• Title/Summary/Keyword: Healthy follicles

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Xenografting of the Human Vitrified Ovarian Tissues into the Immune Deficient Animal (사람 난소조직의 초자화 냉동보존과 면역결핍 동물에의 이식)

  • Lee, Kyung-Ah;Yoon, Se-Jin;Lee, Sook-Hyun;Shin, Chang-Sook;Choi, An-Na;Cho, Yong-Seon;Yoon, Tae-Ki;Cha, Kwang-Yul
    • Clinical and Experimental Reproductive Medicine
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    • v.27 no.2
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    • pp.145-149
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    • 2000
  • Objective: The present study was conducted to evaluate the viability of germ cells from the adult and fetal ovarian tissues after vitrification followed by xenografting. Method: The human adult ovarian tissues were obtained from 33 years old patient, and the fetal ovarian tissues were obtained from 22 weeks and 25 weeks in gestation. Ovarian tissues were cryopreserved by vitrification with 5.5 M ethylene glycol (EG 5.5) and 1.0 M sucrose as cryoprotectants. Adult and fetal ovarian tissues were pre-equilibrated with EG 5.5 at room temperature for 10 and 5 minutes, respectively and plunged into liquid nitrogen immediately. Frozen-thawed tissues were xenografted into NOD-SCID mice to evaluate the viability and capacity for further growth of the primordial follicles. Grafts were recovered from the recipients 4 weeks after transplantation and histological analysis was accomplished. Result and Conclusion: Grafts recovered 4 weeks after transplantation contained less number of oocytes and primordial follicles compared to that of the fresh tissues. Survived follicles were mainly primordial and intermediary with larger diameter and more granulosa cells. It is confirmed that 1) the ovarian tissues were healthy and the germ cells were survived after vitrification, and 2) the survived fetal primordial follicles after vitrification resumed the growth in the xenografts.

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Histopathological Studies of Mice after Administration of Radioactive Iodine($^{131}I$) (방사성동위원소옥소(放射性同位元素沃素)($^{131}I$)가 "마우스"의 주요장기(主要臟器)의 병리조직상(病理組織像)에 미치는 영향(影響)에 관(關)한 연구(硏究))

  • Ro, Chae-Song;Lee, Mun-Ho
    • The Korean Journal of Nuclear Medicine
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    • v.1 no.2
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    • pp.85-97
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    • 1967
  • Histopathological changes of various organs of the mice after intra-peritoneal injections of radioactive iodine ($^{131}I$) were experimentally observed. Sixity healthy female mice, weighing average 25 gm, devided into 6 groups, were used. The various doses of $^{131}I$ were injected intraperitoneally at different intervals. The histopathological changes after these treatments were observed in organs such as thyroids, parathyroids, livers, kidneys and gonads. Following were the results; 1) Thyroid: In the group A given $^{131}I$ with a single dose of $10{\mu}C$ per gm body weight, it was observed that the protoplasms of follicular epithelial cells were destroyed, the nuclei were expanded or dissoluted, showing pyknotic changes of nuclei and vacuolizations of protoplasms. In the group B given $^{131}I$ with a single dose of $5{\mu}C$ per gm body weight, hyperemias, hemorrhages and hyaline degenerations in the whole area were observed. In the group C given $^{131}I$ with 3 doses of $2.5{\mu}C$ per gm body weight every week, the thyroid parenchyms were destroyed and epithelial cells of varing size were observed in the fibrinous tissues. In the group D given $^{131}I$ with 6 doses of $0.5{\mu}C$ per gm body weight every week, some destroyed follicles and new borne follicles were observed. But the histopathological changes resemble the follicles of the normal thyroid gland. In the group E and F given $^{131}I$ with 8 and 10 doses of $0.2{\mu}C\;and\;0.01{\mu}C$ for each group per gm body weight every two days, both pyknotic changes of nuclei and cytoplasmic vacuolizations of the follicular epithelia, hypertrophies of follicles and abnormal irregular follicular structures were observed, and in the group F, lymphocytes appeared around the thyroid glands. 2) Parathyroid: In the group A, hyperemia, proliferations of connective tissues, karyorrhexes and vacuolizations were observed. In other experimental groups, no particular pathological change was observed. 3) Liver: The degnerative changes and acute or chronic inflammatory changes were observed in proportion to the amount of $^{131}I$ injected. Atrophies of the liver cells, dilatations of sinusoids, hyaline degenerations and necrotic pictures were observed. 4) Kidney: In the group A, congestions and infiltrations of mononuclear cells and granulocytes were observed around the cortical arteries, and in the group B, the degenerative changes of cortexes, and, in the group C and D, hydronephrotic changes were observed respectively, and hyaline degenerations were partially observed. 5) Gonad: In the group A, the follicles were degenerated. The ova in the follicles showed irregular figures. The changes in the group B were almost the same as in the group A, but the changes were mild. In the group C, the destructions of whole ova, the hypertrophies of ovarian follicular membranes and pyknotic changes of nuclei were observed. In the group D, the pathological changes were similar to that of group C, but mild in the grade. In the group E, almost none of ovarian follicular fluid was observed, and in the group F, the tissue pictures were almost similar to that of the normal group.

