• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.139-146
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• 1997

This experiment was carried out to evaluate the effect of mouse early embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells(BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined. For a comparative study of in vi패 and in vitro development, the fresh blastocyst which developed in vivo for 120 hours after hCG injection was collected from the uterus, and their numbers of nuclei were also counted. The higher developmental rates of blastocyst formation was a, pp.ared from 91% to 97% when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal eptithelial cells. The number of nuclei in the embryos cultured for 72 hours under each conditions was significantly reduced it than blastocyst in vitro conditions. The number of nuclei in embryos cultured in TCM 199, Ham's F-10 and Medicult IVF medium were counted 68.1$\pm$6.00, 67.3$\pm$4.49, 66.4$\pm$5.64, and 94.3$\pm$8.61, 92.5$\pm$7.60, 92.1$\pm$6.10 with BOEC and 93.3$\pm$5.80, 92.9$\pm$6.53, 92.3$\pm$7.35 with POEC coculture, respectively. These numbers were lowered than 107.2$\pm$7.43 in vivo conditions. In conclusions, the coculture between the mouse early embryos, and oviductal epithelial cells of BOEC and POEC give to improve the developmental and hatching rates of blastocyst but in vivo culture systems for the growth of nuclei were ineligible than in vitro conditions.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.247-268
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• 1996

It has been known for a long time that gonadotropins and steroid hormones play a pivotal role in a series of reproductive biological phenomena including the maturation of ovarian follicles and oocytes, ovulation and implantation, maintenance of pregnancy and fetal growth & development, parturition and mammary development and lactation. Recent investigations, however, have elucidated that in addition to these classic hormones, multiple growth factors also are involved in these phenomena. Most growth factors in reproductive organs mediate the actions of gonadotropins and steroid hormones or synergize with them in an autocrine/paracrine manner. The insulin-like growth factor(IGF) system, which is one of the most actively investigated areas lately in the reproductive organs, has been found to have important roles in a wide gamut of reproductive phenomena. In the present communication, published literature pertaining to the intrauterine IGF system will be reviewed preceded by general information of the IGF system. The IGF family comprises of IGF-I & IGF-II ligands, two types of IGF receptors and six classes of IGF-binding proteins(IGFBPs) that are known to date. IGF-I and IGF-II peptides, which are structurally homologous to proinsulin, possess the insulin-like activity including the stimulatory effect of glucose and amino acid transport. Besides, IGFs as mitogens stimulate cell division, and also play a role in cellular differentiation and functions in a variety of cell lines. IGFs are expressed mainly in the liver and messenchymal cells, and act on almost all types of tissues in an autocrine/paracrine as well as endocrine mode. There are two types of IGF receptors. Type I IGF receptors, which are tyrosine kinase receptors having high-affinity for IGF-I and IGF-II, mediate almost all the IGF actions that are described above. Type II IGF receptors or IGF-II/mannose-6-phosphate receptors have two distinct binding sites; the IGF-II binding site exhibits a high affinity only for IGF-II. The principal role of the type II IGF receptor is to destroy IGF-II by targeting the ligand to the lysosome. IGFs in biological fluids are mostly bound to IGFBP. IGFBPs, in general, are IGF storage/carrier proteins or modulators of IGF actions; however, as for distinct roles for individual IGFBPs, only limited information is available. IGFBPs inhibit IGF actions under most in vitro situations, seemingly because affinities of IGFBPs for IGFs are greater than those of IGF receptors. How IGF is released from IGFBP to reach IGF receptors is not known; however, various IGFBP protease activities that are present in blood and interstitial fluids are believed to play an important role in the process of IGF release from the IGFBP. According to latest reports, there is evidence that under certain in vitro circumstances, IGFBP-1, -3, -5 have their own biological activities independent of the IGF. This may add another dimension of complexity of the already complicated IGF system. Messenger ribonucleic acids and proteins of the IGF family members are expressed in the uterine tissue and conceptus of the primates, rodents and farm animals to play important roles in growth and development of the uterus and fetus. Expression of the uterine IGF system is regulated by gonadal hormones and local regulatory substances with temporal and spatial specificities. Locally expressed IGFs and IGFBPs act on the uterine tissue in an autocrine/paracrine manner, or are secreted into the uterine lumen to participate in conceptus growth and development. Conceptus also expresses the IGF system beginning from the peri-implantation period. When an IGF family member is expressed in the conceptus, however, is determined by the presence or absence of maternally inherited mRNAs, genetic programming of the conceptus itself and an interaction with the maternal tissue. The site of IGF action also follows temporal (physiological status) and spatial specificities. These facts that expression of the IGF system is temporally and spatially regulated support indirectly a hypothesis that IGFs play a role in conceptus growth and development. Uterine and conceptus-derived IGFs stimulate cell division and differentiation, glucose and amino acid transport, general protein synthesis and the biosynthesis of mammotropic hormones including placental lactogen and prolactin, and also play a role in steroidogenesis. The suggested role for IGFs in conceptus growth and development has been proven by the result of IGF-I, IGF-II or IGF receptor gene disruption(targeting) of murine embryos by the homologous recombination technique. Mice carrying a null mutation for IGF-I and/or IGF-II or type I IGF receptor undergo delayed prenatal and postnatal growth and development with 30-60% normal weights at birth. Moreover, mice lacking the type I IGF receptor or IGF-I plus IGF-II die soon after birth. Intrauterine IGFBPs generally are believed to sequester IGF ligands within the uterus or to play a role of negative regulators of IGF actions by inhibiting IGF binding to cognate receptors. However, when it is taken into account that IGFBP-1 is expressed and secreted in primate uteri in amounts assessedly far exceeding those of local IGFs and that IGFBP-1 is one of the major secretory proteins of the primate decidua, the possibility that this IGFBP may have its own biological activity independent of IGF cannot be excluded. Evidently, elucidating the exact role of each IGFBP is an essential step into understanding the whole IGF system. As such, further research in this area is awaited with a lot of anticipation and attention.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.141-148
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• 1999

