-
Xin-Rong PENG, Tao LIU, Yong ZHANG
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
403-407
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: June 26, 2008
JOURNAL
FREE ACCESS
The aim of this study was to reconstruct and cryopreserve somatic cell nuclear transferred (SCNT) ovine embryos and evaluate the effect of α-tocopherol on blastocyst development and subsequent cryosurvival of the SCNT embryos. The α-tocopherol (100 μg/ml) was added into culture medium for the SCNT embryos, the yield and total cell numbers of blastocysts were determined and the apoptosis incidences of the blastocysts were evaluated using the TUNEL assay. The blastocysts from the α-tocopherol and untreated groups were then frozen-thawed, and their cryosurvival was assessed by
in vitro culture for 48 h. The results showed that there were no significant differences in blastocyst yield (26.3
vs. 22.3%) and total cell number (68.2
vs. 64.3) between the α-tocopherol and untreated groups. However, addition of α-tocopherol to the culture medium significantly decreased the apoptotic cell number (3.4
vs. 5.5) and significantly increased the cryosurvival of SCNT blastocysts (66.8
vs. 50.7%). In conclusion, addition of α-tocopherol to SCNT embryo culture medium was beneficial for improving embryo quality by decreasing the apoptotic blastocyst cell number and improving the tolerance of the embryos to cryopreservation.
View full abstract
-
Jae-hyek CHOI, Maho ISHIDA, Takashi MATSUWAKI, Keitaro YAMANOUCHI, Mas ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
408-412
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: July 30, 2008
JOURNAL
FREE ACCESS
The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes progesterone into a biologically inactive steroid, 20α-dihydroprogesterone (20α-OHP). In the corpora lutea of rats and mice, 20α-HSD is considered to be involved in functional luteolysis. It is also distributed in other tissues including the placenta, endometrial epithelia and fetal skin, although the roles it plays in these tissues remain to be elucidated. In the present study, we investigated the role of 20α-HSD in the maintenance of pregnancy using mice with targeted disruption of the 20α-HSD gene. We first confirmed that the number of pups was significantly smaller in
20α-HSD-/- pairs than in
20α-HSD +/+ pairs. We then mated
20α-HSD+/- males and females so that each pregnant female produced
20α-HSD+/+,
20α-HSD+/- and
20α-HSD-/- offspring. The genotype ratio of the offspring did not match the Mendel's law of inheritance, and the numbers of
20α-HSD+/- and
20α-HSD-/- offspring were smaller than expected values. Although the genotype ratio of fetuses on days 13, 15 and 18 of pregnancy matched the Mendel's law, the total number of fetuses on day 18 was significantly smaller than that on day 13, suggesting that fetal loss occurred during late pregnancy. Next, we transferred
20α-HSD+/+ embryos to
20α-HSD+/+ or
20α-HSD-/- females and found that the number of offspring was significantly smaller in
20α-HSD-/- dams than in
20α-HSD+/+ dams. Expression of
20α-HSD mRNA in the fetus, placenta and uterus progressively increased from day 11 to 18 of pregnancy. In addition, concentrations of progesterone were significantly higher in the
20α-HSD-/- fetuses than in the
20α-HSD+/+ fetuses, while those of 20α-OHP were lower in the
20α-HSD-/- fetuses than in the
20α-HSD+/+ fetuses. These results suggest that both maternal and fetal 20α-HSD play a role in maintaining normal pregnancy at least partially by reducing progesterone concentrations in fetuses.