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Effects of FSH and LH on Maturation of Bovine Preantral Follicle (소 Preantral Follicle 성숙에 미치는 FSH와 LH의 영향)

  • 김대진;정학재;김동훈;엄상준;이훈택;정길생
    • Korean Journal of Animal Reproduction
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    • v.25 no.2
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    • pp.101-111
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    • 2001
  • The present study was conducted to develop an in vitro culture system that would support bovine follicle growth from preantral to antral stage, oocyte maturation, fertilization, and embryonic development. Bovine preantral follicles (150$\pm$1.2 ${\mu}{\textrm}{m}$) surrounded by theca cell were isolated ezymatically and mechanically from ovarian cortical slides in Leibovitz L-l5 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium. The survival and growth rates of follicles cultured in the presence of FSH (10~150 ng/$m\ell$) were significantly higher than those of control group (P < 0.001), but no significant differences were observed in survival and growth rates of follicles between the LH treatment groups (1~125 ng/$m\ell$) and the control. The survival (40%) and growth (244 $\pm$ 0.5 $\mu\textrm{g}$) of follicles cultured with FSH (90 ng/$m\ell$) and LH (25 ng/$m\ell$) were higher than those of control (25%, 160 $\pm$1.0 $\mu\textrm{g}$). Finally, 50% percent of healthy antral follicles were obtained, and almost 60% of them has complete meiotic division with 1st polar body (18.1%) and 10.0% have developed to the cleaved embryo and blastocyst stage. These results suggest that bovine preantral follicle with intact theca cell can grow to the antral stage using these culture conditions, and that oocytes from in vitro-matured bovine preantral follicle may acquire meiotic competence and can undergo fertilization and development.

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The Effect of Sulfamonomethoxine on Thyroid Gland of the Dog (Sulfamonomethoxine 이 개의 갑상선(甲狀腺)에 미치는 영향(影響))

  • Lee, Ho Il;Rim, Byung Moo;Lee, Sung Hee
    • Korean Journal of Veterinary Research
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    • v.13 no.2
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    • pp.141-146
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    • 1973
  • In order to investigate the effect of sulfamonomethoxine on the thyroid gland, healthy mongrel dogs were selected at random. The body weights of these animals ranged between 1850 and 2050g at the beginning of the experiment. The 12 dogs used in this work were allotted to groups of two. Dogs in one group served as controls and the others were administered sulfamonomethoxine of 50 mg/kg/day for 15 weeks. The results obtained in this work were summerized as follows: 1. Mean body weights of experimental dogs revelled a slow increasing tendency but weights of thyroid glands were increased highly. 2. Thyroid follicles were atrophied significantly in accordance with experimental term and their colloidal substance was not found on stained sections. 3. It indicated the appearance of typical hyperplastic goiter. New follicular epithelial cells which were changed into hypertrophic cuboidal and columnar in type showed degeneration and necrosis and those cells multiplied difusely and made new small follicles.