This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø＞300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p＜0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p＜0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p＜0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.269-278
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• 2002

Objective s: Chromosome aneuploidy is associated with recurrent abortion and congenital anomaly and genetic diseases occur repeatedly in the specific families. Preimplantation genetic diagnosis (PGD) can prevent aneuploidy or genetic disease by selecting normal embryos before implantation and is an alternative to prenatal diagnosis. The aim of this study is to assess the outcome of PGD cycles by using FISH or PCR, and to determine the clinical usefulness and values in patients with risk of chromosomal aneuploidy or genetic disease. Materials and Methods: From 1995 to Apr. 2001, a total of 108 PGD cycles in 65 patients with poor reproductive outcome were analyzed. The indications of PGD were translocation (n=49), inversion (n=2), aneuploidy screening (n=7), Duchenne muscular dystrophy (n=5) and spinal muscular atrophy (n=2). PGD was applied due to the history of recurrent abortion, previous birth of affected child or risk of aneuploidy related to sex chromosome aneuploidy or old age. Blastomere biopsy was performed in 6$\sim$10 cell stage embryo after IVF with ICSI. In the single blastomere, chromosome aneuploidy was diagnosed by using FISH and PCR was performed for the diagnosis of exon deletion in DMD or SMA. Results: The FISH or PCR amplification was successful in 94.3% of biopsied blastomeres. The rate of transferable balanced emb ryos was 24.0% in the chromosome translocation and inversion, 57.1% for the DMD and SMA, and 28.8% for the aneuploidy screening. Overall hCG positive rate per transfer was 17.8% (18/101) and clinical pregnancy rate was 13.9% (14/101) (11 term pregnancy, 3 abortion, and 4 biochemical pregnancy). The clinical pregnancy rate of translocation and inversion was 12.9% (11/85) and abortion rate was 27.3% (3/11). In the DMD and SMA, the clinical pregnancy rate was 33.3% (3/9) and all delivered at term. The PGD results were confirmed by amniocentesis and were correct. When the embryos developed to compaction or morula, the pregnancy rate was higher (32%) than that of the cases without compaction (7.2%, p<0.01). Conclusions: PGD by using FISH or PCR is useful to get n ormal pregnancy by reducing spontaneous abortion associated with chromosome aneuploidy in the patients with structural chromosome aberration or risk of aneuploidy and can prevent genetic disease prior to implantation.