ groups. Blood urea nitrogen (BUN), albumin (Alb), and total cholesterol (TChol) were significantly higher in the conception group compared with the non-conception group, but no with P4 was observed. We concluded that 1) the conception rate of the P4- group was remarkably low, that 2) the low conception rate and low P4 level was related to a low PL level and that 3) BUN, Alb and TChol were higher in the conception group, although no relation with P4 was found.
The occurrence of apoptosis in a fraction of blastomeres in the preimplantation embryo is well known but the consequences of this phenomenon for the developmental potential of the blastocyst has not been well established. Here we demonstrate that blastocysts with low amounts of activated group II caspase activity have increased potential for development to the hatched blastocyst stage. Bovine blastocysts produced in vitro were assayed using a non-invasive fluoregenic substrate that is cleaved by activated group II caspases (i.e., caspase-2, -3 and -7). Subsequently, blastocysts were cultured until Day 10 post-insemination and the proportion undergoing hatching determined. In Experiment 1, blastocysts were cultured without respect to stage of development (expanded or non-expanded); blastocysts classified as having low caspase activity had higher hatching rates than blastocysts with medium or high caspase activity. In Experiment 2, embryos were categorized as nonexpanded or expanded blastocysts. Caspase activity was lower and hatching rate higher for expanded blastocysts than for nonexpanded blastocysts. For nonexpanded blastocysts, embryos classified as having low caspase activity had higher hatching rates as compared to embryos with medium or high caspase activity. In conclusion, the capacity for blastocysts to undergo further development is related to degree of group II caspase activity. Conditions that enhance the incidence of apoptosis in blastocysts may reduce developmental competence. In addition, determination of caspase activity may be useful for selection of embryos for transfer into recipients.
The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 × g in the presence of 1.0 μg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate snt-GGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts.
Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3β-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.
Lactate dehydrogenase C (LDH-C) has been reported to play a role in the energy metabolism of mammal spermatozoa. However, the functions and expression patterns of LDH-C still remain unclear. In order to elucidate the functions and expression patterns of LDH-C, we cloned the cDNA of yak LDH-C. Total RNA was extracted from yak testes and reverse transcribed and amplified by PCR. The full length open reading frame (ORF) of LDH-C and its five splice variants were obtained. The full length ORF contained 999 bp encoding a 332-amino-acid protein that showed 100% identity with bovine LDH-C. Compared with the full length ORF of LDH-C, the five variants used the same start codon as the full length ORF and encoded 5 putative proteins. In detail, variants 1 (missing the coding sequence of exon 6 and 7) and 2 (missing the coding sequence of exon 7) bear the entire nicotinamide-adenine dinucleotide (NAD) binding domain and an active site. Variants 3 (missing the first 42 nuleotides of exon 4) and 4 (missing the coding sequences of exons 5, 6 and 7) lack part of the NAD binding domain but contained the entire active site. Variant 5 (missing the coding sequence of exons 4 and 7) lacks a large part of the NAD binding domain and the entire active site. Native polyacrylamide gel electrophoresis was performed to determine if the splice variants can be translated into proteins. However, native PAGE detected no specific bands from yak testes and bovine spermatozoa. This study suggests the alternative splicing of LDH-C is ubiquitous in bovine testes and might be involved in regulation of LDH-C expression. The findings also help to elucidate the functions of LDH-C.
Successful cloning requires reprogramming of epigenetic information of the somatic nucleus to an embryonic state. However, the molecular mechanisms regarding epigenetic reprogramming of the somatic chromatin are unclear. Herein, we transferred NIH3T3 cell nuclei into enucleated mouse oocytes and evaluated the histone H3 dimethyl-lysine 4 (H3K4me2) dynamics by immunocytochemistry. A low level of H3K4me2 in the somatic chromatin was maintained in pseudo-pronuclei. Unlike in vitro fertilized (IVF) embryos, the methylation level of nuclear transfer (NT) embryos was significantly increased at the 8-cell stage. NT embryos showed lower H3K4me2 intensity than IVF embryos at the 2-cell stage, which is when the mouse embryonic genome is activated. Moreover, the H3K4me2 signal was weak in the recloned embryos derived from single blastomeres of the NT embryos, whereas it was intense in those from IVF embryos. Two imprinted genes, U2afbp-rs and Xist, were abnormally transcribed in cloned embryos compared with IVF embryos, and this was partly correlated to the H3K4me2 level. Our results suggest that abnormal reprogramming of epigenetic markers such as histone acetylation and methylation may lead to dysregualtion of gene expression in cloned embryos.