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The Effect of Estrogen Pretreatment on Ovarian Morphology and Ovulation, Fertilization of the Oocytes Following Super Ovulation in Immature Mice (미숙 흰쥐의 과도배란에 따른 난소의 조직학적 형태와 난모세포의 배란 및 수정에 estrogen의 전처치가 미치는 영향)

  • Kim, Moon-Hwoe;Suh, Byung-Hee;Lee, Jae-Hyun
    • Clinical and Experimental Reproductive Medicine
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    • v.12 no.2
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    • pp.71-79
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    • 1985
  • Systemic extrogen therapy promotes multiple preantral follicular development in immature mice. Estrogen pretreated ovaries might therefore be a useful source of cells for in vitro studies of oocytes maturation. Silastic capsules (5.0 mm length; 3.18 mm outer diameter, 1.57 mm inner diameter) filled with diethylstilbesterol were implanted subcutaneously in experimental mice (ICR) for up to 6 days. Ovarian weight and histology in diethylstilbesterol pretreated and control animal were assessed before and after pregnant mare serum gonadotrophin treatment and after human chorionic gonadotrophin. The following results were obtained; 1. Ovarian weight was significantly increased by 6 days of diethylstilbesterol pretreatment. Subsequent ovarian weight gain in response to pregnant mare serum gonadotrophin and human chorionic gonadotrophin was increased. 2. Diethylstilnbesterol pretreatment stimulated the developed healthy preantral follicles. 3. Forty eight hours after pregnant mare serum gonadotrophin treatment, a larger number of the antral follicles which developed in diethylstilbesterol pretreated animals showed signs of atresia, whereas in the control ovaries there was a higher incidence of premature luteinization. 4. Forty eight hours after human chorionic gonadotrophin, numerous corpora lutea and occasional luteinized unruptured follicles were present in both control and diethylstilbesterol ovaries. 5. Ovulation rate, fertilization rate and subsequent preimplantation development in vitro were not adversely affected by diethylstilbesterol pretreatment. However, there was considerable variation in the ovulation rate the number of animals with more than 60 ovulations was greater in the diethylstilbesterol gorup (52.4%) as compared to the control (33.3%).

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Antibody Response to Crude Cell Lysate of Propionibacterium acnes and Induction of Pro-Inflammatory Cytokines in Patients with Acne and Normal Healthy Subjects

  • Basal, E.;Jain, A.;Kaushal, G.P.
    • Journal of Microbiology
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    • v.42 no.2
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    • pp.117-125
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    • 2004
  • Propionibacterium acnes (P. acnes) plays an important role in the disease pathogenesis of acne vulgaris, a disorder of pilosebaceous follicles, seen primarily in the adolescent age group. In the present study, the presence of antibodies against P. acnes (MTCC1951) were detected in acne patient (n=50) and disease free controls (n=25) using dot-ELISA and Western blot assay. The ability of P. acnes to induce pro-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), obtained from acne patients and healthy subjects, were also analysed. The patients (n=26) who were culture positive for skin swab culture, were found to have a more advanced disease and higher antibody titres (1:4000 to >1:16000) compared to the P. acnes negative patients (n=24) and normal controls (n=25). An analysis of patients' sera by western blot assay recognized a number of antigenic components of P. acnes, rang-ing from 29 to 205 kDa. The major reactive component was an approximately 96 kDa polypeptide, which was recognised in 92% (24 of 26) of the patients sera. Further, the P. acnes culture supernatant, crude cell lysate and heat killed P. acnes whole cells, obtained from 72-h incubation culture, were observed to be able to induce significant amounts of IL-8 and tumor necrosis factor alpha (TNF-${\alpha}$) by the PBMCs in both the healthy subjects and patients, as analysed by cytokine-ELISA. The levels of cytokines were significantly higher in the patients than the healthy subjects. A major 96 kDa polypep-tide reactant was eluted from the gel and was found to cause dose dependent stimulation of the pro-ductions of IL-8 and TNF-${\alpha}$. Thus, the above results suggest that both humoral and pro-inflammatory responses play major roles in the pathogenesis of acne.

