• Clinical and Experimental Reproductive Medicine
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• v.40 no.3
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• pp.107-114
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• 2013

Homeobox genes play essential roles in embryonic development and reproduction. Recently, a large cluster of homeobox genes, reproductive homeobox genes on the X chromosome (Rhox) genes, was discovered as three gene clusters, ${\alpha}$, ${\beta}$, and ${\gamma}$ in mice. It was found that Rhox genes were selectively expressed in reproduction-associated tissues, such as those of the testes, epididymis, ovaries, and placenta. Hence, it was proposed that Rhox genes are important for regulating various reproductive features, especially gametogenesis in male as well as in female mammals. It was first determined that 12 Rhox genes are clustered into ${\alpha}$ (Rhox1-4), ${\beta}$ (Rhox5-9), and ${\gamma}$ (Rhox10-12) subclusters, and recently Rhox13 has also been found. At present, 33 Rhox genes have been identified in the mouse genome, 11 in the rat, and three in the human. Rhox genes are also responsible for embryonic development, with considerable amounts of Rhox expression in trophoblasts, placenta tissue, embryonic stem cells, and primordial germ cells. In this article we summarized the current understanding of Rhox family genes involved in reproduction and embryonic development and elucidated a previously unreported cell-specific expression in ovarian cells.

• Korean Journal of Poultry Science
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• v.41 no.4
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• pp.249-259
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• 2014

Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps and freezing media on the rates of viability of cryopreserved chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos of Korean Ogye (KO) and Commercial breeds (C), using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). Gonads were harvested from stage 28 chick embryos and pooled in groups of 5, 10, 15, 20E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15% and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to slow freezing and vitrification, with different concentrations of the cryoprotectant solution, were examined. After vitrification and slow freezing, survival rates of the frozen-thawed PGCs from the 10% EG plus FBS treatment were 85.63%, and 66.14% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05) (85.63% vs 66.81%) by vitrification. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at a germplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.207-216
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• 2005

Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Müllerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

• Korean Journal of Ichthyology
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• v.21 no.3
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• pp.141-148
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• 2009

Sex differentiation and gonad development were investigated in a marine medaka species, Oryzias dancena (Beloniformes; Teleostei). The average time to hatch was 11 days post-fertilization (dpf) at $25^{\circ}C$. Primordial germ cell (PGC) was first observed at 5 dpf and migrated to presumptive gonadal area between the gut and pronephric duct at 9 dpf. Male and female gonads were morphologically differentiated at 12 days post-hatching (dph). Early oocytes at perinucleolus stage as well as the formation of spermatid and efferent duct were observed at 28 dph. At 6 weeks of age, the ovary exhibited yolk granulation in many oocytes, while testis possessed a considerable number of spermatogonia and spermatids. The first ovulation was observed in 9-week-old females, and at the same age, males contained fully-matured spermatozoa. Data obtained in this study indicate that the gonad differentiation of O. dancena is the typical type of differentiated gonochorism.

• Development and Reproduction
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• v.2 no.1
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• pp.69-74
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• 1998

The early gonadal development and sexual differentiation of Rhynchocypris oxycephalus are described from the stage of hatching to 150 days after hatching. During this peroid, the average length of the body grew from 0.64 cm to 5.96 cm. the primordial germ cells (PGCs), which could be recognized at the time of hatching, began to protrude into peritoneal cavity at a standard total length of 1.91 cm. At a standard total length of 2.29cm, initial ovarian differentiation wasidentified by the transformation of PGCs to meiotic oocytes. Finally, at the standard total length of 5.96 cm, the female gonads gradually developed towards migratory nucleus oocytes, characterisiing the maturation. Oocytes proliferated rapidly after sex differentiation while the testis entered a period of quiescence, as they continued to multiply but did not undergo growth until the standard total length of 4.00 cm. At a standard total length of 4.00 cm, spermatocytes arrested in thephase of interkinesis, Sertoli-like cells and sperm duct formation, with signs of meiotic activity, were observed. Therefore it may be concluded that R. oxycephalus belongs to the differentiated type of gonochoristic teleosts.

• Korean Journal of Poultry Science
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• v.23 no.1
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• pp.9-17
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• 1996

This study was conducted to compare the survival rate of chick embryos among different eggshell window positions and to search for the most appropriate injection position. The eggshells were punctured at blunt-end, sharp-end and side-up with a sterilized fine forceps, respectively. The survival rate of sharp-end window was higher than the other window positions. Injection of Dulbecco’s modified eagle’s medium (DMEM) through blunt-end window (BE1) was impossible because inner cell membrane was obscure. The 2 ${\mu}$L DMEM was injected into 2.5 d-old embryo blood vessel through sharp end window. To prevent hemorrhages at the point of injection, the air bubbles were injected into the embryo blood vessel. The survival rate of chicks embryo in sharp end window was about 17.0％. Therefore, this sharp-end window system will be helpful for the production of germline chimera or transgenic chicken using primordial germ cells ( PGCs ).