View full abstract
-
Fang WU, Cécile CARON, Christine DE ROBERTIS, Saadi KHOCHBIN, S ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
413-417
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: August 12, 2008
JOURNAL
FREE ACCESS
Before fertilization, the genome packaging of male and female gametes is very different. Indeed, whereas the female haploid genome is associated with histones in a somatic-like chromatin structure, most of the male genome is tightly bound to protamines. However, it has recently been demonstrated that the pericentric heterochromatin regions of the male genome are associated with specific H2A-like histone variants, named H2AL1 and H2AL2, suggesting a heterogeneous organization. The fate and role of the sex-specific genome packaging transmitted by germinal cells to the embryo are not well understood. The aim of the present study was to follow reprogramming of the parental genomes in early embryos after
in vivo fertilization. We show here that two typical epigenetic markers, trimethylated lysine 9 of histone H3 (TriMethylH3K9) and acetylated H4, are asymmetrically distributed between the parental genomes in one-cell mouse embryos, confirming data from embryos obtained after intracytoplasmic sperm injection (ICSI) or
in vitro fertilization (IVF). Indeed, whereas the maternal genome is highly enriched with trimethylH3K9, this mark is not detected in the paternal genome. On the contrary, histone H4 incorporated in the paternal genome is highly acetylated at an early stage, while in the maternal pronucleus, the level of acetylated H4 remains low in early one-cell embryos and becomes enriched at a later stage. Moreover, our results suggest a very quick disappearance of histone H2A variants H2AL1 and H2Al2 from the paternal pericentric heterochromatin regions after sperm-egg fusion.
View full abstract
-
Hiroe KON, Hisashi KISHI, Koji Y. ARAI, Motoo SHINODA, Gen WATANABE, K ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
418-423
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 02, 2008
JOURNAL
FREE ACCESS
The present study was conducted to evaluate the endocrinological effects of the pituitary on luteal maintenance and regression in the cyclic golden hamster (
Mesocritus auratus). After hypophysectomy (Hypox) at 0900 h on day 1 of the estrous cycle (the day of ovulation), the animals received injection of prolactin (PRL) or PRL plus equine chorionic gonadotropin (eCG). They were decapitated at 1500 h on day 3 of the cycle, and trunk blood was collected for measurement of progesterone (P4). Corpora lutea (CLs) were dissected from one ovary for DNA ladder detection by electrophoresis, determination of DNA fragmentation ratio by fluorometric measurement method and measurement of P4. The other ovary was used for histological observation. After the Hypox, the daily injection of 1 mg ovine PRL restrained the DNA fragmentation ratio and number of apoptotic cell in the CLs. The PRL treatment maintained the luteal morphology and increased the luteal P4 concentration, but not in the plasma P4 concentration. In addition to PRL, injection of 2 IU eCG after the Hypox also restrained the DNA fragmentation ratio and number of apoptotic cells in the CLs to the level of a pregnant animal. The PRL plus eCG treatment maintained the luteal morphology in the same manner as the PRL only treatment and increased not only the luteal but also the plasma P4 concentration. These results suggest that PRL restrains luteal apoptosis and maintains luteal morphology and that the combination of PRL and eCG restrains not only structural but also functional luteal regression in the cyclic hamster.
View full abstract
-
Jun TAKAHASHI, Akio ISHIKAWA, Takao SUSA, Takako KATO, Yukio KATO
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
424-430
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 02, 2008
JOURNAL
FREE ACCESS
CArG binding factor A (CBF-A) is a transcription factor first isolated from mouse C2 myogenic cells. Several lines of evidence indicate that CBF-A is also present in the anterior pituitary lobe and participates in the process of development and cell transformation. This study was performed to clone porcine CBF-A and to investigate its roles in the porcine anterior pituitary lobe. The predicted amino acid sequence of porcine CBF-A showed a unique insertion of five TG-repeats in the N-terminal region in comparison with those of other mammals, whereas the other regions appeared to be mostly conserved including two RNA recognition motifs in the middle region. Investigation of the expression of CBF-A gene during porcine pituitary development by RT-PCR showed an exclusive and temporary decrease in expression level shortly after birth in both sexes that was gradually but insufficiently restored. The expression of fluorescence protein-fused CBF-A in CHO cells demonstrated that CBF-A is located in the nuclei. We examined whether CBF-A regulates the expression of pituitary hormone genes in CHO cells and found that CBF-A significantly stimulated the promoter activity of growth hormone and prolactin by about 2-fold but did not stimulate the LHβ gene. The specific DNA binding ability of porcine CBF-A was examined using serial oligonucleotides, CArG box and CC(W)
0-6GG (W=A or T). As a result, porcine CBF-A was shown to have a high binding affinity for double- and single-stranded CC(W)
6GG but no affinity for the known sequences of the CBF-A-target genes. Accordingly, this study demonstrated that porcine CBF-A may play a role in regulating at least two pituitary hormone genes, GH and PRL.