Effects of Melatonin on the Meiotic Maturation of Mouse Oocytes in vitro (생쥐 난자의 체외 성숙에 미치는 Melatonin의 영향)

  • Ahn, Hee-Jin;Bae, In-Ha
    • Clinical and Experimental Reproductive Medicine
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    • v.31 no.3
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    • pp.155-168
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    • 2004
  • Objective: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. Material and Method: Immature mouse oocytes were obtained from the ovarian follicles of $3{\sim}4$ weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at $37^{circ}C$, 5% $CO_2$ and 21% $O_2$ (95% air) or 5% $CO_2$, 5% $O_2$ and 90% $N_2$. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of $0.0001{\mu}M$, $0.01{\mu}M$ or $1.0{\mu}M$. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. Results: Under 21% oxygen condition, oocytes cultured in the presence of $0.01{\mu}M$ melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with $0.0001{\mu}M$ or $1.0{\mu}M$ melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of $0.01{\mu}M$ melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. Conclusion: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

Effects of Aromatase Inhibitor on Reproductive Hormone Profiles and Ovulation Induction (방향화효소억제제의 생식호르몬 분비와 배란유도에 대한 효과)

  • Kim, Sook-Hyun;Kim, Jeong-Ah;Park, Joon-Cheol;Bae, Jin-Gon;Shin, So-Jin;Kwon, Sang-Hoon;Cho, Chi-Heum;Yoon, Sung-Do;Cha, Soon-Do;Kim, Jong-In;Rhee, Jeong-Ho
    • Clinical and Experimental Reproductive Medicine
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    • v.35 no.2
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    • pp.143-153
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    • 2008
  • Objective: To investigate the effects of aromatase inhibitor on reproductive hormone profiles and evaluate it's ovulation inducing capability in anovulatory infertile women. Methods: We quantified the blood levels of reproductive hormones from 30 healthy normal cycling women in natural cycle (control) and letrozole medicated cycle (study). LH, FSH, estradiol, testosterone, DHEA-S were quantified on third, 11th, 21th day in both cycles, and on 21th day, progesterone was added. Sixth anovulatory infertile women received either letrozole or clomiphene citrate for ovulation induction (n=30 in each groups). We compared the clinical parameters such as ovulation rate, pregnancy rate, the day of LH surge, number of follicles and endometrial thickness, cervical mucus amount, spinnbarkeit, mean diameter of follicles on the day of LH surge. Results: Letrozole had no effect on the LH, FSH, estradiol, DHEA-S secretion but there were significant increase in testosterone level on day 11 and progesterone level on day 21 in letrozole medicated cycle compared than control cycle ($0.40{\pm}0.16$ vs $0.28{\pm}0.11\;ng/ml$, p=0.002, $18.18{\pm}13.07$ vs $8.38{\pm}7.64\;ng/ml$, p=0.001, respectively). In comparison between letrozole and clomiphene groups, there were no significant difference in ovulation rate, pregnancy rate, number of mature follicle, mean diameter of follicles, but showed earlier LH surge, thicker endometrium, more cervical mucus, and higher spinnbarkeit in letrozole group ($12.12{\pm}2.46$ vs $14.52{\pm}3.18$ days, p=0.006, $10.48{\pm}1.23$ vs $8.52{\pm}0.93\;mm$, p=0.000, $2.04{\pm}0.61$ vs $1.57{\pm}0.59$, p=0.012, $6.00{\pm}1.12$ vs $4.95{\pm}1.61\;cm$, p=0.003, respectively). Conclusion: Letrozole may augment folliculogenesis and improve the endometrial condition for implantation in normal cycling women. Ovulation efficacy of letrozole in anovulatory women was comparable to clomiphene citrate and letrozole may be more physiological in ovulation induction.

