• Molecules and Cells
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• v.44 no.12
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• pp.911-919
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• 2021

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M₁ progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

• Development and Reproduction
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• v.26 no.2
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• pp.37-47
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• 2022

Photoperiod has well been established to regulate testicular activities in golden hamsters. These animals breed actively around summer but become infertile in winter. In males, testicles are full of multistep germ cells including spermatozoa in summer. But in winter only fundamental cells consisting of the testicles are detected. The testicular degeneration is accompanied by the reduced levels of blood gonadotropins and testosterone. In this study, the expressions of gonadotropin subunit genes were investigated in the reproductive active and inactive testicles. And parts of sequences of the gonadotropin subunits were identified and compared with those of other rodents. As results, common gonadotropin alpha (CGa), follicle-stimulating hormone (FSH) β, and luteinizing hormone (LH) β genes were equivalently detected in pituitaries of both sexually active and inactive animals. In considering low concentrations of gonadotropin hormones determined in pituitary, the present findings imply that the processes involved in translation and/or formation of functional hormones could be impeded in the sexually inactive hamsters. All the nucleotide sequences of gonadotropin subunits identified in this study were same as those reported previously except for one base in CGa. An unsure amino acid deduced from the CGa sequence was confirmed from mRNA sequencing. The outcomes mentioned above suggest that animals with regressed testes prepare for the sexually active period forthcoming in the future.

• Development and Reproduction
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• v.26 no.3
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• pp.107-115
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• 2022

Kisspeptins, products of KISS1 gene, are ligands of the G-protein coupled receptor (GPR54), and the kisspeptin-GPR54 signaling has an important role as an upstream regulator of gonadotropin releasing hormone (GnRH) neurons. Interestingly, extrahypothalamic expressions of kisspeptin/GPR-54 in gonads have been found in primates and experimental rodents such as rats and mice. Hamsters, another potent experimental rodent, also have a kisspeptin-GPR54 system in their ovaries. The presence of testicular kisspeptin-GPR54 system, however, remains to be solved. The present study was undertaken to determine whether the kisspeptin is expressed in hamster testis. To do this, reverse transcription-polymerase chain reactions (RT-PCRs) and immunohistochemistry (IHC) were employed. After the nest PCR, two cDNA products (320 and 280 bp, respectively) were detected by 3% agarose gel electrophoresis, and sequencing analysis revealed that the 320 bp product was correctly amplified from hamster kisspeptin cDNA. Modest immunoreactive (IR) kisspeptins were detected in Leydig-interstitial cells, and the weak signals were detected in germ cells, mostly in round spermatids and residual bodies of elongated spermatids. In the present study, we found the kisspeptin expression in the testis of Syrian hamster. Further studies on the local role(s) of testicular kisspeptin are expected for a better understanding the physiology of hamster testis, including photoperiodic gonadal regression specifically occurred in hamster gonads.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.149-154
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• 2022

Busulfan is the most commonly used drug for preconditioning during the transplantation of hematopoietic stem cells and male germ cells. Here, we describe side effects of high doses of busulfan in male mongrel dogs. Busulfan was intravenously administered to three groups of dogs at doses of 10, 15, and 17.5 mg/kg body weight. The total white blood cell, neutrophil, eosinophil, lymphocyte, monocyte, and platelet counts steadily reduced in a dose-dependent manner following busulfan treatment. The white blood cell, neutrophil, and monocyte counts recovered after 6 weeks of busulfan treatment, however, the eosinophil, lymphocyte, and platelet counts remained unaltered. Additionally, there was one fatality in the each of the groups that were administered 15 and 17.5 mg/kg busulfan. The gross lesions included severe hemorrhage in the stomach, intestinal tracts, mesentery and urinary bladder. Microscopic investigation revealed severe pulmonary edema and hemorrhage in the lungs, and severe multifocal to coalescing transmural hemorrhage in the intestines and urinary bladder. These results indicated that treatment with busulfan at doses higher than 15 mg/kg initiates severe bleeding in the internal organs and can have fatal results.

• Applied Microscopy
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• v.33 no.3
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• pp.243-250
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• 2003

Ultrastructural changes of the male germ cells and structure of spermatozoa in Paralichthys olivaceus were examined by means of the light and transmission electron microscopes. The spermatogonium has a large nucleus with a single nucleus with a single nucleolus in the interphase. Primary spermatocytes are identified by the formation of the synaptonemal complex in the karyoplasm. The secondary spermatocytes are more concentrated and contains numerous cell organelle in the cytoplasm. The nucleus of spermatid in spermiogenesis is more condensed in the karyoplasm, and show spherical structure in shape. Mitochondria of the spermatids are observed in the lower portion of the nucleus. The spermatozoon consists of the head, mid piece and tail. The acrosome is not observed in the head. Axial filaments of the flagellum consists of nine pairs of the peripheral microtubules and one pair of the central microtubules.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2004.11a
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• pp.9-10
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• 2004