View full abstract
-
Klaus-Peter BRÜSSOW, Falk SCHNEIDER, Armin TUCHSCHERER, Istvan EG ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
431-438
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 12, 2008
JOURNAL
FREE ACCESS
The objective of this study was to evaluate luteinizing hormone (LH) and luteal progesterone (P4) secretion in systemic blood and blood near the ovaries in Mangalica (M) and Landrace (L) gilts by implanting catheters into the
Vena jugularis and the
Vena cava caudalis via the
Vena saphena, respectively. Furthermore, leptin was analyzed in jugular vein blood. Blood was collected twice daily from day 7 to day 19 of the oestrous cycle and frequently (10-min intervals for 6 h) on day 9, day 12 and day 15 in M (n=3) and L gilts (n=4). L gilts had congruent pulsatile LH secretion in both veins, but the LH concentrations in M were always below the assay sensitivity during the luteal phase. In both breeds, episodic P4 secretion was found in the jugular and caval veins, and both sampling site and breed had an influence on P4 secretion (P<0.05). The mean concentration of P4 was higher (P<0.01) in utero-ovarian blood (75.8 ± 5.3 in M; 49.6 ± 4.2 ng/ml in L) than in the periphery (31.3 ± 2.0 in M; 21.2 ± 1.8 ng/ml in L). M pigs had a lower number of corpora lutea (9.7 ± 2.3
vs. 20.5 ± 4.4), and analysis of the P4 secretion ratio per corpus luteum revealed an influence of breed (P<0.01). This ratio was significantly higher in M (3.8 ± 0.3 and 8.7 ± 0.7 ng/ml) compared with the L gilts (1.4 ± 0.1 and 2.8 ± 0.3 ng/ml) in the jugular and caval veins, respectively. Blood sampling from the
Vena cava caudalis is potentially more precise than from the
Vena jugularis for evaluation of ovarian P4 secretion. Both the higher P4 concentration and increased leptin secretion (11.3 ± 0.6
vs. 3.0 ± 0.1 ng/ml, P<0.05) and consequently the altered LH secretion pattern in the Mangalica may contribute to the lower fecundity of this breed.
View full abstract
-
Govindarajan THANGAVELU, Marcos Germán COLAZO, Masahito OBA, Mi ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
439-446
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 02, 2008
JOURNAL
FREE ACCESS
We hypothesized that the inclusion of flaxseed in the diets of lactating dairy cows will increase urinary and fecal concentrations of the lignans, secoisolariciresinol diglycoside (SDG), enterolactone and enterodiol, reduce intrafollicular concentrations of IGF-I and estradiol, and subsequently reduce estradiol and oxytocin receptor expression in the endometrium. To test this hypothesis, 27 cycling, lactating Holstein cows were assigned to 1 of 3 diets supplemented with saturated fatty acids (SAT), flax (FLX), or sunflower (SUN) seed. Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized, and 5 d later, follicles > 8 mm were ablated by an ultrasound-guided procedure in all cows. Samples of blood (Days 0 to 14), follicular fluid (Day 5 and 15), endometrium (Day 15), as well as urine and feces were collected in a subset of the animals. The fecal concentrations of SDG and enterodiol were higher (P<0.05) in cows fed FLX than in those fed SAT or SUN. Enterodiol increased (P<0.05) in urine samples of cows fed FLX, compared to those of cows fed SUN. However, follicular estradiol concentrations on Day 5 and 15 and endometrial concentrations of estradiol and oxytocin receptors on Day 15 did not differ among the dietary groups. Mean plasma progesterone concentrations were higher (P<0.05) in cows fed FLX and SUN than in those fed SAT. In summary, a diet supplemented with flaxseed increased the concentrations of SDG and enterodiol in feces, as hypothesized, but did not alter intrafollicular concentrations of IGF-I or estradiol, or endometrial populations of oxytocin or estrogen receptors in lactating dairy cows.
