• International Journal of Industrial Entomology and Biomaterials
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• v.46 no.1
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• pp.16-23
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• 2023

The silkworm's dormancy and embryonic development are accomplished through the interaction of various genes. Analysis of the expression of several interacting genes can predict the embryonic stage of silkworms. In this study, we analyzed the changes in the expression level of genes at each stage during the embryonic development of dormant silkworm eggs and selected genes that can predict the hatching time. Jam123 and Jam124 silkworms were collected after egg laying, and the silkworm eggs were preserved using a double refrigeration method and expression analysis was performed for 23 genes during embryogenesis. There were 5 genes showing significant changes during embryogenesis: UDP-glucuronosyltransferases (BmUGTs), heat shock protein hsp20.8 (BmHsp20.8), Cytochromes b5-like proteins (BmCytb5), Krüppel homolog 1 (BmKr-h1), and cuticular protein RR-1 motif 41 (BmCpr41). As a result of quantitative comparison of the expression levels of these 5 genes through real-time PCR, the BmUGTs gene showed a difference between Jam123 and Jam124, making it difficult to see it as an indicator for predicting hatching time. However, the BmHsp20.8 gene had a common expression decreased at the imminent hatching stage. In addition, it was confirmed that the expression level of the BmCytb5 gene decreased to the lowest level at the time of imminent hatching, and the expression of the BmKr-h gene was made only at the time of imminent hatching. The expression of the last BmCpr41 gene can be confirmed only at the time of imminent hatching, and it was confirmed that it shows a rapid increase right before hatching. Taken together, these results suggest that expression analysis of BmHsp20.8, BmCytb5, BmKr-h1, and BmCpr41 genes can determine the stage of embryogenesis, predict hatching time, which facilitate better management of silkworm eggs.

• Journal of Sericultural and Entomological Science
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• v.46 no.2
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• pp.65-71
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• 2004

Long term preservation experiment through refrigeration was conducted for 2 year on 300 lines of silkworm races preserved, as one method for the development of long term safe preservation technique. Experiment with 6 treatments was conducted for 680 days from July 1 st 2000 to May 1st 2002. Embryonic development was conducted to each treatment. There are no differences among treatments and races in 400 days preservation, the stage of whole embryonic development was Eul B and condition of eggs was good. In 650 days preservation experiment, differences were reveled among races and treatment, the level of whole embryonic development was Byeong A and condition of eggs was good. The order of embryonic development is European races >Tropical, Korean races >Japanese, Chinese races, thus European races showed fast embryonic development. Control(treatment A) and treatment C showed faster development than other treatments. And treatment D and F showed stable individual stage among all treatments. The test of shape characteristics and embryo which were conducted in hatching period showed 61% of high line succession possibility in average of 6 treatments. But treatment A and B showed no hatching, 3 lines of treatment C, 48 lines of treatment D, 1 line of treatment E, and 29 lines of treatment F showed slow development. And treatments D and F which showed stable embryo condition had highest possibility. The two treatments D and F showed good result among six treatments, and the preservation period of treatment D and F are 235 days and 310 days, and exposure period in $-2.5^{\circ}C$...was longer than other treatments. Numbers of hatched lines of treatment D and F are 48 and 29, and occupied 15.6% and 9.4% of all tested lines, respectively. Average hatching ratio of treatment D and F were 54.% and 71.6%, and average dead egg ratio were 12.6% and 2.4%, respectively. These results show that average ratio of hatching dead eggs in treatment D and F are higher than general line. Thus reconsideration of hatching condition on treatment D and F is needed.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.163-169
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• 2013

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

• Journal of Plant Biotechnology
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• v.33 no.3
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• pp.195-207
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• 2006