We developed a new panel of markers for the characterization of chicken PGCs. The results of immunostaining demonstrated that anti-SSEA-3, anti-SSEA-4, anti-integrin 6, and anti-integrin 1 antibodies. and STA and DBA bound specifically to chicken PGCs. These reagents could be used to characterize chicken PGCs together with conventional marker reagents such as PAS and anti-SSEA-1 antibody. We also showed that double staining of PGCs with the newly developed markers was feasible, which might contribute to rapid detection and accurate characterization of chicken PGCs.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.255-260
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• 1994

원시생식세포(primordial germ cell; PGC)는 성성숙 이후에 기능을 갖는 생식세포의 근원이 되는 세포로서, 다능성을 갖고 있는 것으로 알려져 있다. 그러므로 chimera 및 유전자 변환동물 생산을 위해 널리 사용되어 온 배아주(embrynic stem; ES)세포를 대신할 다른 세포계라고 생각되어져 많은 연구가 진행되고 있다. 본 실험은 체외배양을 통하여 원시생식세포의 증식과 확립을 위해 배양조건을 구명하고, 또한 성장인자의 효과를 검증하기 위하여 실시되었다. 원시생식세포는 12.5일째의 ICR 생쥐태아의 원시생식선 융기조직으로부터 추출하였으며, DMEM + 20% FCS + nucleosides + antibiotics로 조성된 sDMEM 배양액을 사용하여 mitomycin C로 전처리한 되먹임세포단층(feeder layer)위에서 체외배양하였다. bFGF 및 LIF를 20, 40ng/ml농도로 각각 또는 함께 첨가하여 성장인자의 효과를 검토하였다. 원시생식세포는 성에 따라 유의적인 colony 형성율을 보였고(♂:1.9 colonies / genital ridge, ♀:1.3 colonies / genital ridge), bFGF 및 LIF의 첨가 및 첨가농도에 따라서도 유의성 있는 결과를 보였다(0.3~1.9 colonies / genital riege). 그러나 3회 이상 계대배양을 할 경우, 원시생식세포의 colony를 4% prarformaldehyde로 20분간 고정한 후, tris-maleate buffer(pH 9.0)로 10분간 3회 세정하였다. Fast Red로 염색을 실시한 결과, 대부분의 colony가 염색반응을 보여 다능성을 갖는 원시생식세포의 colony임이 입증되었다. 그러나 대부분의 colony가 3회 이상의 계대배양시 생종율이 급격히 떨어지는 것을 감안하면, 또 다른 미지의 성장인자나 보다 적절한 배양조건이 요구된다고 생각된다.

• Korean Journal of Poultry Science
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• v.16 no.1
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• pp.1-8
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• 1989

This experiment was conducted to improve the performance of chickens by the precise separation and analysis of chromosomes which are integrated genetic materials, and by the use of gene manipulation techniques. Following are the main results obtained. 1. When the chromosomes were separated through the leucocyte culture and analyzed by Giemsa banding techniques (especially by the method in which 20 layers of banding patterns could be found in chromosome ＃1), the normal Patterns of chromosomes ＃l-9 and sex chromosomes, and the location of constitutive heterochromatin without any gene activities in all chromosomes were discovered. 2. To utilize the primodial germ cells (PGC) as the genetic vector which is one of the most important gene manipulation techniques, PGC's from triploid were transplanted to normal host embryos. Since the donor PGC's(3n) were found in the gonads of growing host embryos gene manipulation in poultry using PGC's, seemed to be possible.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.193-199
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• 2005

The cryopreservation of germ cells, sperm and embryos, has been largely used to increase the effect of artificial reproductive techniques for human infertility, but the efficiency of germ cell cryopreservation has been conkoversial till now. Thus, the effect of the cryopreservation of human sperm used for ICSI and the effect of the cryopreservation of embryos produced by ICSI on fertilizatiof development and pregnancy were investigated. Sperm freezing did not affect fertilizatiort development and pregnancy rates. Also, there was no significant difference between ejaculated and testicular sperm in ferclizatiort development and pregnancy. Embryo freezing methods, slow freezing and vitrificatior did not differ each other in viability and pregnncy rates. However, ICSI embryo freezing significantly decreased pregnancy rate compared to fresh embryos freezing (p<0.05). In conclusiof this result suggested that cryopreservation of sperm for ICSI did not affect on the resulted embryo development and pregnancy, but ICSI embryo cryopreservation would significantly inhibit pregnancy.

• Animal cells and systems
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• v.3 no.3
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• pp.243-246
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• 1999

A type of symbiosis was previously described from nature in which the gametophytes of Laminariales were endophytic in filamentous red algae. Here we reconstruct this symbiosis for the first time in laboratory culture using zoospores of the kelp, Undaria pinnatifida, and the red alga, Aglaothamnion oosumiense. Zoospores of U. pinnatifida readily attached to A. oosumiense. In 48 h these spores germinated and the initial germ tube penetrated into the host cell wall leaving only an empty zoospore wall outside the host. Within ten days, four to five-celled endophytic gametophytes were present. Zoospores of Laminaria religiosa which were also inoculated into cultures of A. oosumiense rarely attached to the red alga and never became endophytic. Within ten days the free-living gametophytes of L. religiosa on cover slips became fertile and produced young sporophytes. These observations demonstrate the ability of U. pinnatifida to become endophytic, and show differences in host specificity among kelp species.