Development of Isolation and Cultivation Method for Outer Root Sheath Cells from Human Hair Follicle and Construction of Bioartificial Skin

  • Sin, Yeon-Ho;Seo, Yeong-Gwon;Lee, Du-Hun;Yu, Bo-Yeong;Song, Gye-Yong;Seo, Seong-Jun;Hwang, Seong-Ju;Kim, Yeong-Jin;Yang, Eun-Gyeong;Park, Jang-Seo;Jang, Lee-Seop;Park, Jeong-Geuk
    • 한국생물공학회:학술대회논문집
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    • 2003.04a
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    • pp.302-305
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    • 2003
  • It is difficult to obtain sufficient healthy skin for coverage of a wide area of skin wound. In the skin, an additional population of living epithelial cells is located in the outer root sheath (ORS) of hair $follicles.^{1),2)}$ ORS cells should be a good source of epithelium because they are easily obtainable and patients do not have to suffer from scar formation at donor sites. We modified ordinary primary culture technique for the purpose of solving such problem that epithelial cells have a low propagation and easy aging during culture periods. First of all, we improved primary cultivation methods. In the ordinary primary culture, average yield of human ORS cells was $2\;{\times}\;10^3$ cells/follicle by direct incubation with trypsin (0.1%)/EDTA (0.02%) solution for 15 min at $37^{\circ}C$ but we could obtain about $6.5\;{\times}\;10^3$ cells/follicle by two step enzyme digestion method with dispase (1.2 U/ml) and trypsin (0.1%)/EDTA (0.02%) solution. So we could achieve three times higher primary cultured ORS cell yield. Secondly, we could obtain total $2\;{\times}\;10^7$ cells in serum free medium and even more total $6\;{\times}\;10^7$ cells in modified E-medium with mitomycin C-treated feeder cells during 17 days. Using the cultured ORS cells, and we could make bioartificial skin equivalent in vitro and concluded that ORS cells were progenitor cells for skin epithelial cell.

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Determination of Apoptosisin Granulosa-Luteal Cells Obtained from Hyperstimulated Human Ovaries (과배란 유도시 인간 난소로부터 얻어진 과립-황체화 세포의 자연세포사 검정)

  • 양현원
    • Development and Reproduction
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    • v.1 no.1
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    • pp.79-89
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    • 1997
  • Recent studies have demonstrated that apoptotic cell death plays an important role in the mechanism underlying follicular atresia and luteolysis. However, the mechanisms responsible for initiating these processes have not been elucidated. In in vitro fertilization (IVF) programs, it is highly possible that continuous and repeated administration of FSH/hMG and GnRH agonists for the usage of ovarian hyperstimulation may induce apoptotic death of granulosa cells leading to atresia in the human ovarian follicles. The present study was performed to investigate whether FSH/hMG and GnRh agonists used for a longer period in controlled ovarian hyperstimulation has any effect on the apoptosis of granulosa-luteal (GL) cells obtained from hyperstimulated ovaries. To examine apoptotic cell death in the GL cells, cells were stained with acridie orange followed by observed in some of GL cells. Similar but distinct staining of apoptotic GL cells was observed when the cells were examined by using in situ TUNEL method. The healthy-looking cells with normal nuclear morphology were not stained, whereas cells with pyknotic nuclei or with apoptotic nuclei were intensively stained. After examining the ultrastructural features of GL cells by TEM, it was confirmed that the majority of cells seemed to have normal nuclei while GL cells undergoing apoptotic cel death were rarely found. The DNA extracted from GL cells showed a typical pattern of fragmentation following DNA electrophoretic analysis. We have confirmed that the apoptosis occurs in granulosa-luteal cells obtained from hyperstimulated ovaries. Technically, in situ apoptosis detection method is simple and reproducible and is well suited to identify the quality of oocytes retrieved from hyperstimulated ovaries.

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