View full abstract
-
Alexandra RAUCH, Lars KRÜGER, Akio MIYAMOTO, Heinrich BOLLWEIN
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
447-453
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 12, 2008
JOURNAL
FREE ACCESS
The goals of the present study were to investigate whether colour Doppler sonography can be used to differentiate temporary from persistent ovarian follicles and follicles with luteal tissue from follicles without luteal tissue and to assess the response of follicular cysts to administration of a gonadotropin releasing hormone (GnRH) analogue. Fifty-four cows having ovarian follicular structures with a diameter of >15 mm but no corpus luteum were included. These cows were examined via B-mode and colour Doppler sonography. The same examinations were repeated 10 to 12 days later, and the cows with follicular cysts (n=17) received a GnRH analogue. Blood flow was measured before and 30 min after treatment. Ten to 12 days later, the response to treatment was assessed using B-mode sonography. While 31 of 54 follicles disappeared spontaneously (temporary follicles), 23 follicles persisted and were diagnosed as cystic ovarian follicles (COFs). There was no difference between temporary follicles and COFs in regard to total area, wall thickness or the perfused area. In the luteinized follicles (n=13), based on the plasma progesterone concentration, total area was twice as large, wall thickness was three times greater and the perfused area was 4.5 times larger than those of the non-luteinized follicles (n=41). The sensitivity of diagnosing luteinized follicles was 61.5% using B-mode sonography and 92.3% using colour Doppler sonography. Twelve cows responded to GnRH, and five cows did not. There was a trend (P=0.07) toward higher (59.3%) blood flow in the cyst wall 30 min after treatment in the responding cows compared with the non-responding cows. Our results showed that the perfused area more accurately reflects active luteal tissue than wall thickness. Thus, colour Doppler sonography is superior to B-mode sonography for differentiating follicular and luteal cysts and aids in the selection of treatment. However, exact prediction of COFs destined to regress or persist and the response of COFs to treatment with a GnRH analogue were not possible using colour Doppler sonography.
View full abstract
-
Yuan CHENG, Akihisa MAEDA, Yasufumi GOTO, Fuko MATSUDA, Takashi MIYANO ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
454-459
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 26, 2008
JOURNAL
FREE ACCESS
Follicular selection predominantly depends on granulosa cell apoptosis in porcine ovaries, but the molecular mechanisms regulating the induction of apoptosis in granulosa cells during follicular selection remain incompletely understood. To determine the role of X-linked inhibitor of apoptosis protein (XIAP), which suppresses caspase-3, -7 and -9 activities and acts as an endogenous inhibitor of apoptotic cell death, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression level and localization of XIAP mRNA and protein in granulosa cells during follicular atresia using reverse transcription-polymerase chain reaction (RT-PCR),
in situ hybridization, Western blotting and immunohistochemistry, respectively. High levels of XIAP mRNA and protein were noted in the granulosa cells of healthy follicles, and decreased levels were noted during follicular atresia.
In situ hybridization and immunohistochemistry demonstrated that XIAP mRNA and protein were strongly expressed in the granulosa cells of healthy follicles, but negative/trace stainings were noted in those of atretic follicles. The present findings strongly indicate that XIAP is a candidate molecule which acts as an anti-apoptotic/pro-survival factor by inhibiting intracellular apoptosis signaling and is involved in the regulation of apoptosis in porcine granulosa cells.
View full abstract
-
Xia SHENG, Haolin ZHANG, Wei ZHANG, Moshi SONG, Mengyuan ZHANG, Ben LI ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
460-464
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: September 26, 2008
JOURNAL
FREE ACCESS
The objective of this study was to investigate the seasonal changes in spermatogenesis and the immunolocalization of the inhibin α and inhibin/activin (β
A and β
B) subunits during the breeding and non-breeding seasons in the wild male ground squirrel. The testicular weight and size and seminiferous tubule diameter were measured, and histological observations of testes were performed. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin β
A and inhibin/activin β
B during the breeding and non-breeding seasons. There were marked variations in testicular weight and size and seminiferous tubule diameter between the breeding and non-breeding seasons, and all types of spermatogenic cells, including spermatozoa, were found in the breeding season. In addition, immunoreactivity was also detected for the inhibin α, β
A and β
B subunits in Sertoli and Leydig cells during the breeding season, but immunostaining was only present for the inhibin α and inhibin/activin β
B subunits in Sertoli cells during the non-breeding season. These results suggest that seasonal changes in testicular weight and size and seminiferous tubule diameter of wild ground squirrels are correlated with changes in spermatogenesis, and the cellular localization of the inhibin/activin subunits showed season related changes in the breeding and non-breeding seasons.
