scholarly journals Plasma Steroid Dynamics in Late- and Near-term Naturally and Artificially Conceived Bovine Pregnancies as Elucidated by Multihormone High-resolution LC-MS/MS

Endocrinology ◽  
2014 ◽  
Vol 155 (12) ◽  
pp. 5011-5023 ◽  
Author(s):  
Helio A. Martins-Júnior ◽  
Fábio L. V. Pinaffi ◽  
Rosineide C. Simas ◽  
Adriana K. Tarouco ◽  
Christina R. Ferreira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Steroid Hormones ◽  
Sex Steroids ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Term Pregnancy ◽  
Near Term

The plasma levels of corticosteroids and sex steroids during pregnancy are key indicators of mammalian placental function and the onset of parturition. Steroid hormones are believed to be disturbed in pregnancies produced using assisted reproductive technologies (ARTs) due to placental dysfunction and the frequently observed lack of parturition signals. To elucidate the plasma steroid dynamics, a liquid chromatography-tandem mass spectrometry method was developed and used to determine the levels of corticosteroids (corticosterone, 11-deoxycortisol, and cortisol) and their direct precursors (progesterone and 17α-OH-progesterone) as well as sex steroids (androstenedione, estrone, estrone sulfate, testosterone, and 17β-estradiol) in bovine plasma. The levels of these 10 steroids in recipient cows carrying naturally conceived (control), in vitro fertilized (IVF), or cloned (somatic cell nuclear transfer) conceptuses were compared during late-term pregnancy (30 days before parturition), during near-term pregnancy (1 day before parturition), and on the day of parturition (day 0). Significant differences were observed among the corticosteroid levels: higher levels of corticosterone, 11-deoxycortisol, and cortisol were detected in cloned pregnancies at day 30; lower levels of corticosterone were observed in ART-derived pregnancies at days 1 and 0; and estrone and estradiol levels were higher in IVF pregnancies throughout the final development. These results suggested an upregulation of the P450C11 and P450C21 enzymes 30 days before parturition in somatic cell nuclear transfer pregnancies and an overactivation of the aromatase enzyme in IVF pregnancies. Taken together, the monitoring of multiple steroid hormones revealed that the pregnancies obtained using ART exhibited plasma steroid concentration dynamics compatible with the dysregulation of steroidogenic tissues.

scholarly journals 56 SOMATIC CELL NUCLEAR TRANSFER IN NON-HUMAN PRIMATES: THE POSSIBILITY OF USING OOCYTES MATURED IN VITRO FOR UP TO 3 DAYS AS HOST OOPLASTS

2005 ◽  
Vol 17 (2) ◽  
pp. 177
Author(s):  
N.T. Uoc ◽  
B.D. Bavister ◽  
N.V. Hanh ◽  
L.C. Bui ◽  
N.T. Thanh ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Polar Body ◽  
Reproductive Technologies ◽  
High Dose ◽  
Cumulus Expansion ◽  
First Polar Body

Production of cloned nonhuman primate embryos has been reported using mature oocytes obtained from donors treated in vivo with a high dose of recombinant human FSH (r-hFSH, 35 IU per day for 10 days). The disadvantages of this approach are the high cost of hormones and the need to use the oocytes shortly after collection. Our study aimed to investigate the possibility of using initial in vivo treatment with a reduced FSH dose followed by in vitro culture for long periods of up to 3 days to produce mature monkey oocytes as host ooplasts for somatic cell nuclear transfer (SCNT). Adult female long-tailed Macaque (Macaca fascicularis) monkeys were treated with r-hFSH (Serono, Aubonne, Switzerland, 35 IU per day, i.m.) either for 10 days with an injection of hCG (1000 IU, i/m) 34 h before oocyte collection (G.I) or with only r-hFSH for 7 days (G.II). Cumulus oocyte complexes (COCs) were collected by follicular aspiration and then cultured in TCM-199 medium (GIBCO) supplemented with estradiol-17β, FSH, LH, and 10% FCS at 39°C in an incubator with 5% CO2 in air. The maturation rate based on the level of cumulus expansion and the presence of the first polar body was recorded at the moment of collection and during 24 h, 48 h, and 72 h of in vitro maturation (IVM). For SCNT, the mature Metaphase II oocytes were separated from cumulus cells and selected for enucleation in the presence of cytochalasin B (Sigma, St. Louis, MO, USA). Skin fibroblasts obtained from adult monkeys were cultured in DMEM+ 10% FCS and induced to quiescence in DMEM 0% FCS 2 days before use. A single cell was transferred under the zona of each enucleated oocyte. Couplets were fused with two direct current (DC) pulses of 220 V/mm for 25 μs in Zimmerman medium. Fused oocytes were cultured in medium containing cyclohexamide for 6 h before placing them into monkey culture medium (Cook, Brisbane, Australia). The average number of oocytes collected per animal were 21.2 (n = 18) and 18.6 (n = 12) for the G.I and G.II treatments, respectively. For G.I, the rate of COCs with fully expanded cumulus was 42% at collection and was maximal (80%) at Day 1 of IVM. For G.II, fully expanded cumulus was not observed at the time of collection and during the first 2 days of IVM, but 75% of COCs had full cumulus expansion by Day 3 of IVM. The rates of intact and fused oocytes were 50.3% for G.I and 55.4% for G.II. From the fused oocytes, 67.8% and 64.4% developed to the 4- to 8-cell stages at Days 2–3 after nuclear transfer for G.I and G.II, respectively. From these data, it can be concluded that this approach can be applied to optimize production of mature oocytes for non-human primate SCNT and ART (assisted reproductive technologies) programs. This work was supported by AIRE-Development.