Stem cells are the primordial, initial cells which usually divide asymmetrically giving rise to on the one hand self-renewals and on the other hand progenitor cells with potential for differentiation. Zygote (fertilized egg), with totipotency, deserves the top-ranking stem cell - he totipotent stem cell (TSC). Both the ICM (inner cell mass) taken from the 6 days-old human blastocyst and ESC (embryonic stem cell) derived from the in vitro cultured ICM have slightly less potency for differentiation than the zygote, and are termed pluripotent stem cells. Stem cells in the tissues and organs of fetus, infant, and adult have highly reduced potency and committed to produce only progenitor cells for particular tissues. These tissue-specific stem cells are called multipotent stem cells. These tissue-specific/committed multipotent stem cells, when placed in altered environment other than their original niche, can yield cells characteristic of the altered environment. These findings are certainly of potential interest from the clinical, therapeutic perspective. The controversial terminology 'somatic stem cell plasticity' coined by the stem cell community seems to have been proved true. Followings are some of the recent knowledges related to the stem cell. Just as the tissues of our body have their own multipotent stem cells, cancerous tumor has undifferentiated cells known as cancer stem cell (CSC). Each time CSC cleaves, it makes two daughter cells with different fate. One is endowed with immortality, the remarkable ability to divide indefinitely, while the other progeny cell divides occasionally but lives forever. In the cancer tumor, CSC is minority being as few as 3-5% of the tumor mass but it is the culprit behind the tumor-malignancy, metastasis, and recurrence of cancer. CSC is like a master print. As long as the original exists, copies can be made and the disease can persist. If the CSC is destroyed, cancer tumor can't grow. In the decades-long cancer therapy, efforts were focused on the reducing of the bulk of cancerous growth. How cancer therapy is changing to destroy the origin of tumor, the CSC. The next generation of treatments should be to recognize and target the root cause of cancerous growth, the CSC, rather than the reducing of the bulk of tumor, Now the strategy is to find a way to identify and isolate the stem cells. The surfaces of normal as well as the cancer stem cells are studded with proteins. In leukaemia stem cell, for example, protein CD 34 is identified. In the new treatment of cancer disease it is needed to look for protein unique to the CSC. Blocking the stem cell's source of nutrients might be another effective strategy. The mystery of sternness of stem cells has begun to be deciphered. ESC can replicate indefinitely and yet retains the potential to turn into any kind of differentiated cells. Polycomb group protein such as Suz 12 repress most of the regulatory genes which, activated, are turned to be developmental genes. These protein molecules keep the ESC in an undifferentiated state. Many of the regulator genes silenced by polycomb proteins are also occupied by such ESC transcription factors as Oct 4, Sox 2, and Nanog. Both polycomb and transcription factor proteins seem to cooperate to keep the ESC in an undifferentiated state, pluripotent, and self-renewable. A normal prion protein (PrP) is found throughout the body from blood to the brain. Prion diseases such as mad cow disease (bovine spongiform encephalopathy) are caused when a normal prion protein misfolds to give rise to PrP$^{SC}$ and assault brain tissue. Why has human body kept such a deadly and enigmatic protein? Although our body has preserved the prion protein, prion diseases are of rare occurrence. Deadly prion diseases have been intensively studied, but normal prion problems are not. Very few facts on the benefit of prion proteins have been known so far. It was found that PrP was hugely expressed on the stem cell surface of bone marrow and on the cells of neural progenitor, PrP seems to have some function in cell maturation and facilitate the division of stem cells and their self-renewal. PrP also might help guide the decision of neural progenitor cell to become a neuron.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.2
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• pp.79-92
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• 1992

This study was carried out to develop new techniques useful for cryopreservation, thawing and artificial insemination, and ultrastructural changes of cryopreserved spermatozoa in rainbow trout(Oncorhynchus mykiss) . Two extenders, such as Tyrode solution and Whittingham's $T_6$ solution, were used to preserve rainbow trout sperm in refrigerator $(-20,\;-40\;and\;-70^{\circ}C)$ or liquid nitrogen $%(-196^{\circ})$. Hand-stripped semen was diluted to 1:16 with two extenders, an then the semen were frozen after mixing semen and each extender containing 1M or 1.5M DMSO solution to 1:1. After 60 days cryopreserved semen was thawed in a $13^{\circ}$ water bath, and subsequently centrifugated. After centrifugation at 1,000 rpm for 5 min thawed semen was washed with extenders, and then fertilized with fresh eggs. The results obtained in these experiments were summarized as follows: After cryopreservation, over 75% of spermatozoa were appeared motile and the survival rate was high. Following cryopreservation by the addition of cryoprotectant such as DMSO, methanol and glycerol, the fertilization rate of the thawed spermatozoa appeared over $99\%$ compared with the control having $99\%$ of fertilization rate. There was no difference between the control and experimental groups such as $(-20^{\circ}C\;-40^{\circ}C\;and\;-70^{\circ}C)$ and $-196^{\circ}$ in fertilization rate. Following cryopreservation at $-196^{\circ}$ by the addition of 1M DMSO of cryoprotectant, each fertilization rate following 24 hours and hatching rate following 24 days showed $96\%$ and $8\%$ by the addition of BSA, but showed $98\%\;and\;10%$ by no addition of BSA. Following 2 months of cryopreservation by the addition of 1M DMSO of cryoprotectant, there were $10%$ of hatching rate at $-196^{\circ}\;and\;10\%\;and\;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1M methanol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C,\;and\;28\%,\;at\;-70^{\circ}C$ Following 2 months of cryopreservation by the addition of 1M glycerol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C$, and $33\%,\;at\;-70^{\circ}C$. pollowing 2 months of cryopreservation by the addition of 1.5M DMSO of cryoprotectant, there were $27\%$ of fertilization rate at $-20^{\circ}C,\;an\;36\%\;and \;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1.5M glycerol of cryoprotectant, there were $34\% \;of\;fertilization\;rate\;at\;-20^{\circ}C, \;and\;31\%\;and\;31\%,\;respectively,\;at \;-40^{\circ}C\;and\;-70^{\circ}$. Following 2 months of cryopreservation by the addition of 1.5M methanol of cryoprotectant, there were $28\%$ of fertilization rate at $-20^{\circ}C,\;and\;29\%\;and\;28\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C.$ From 10 days and 15 days following fertilization at $13^{\circ}C\;and\;10^{\circ}C$, respectively, the mortality rate of fertilized ova was markedly increased. The middle piece of spermatozoa had two set of central doublets, nine set of outer coarse fibres, and mitochondrial sheath. Spermatozoa went through morphological changes during storage, e.g. winding of flagella, detachment of the nuclear envelope and the plasma membrane from the nucleus of the sperm head. There were $1\%$ abnormal spermatozoa in fresh sperm and about $15\%$ during storage.