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.4
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.207-216
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• 2013

This study was conducted to establish the method for preserving PGCs that enables long-term storage in liquid nitrogen for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of freeze-thaw treatment on viability of PGCs in chickens. PGCs were collected separately from a germinal gonad of an early embryo of 5.5~6 day (stage 28) of Isa brown, Korean Oge (KO), White Leghorn and Commercial breeds. PGCs separated from a germinal gonad of an early embryo of 5.5~6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The PGCs were then purified using magnetic activated cell sorting (MACS) method. The viability of PGCs after thawing was $87.4{\pm}0.4%$ and $89.4{\pm}0.2%$ with the 10% EG treatments with no significant difference between the Isa brown and Commercial breeds. The viability of PGCs after freeze- thawing was significantly higher for Isa brown ($87.4{\pm}0.4%$) and Commercial breeds ($89.4{\pm}0.2%$) than Korean Oge (KO) ($77.6{\pm}1.1%$) and White Leghorn ($76.2{\pm}0.9%$)(p<0.05) using 10% EG cryoprotectant. This study established a method for pre- serving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at agermplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.225-230
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• 2012

Interferon induced transmembrane protein-1 (Ifitm-1) has been reported to have an important role in primordial germ cell formation, and it has expressed in female reproductive organ. In the present study, Ifitm-1 gene expression was identified in testes and all part of epididymis using western immunoblot and immunohistochemistry. Interestingly, Ifitm-1 expression was observed on the head of spermatozoa. To investigate the role of Ifitm-1 gene expression in behavior of spermatozoa after acrosome reaction, fresh sperm was incubated with calcium ionophore to induce acrosome reaction, whereas the expression of Ifitm-1 was not altered after the acrosome reaction. Then to identify the effect of Ifitm-1 in sperm motility and other seminal parameters, different concentration of Ifitm-1 antibody was incubated with spermatozoa, and seminal parameters were assessed using computer-assisted semen analysis (CASA). Interestingly, motility, progressive, and VAP were increased in the sperm with Ifitm-1 antibody treated compared to rabbit serum, however other parameters such as straightness were not changed. In order to identify the functional significance of Ifitm-1 in fertilization, capacitated spermatozoa were pre-incubated with anti-Ifitm-1 antibody and subsequently examined the ability to adhere to mouse oocytes. However, any defection or alteration in sperm-egg fusion was not found, Ifitm-1 antibody treated or non-treated spermatozoa showed a normal penetration. Although the precise role of Ifitm-1 in sperm motility and following fertilization need to be elucidated, this study suggests that the activation of Ifitm-1 on the sperm may enhance the motility of spermatozoa in mice.

• Development and Reproduction
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• v.20 no.4
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• pp.289-296
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• 2016

This report describes the sex differentiation of the Korean rose bitterling, Rhodeus uyekii, from hatching to 170 days post-hatch (DPH) in relation to total length (TL), body weight (BW), and integral water temperature (IWT). The growth curve of TL from just hatching to 83 DPH was $5.144e^{0.045t}$ ($R^2=0.961$; t, time), and that of BW was $2.398e^{0.086t}$ ($R^2=0.725$). Primordial germ cells (PGCs) were observed at 17 DPH (7.9 mm TL, 3.74 mg BW, $374^{\circ}C$ IWT), and thereafter began to protrude into the peritoneal cavity. At 21 DPH ($9.2{\pm}0.14mm$ TL, $4.8{\pm}0.07mg$ BW, $462^{\circ}C$ IWT), some PGCs contained condensed chromatin and oocyte were observed in meiotic prophase. In contrast to the ovaries, which grew gradually after sexual differentiation, testes began multiplying at 25 DPH (10.1 mm TL, 5.42 mg BW, $550^{\circ}C$ IWT), when testicular differentiation was first identified, and multiplied continuously thereafter. At 33 DPH (11.2 mm TL, 10.5 mg BW, $726^{\circ}C$ IWT), the developing testes contained spermatogonia that exhibited mitotic activity. No spermatocyte or sperm cell was observed until 83 DPH (18.9 TL, 48.2 mg BW, $1,826^{\circ}C$ IWT). At 170 DPH (32.5 mm TL, 270.1 mg BW, $3,740^{\circ}C$ IWT), which was the end point of this study, the mature ovaries showed germinal vesicle breakdown, while the mature testes contained observable spermatocytes and sperm cells. These results allow us to identify the sex differentiation type of the Korean rose bitterling as differentiated gonochoristic.