32 CLONING OF ELITE QUARANTINE SNIFFING DOG BY SOMATIC CELL NUCLEAR TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 164
Author(s):  
H. J. Oh ◽  
M. J. Kim ◽  
G. A. Kim ◽  
J. Choi ◽  
E. J. Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Donor Cells ◽  
Wide Range ◽  
Sound Sensitivity

Somatic cell nuclear transfer (SCNT) in assisted reproductive technologies has been considered for the conservation of valuable or endangered animals. Dogs that were originally bred for hunting, such as beagles, have an exceptional ability to detect a particular smell from many others. For that reason, the beagles have been used to detect quarantine risk items from a wide range of goods in assorted luggage without scaring or disrupting the passengers. Though very useful and highly in need, elite quarantine sniffing beagles with excellent abilities are rare; much time, effort, and money are required in producing them. Here, we have applied SCNT for propagation of elite quarantine sniffing dogs to save time and economic burden. Ear fibroblasts from a 10-year-old adult male elite quarantine sniffing beagle were isolated and cultured in vitro as donor cells. For SCNT, in vivo-matured oocytes, obtained by flushing the uterine tubes of oocyte donors (mixed breed), were used. The oocytes were enucleated, microinjected with donor cells, fused by electrical stimulation, and activated chemically. Reconstructed oocytes were surgically transferred into the uterine tube of naturally synchronous recipient females. A total of 212 activated cloned embryos were transferred into 12 female recipient dogs and 4 recipients became pregnant. The 4 pregnant recipients delivered 4 pups through caesarean section or natural delivery, but 1 died right after birth and did not show an abnormality. Other live puppies exhibited normal phenotypes; their appearance was similar to that of the donor dog. All cloned pups were genetically identical to the donor dog and their mitochondrial DNA was from their oocyte donor dogs. When the cloned pups were 16 weeks old, we conducted a Volhard test, which is commonly used to describe the following puppy aptitudes: social attraction, following, restraint, social dominance, elevation dominance, retrieving, touch sensitivity, sound sensitivity, and sight sensitivity. Dog behavior data on differences in transcript abundance were analyzed by a general linear mixed model. The 3 cloned pups showed similar behavioral tendencies. The present study demonstrates that NT technique using donor cell derived from 1 elite quarantine sniffing dog is useful to produce a large number of quarantine sniffing dogs. This study was supported by RDA (no. PJ0089752012), RNL Bio (no. 550-20120006), IPET (no. 311062-04-1-SB010), Research Institute for Veterinary Science, Nestlé Purina Korea, and TS Corporation.

scholarly journals Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 433-445 ◽  
Author(s):  
Natalie I Alexopoulos ◽  
Poul Maddox-Hyttel ◽  
Pernille Tveden-Nyborg ◽  
Nancy T D'Cruz ◽  
Tayfur R Tecirlioglu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Reproductive Technologies ◽  
Embryonic Mortality ◽  
High Rate ◽  
Morphological Examination ◽  
Embryonic Loss ◽  
The Impact

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by eitherin vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establishin vivodevelopmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.

scholarly journals Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer

2008 ◽  
Vol 33 (1) ◽  
pp. 65-77 ◽  
Author(s):  
Robin E. Everts ◽  
Pascale Chavatte-Palmer ◽  
Anthony Razzak ◽  
Isabelle Hue ◽  
Cheryl A. Green ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Pathological Finding ◽  
Culture Methods ◽  
Vitro Fertilization ◽  
Near Term