View full abstract
-
Carlos Manuel ULLOA ULLOA, Midori YOSHIZAWA, Akira YAMASHITA, Satoshi ...
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
465-472
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: October 28, 2008
JOURNAL
FREE ACCESS
The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8 ± 42.4, P<0.05), largest number of metaphases per blastocyst (9.5 ± 4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6 ± 33.4, P<0.05), a large number of metaphases per blastocyst (11.9 ± 5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.
View full abstract
-
Seiji KATAGIRI, Yoshiyuki TAKAHASHI
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
473-479
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: October 15, 2008
JOURNAL
FREE ACCESS
The objective of the present study was to examine the efficacy of a progestin-based treatment with a high dose of estradiol benzoate (EB) to normalize the epidermal growth factor (EGF) profile in the uterine endometrium and restore fertility in repeat breeder cows. Repeat breeder cows without peaks in their endometrial EGF concentrations on Days 3 and 14 of the estrous cycle were used throughout the study. The effect of 1 (standard dose), 2.5 and 5 mg of EB in a progestin-based treatment protocol (EB1, EB2.5 and EB5 treatments, respectively; n=5 for each group) on endometrial EGF concentrations was first examined. The EB1 and EB2.5 treatments in the repeat breeder cows produced a suppressed response in endometrial EGF compared with EB1 treatment in the fertile controls (n=5) and failed to restore the normal EGF profile during the next estrous cycle. However, EB5 treatment produced an increase in EGF concentrations similar to the fertile controls and normalized the endometrial EGF profile. The effects of the EB1 and EB5 treatments (n=30 for each group) on the endometrial EGF profile and fertility were then examined in the repeat breeder cows. The proportion of cows, with an EGF profile normalized by the treatments was higher in the EB5 group (66.7%) than in the EB1 (30.0%) and untreated control (13.3%; n=30) groups (P<0.01). The pregnancy rates of the cows having a normal EGF profile after treatment in the EB1 and EB5 groups were similar (88.9 and 85.0%, respectively) and higher than those of the cows having an abnormal profile within the same groups (19.0 and 30.0%, respectively, P<0.01). In summary, the endometrial response to EB in terms of the EGF concentration was suppressed in repeat breeder cows. A high dose (5 mg) of EB in a progestin-based treatment was found to be effective for restoration of a normal EGF profile and fertility in repeat breeder cows having lesser endometrial EGF concentrations on Days 3 and 14.
View full abstract
-
Hiroyuki KANEKO, Kazuhiro KIKUCHI, Michiko NAKAI, Junko NOGUCHI
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
480-485
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: October 15, 2008
JOURNAL
FREE ACCESS
Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin α subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.
View full abstract
-
Tetsuo ONO, Eiji MIZUTANI, Chong LI, Teruhiko WAKAYAMA
Article type: -Full Paper-
2008 Volume 54 Issue 6 Pages
486-491
Published: 2008
Released on J-STAGE: January 07, 2009
Advance online publication: October 15, 2008
JOURNAL
FREE ACCESS
Mouse spermatozoa can be freeze dried without losing genetic integrity and reproductive potential. However, it is not known if freeze-dried mouse cells similarly maintain their genetic integrity and developmental potential following nuclear transfer. Here, we investigated the developmental capacity and embryonic stem (ES) cell derivation of reconstructed oocytes by nuclear transfer using freeze-dried cumulus or ES cells. Cumulus and ES cells were lyophilized overnight and stored at 4 C for up to 1 week. After rehydration, all cells showed membrane damage and were unviable. However, following nuclear transfer, 1-4% of the reconstructed oocytes developed to the blastocyst stage. A total of five nuclear transfer ES (ntES) cell lines were generated from blastocysts and morulae. All ntES cell lines had normal karyotypes and were positive for the ES-cell-specific markers (alkaline phosphatase, Oct3/4 and Nanog). After aggregation of ntES cells with fertilized embryos, chimeric mice with a high level of coat color chimerism were generated. Our findings show that the genomic integrity of cells can be maintained after freeze-drying and that it is possible to produce offspring from the cells using nuclear transfer techniques.
View full abstract