Transcription profiling of placentomes derived from somatic cell nuclear transfer (SCNT, n = 20), in vitro fertilization (IVF, n = 9), and artificial insemination (AI, n = 9) at or near term development was performed to better understand why SCNT and IVF often result in placental defects, hydrops, and large offspring syndrome (LOS). Multivariate analysis of variance was used to distinguish the effects of SCNT, IVF, and AI on gene expression, taking into account the effects of parturition (term or preterm), sex of fetus, breed of dam, breed of fetus, and pathological finding in the offspring (hydrops, normal, or other abnormalities). Differential expression of 20 physiologically important genes was confirmed with quantitative PCR. The largest effect on placentome gene expression was attributable to whether placentas were collected at term or preterm (i.e., whether the collection was because of disease or to obtain stage-matched controls) followed by placentome source (AI, IVF, or SCNT). Gene expression in SCNT placentomes was dramatically different from AI ( n = 336 genes; 276 >2-fold) and from IVF ( n = 733 genes; 162 >2-fold) placentomes. Functional analysis of differentially expressed genes (DEG) showed that IVF has significant effects on genes associated with cellular metabolism. In contrast, DEG associated with SCNT are involved in multiple pathways, including cell cycle, cell death, and gene expression. Many DEG were shared between the gene lists for IVF and SCNT comparisons, suggesting that common pathways are affected by the embryo culture methods used for IVF and SCNT. However, the many unique gene functions and pathways affected by SCNT suggest that cloned fetuses may be starved and accumulating toxic wastes due to placental insufficiency caused by reprogramming errors. Many of these genes are candidates for hydrops and LOS.

scholarly journals Birth of offspring from spermatid or somatic cell by co-injection of PLCζ-cRNA

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 319-330
Author(s):  
Naoki Hirose ◽  
Sayaka Wakayama ◽  
Rei Inoue ◽  
Junya Ito ◽  
Masatoshi Ooga ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Activation Process ◽  
Oocyte Activation ◽  
Basic Study ◽  
Injection Method ◽  
Significant Difference

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

scholarly journals Generation of monogenetic cattle by different techniques of embryonic cell and somatic cell cloning - their application to biotechnological, agricultural, nutritional, biomedical and transgenic research

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Maria Skrzyszowska ◽  
Marcin Samiec
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Embryonic Cell ◽  
Cell Cloning ◽  
Genetic Rescue ◽  
The Family ◽  
Current Article

AbstractThe development of effective approaches for not only the in vitro maturation (IVM) of heifer/cow oocytes and their extracorporeal fertilization (IVF) but also the non-surgical collection and transfer of bovine embryos has given rise to optimizing comprehensive in vitro embryo production (IVP) technology and improving other assisted reproductive technologies (ARTs), such as cattle cloning by embryo bisection, embryonic cell nuclear transfer (ECNT) and somatic cell nuclear transfer (SCNT). The primary goal of the present paper is to demonstrate the progress and achievements in the strategies utilized for embryonic cell cloning and somatic cell cloning in cattle. Moreover, the current article is focused on recognizing and identifying the suitability and reliability of bovine cloning techniques for nutritional biotechnology, agri-food and biopharmaceutical industry, biomedical and transgenic research and for the genetic rescue of endangered or extinct breeds and species of domesticated or wild-living artiodactyl mammals (even-toed ungulates) originating from the family Bovidae.

Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25)

2020 ◽  
Vol 160 (11-12) ◽  
pp. 688-697
Author(s):  
Sharmila Ghosh ◽  
Candice F. Carden ◽  
Rytis Juras ◽  
Mayra N. Mendoza ◽  
Matthew J. Jevit ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Congenital Abnormalities ◽  
Assisted Reproductive Technologies ◽  
De Novo ◽  
Reproductive Technologies ◽  
Balanced Translocation ◽  
Translocation Breakpoints ◽  
Normal Offspring ◽  
Embryonic Death

We report 2 novel autosomal translocations in the horse. In Case 1, a breeding stallion with a balanced t(4p;30) had produced normal foals and those with congenital abnormalities. Of his 9 phenotypically normal offspring, 4 had normal karyotypes, 4 had balanced t(4p;30), and 1 carried an unbalanced translocation with tertiary trisomy of 4p. We argue that unbalanced forms of t(4p;30) are more tolerated and result in viable congenital abnormalities, without causing embryonic death like all other known equine autosomal translocations. In Case 2, two stallions produced by somatic cell nuclear transfer from the same donor were karyotyped because of fertility issues. A balanced translocation t(12q;25) was found in one, but not in the other clone. The findings underscore the importance of routine cytogenetic screening of breeding animals and animals produced by assisted reproductive technologies. These cases will contribute to molecular studies of translocation breakpoints and their genetic consequences in the horse.

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

MicroRNA-145 Inhibitor Significantly Improves the Development of Bovine Somatic Cell Nuclear Transfer Embryos In Vitro

2016 ◽  
Vol 18 (4) ◽  
pp. 230-236 ◽  
Author(s):  
Wenzhe Li ◽  
Yongjie Xiong ◽  
Fengyu Wang ◽  
Xin Liu ◽  
Yang Gao ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer
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