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The Effect of L-carnitine and Acetylcarnitine on Sperm Parameters in vitro.

Korean Journal of Fertility and Sterility.2002;29(2):149-157.

OBJECTIVE: To assess the scavenging effect of carnitine derivatives on oxidative damage to sperm during sperm processing cryopreservation and thawing. MATERIALS AND METHODS: Fresh semen samples from 20 normal healthy volunteers were collected by masturbation after at least 48 hours abstinence. After liquefaction of semen samples at room temperature, the specimens were diluted with sperm wash media (Ham's F-10, Life technologics) to a uniform density of 20x106/ml. L-carnitine or acetylcarnitine were added with various concentration of 0 micrometer, 10 micrometer, 30 micrometer in semen sample or cryoprotectant. All specimens were cryopreservated at -196degrees C LN2 for 3 days. Sperm motility, vitality, fertilizing capacity, reactive oxygen species formation and the level of lipid peroxidation were analyzed by computer assisted semen analyzer, eosin-nigrosin stain, hypo-osmotic swelling test, chemiluminescence and thiobarbituric acid method, respectively, during sperm processing, cryopreservation and thawing. RESULTS: The sperm motility was only increased in proportion to the concentration of acetylcarnitine with no statistical significance (p>0.05). The sperm vitality was also significantly improved in proportion to the concentration of acetylcarnitine with statistical significance (p<0.05). The sperm fertilizing capacity was significantly increased in proportion to the concentration of L-carnitine and acetylcarnitine and reactive oxygen species generation and lipid peroxidation were significantly decreased with same fashion(p<0.05). On comparison of effects between L-carnitine and acetylcarnitine, acetylcarnitine was superior to L-carnitine on the improvement of sperm motility and vitality as well as the suppression of reactive oxygen species generation and lipid peroxidation. CONCLUSION: These results suggest that carnitine derivatives have a scavenging effect against oxidative damages during sperm processing, cryopreservation and thawing. Therefore, carnitine derivatives may be useful as an oral antioxidant in patients with male infertility due to increased ROS generation.
Acetylcarnitine* ; Carnitine* ; Cryopreservation ; Healthy Volunteers ; Humans ; Infertility, Male ; Lipid Peroxidation ; Luminescence ; Male ; Masturbation ; Reactive Oxygen Species ; Semen ; Sperm Motility ; Spermatozoa*

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Functional Cardiomyocytes Formation Derived from Parthenogenetic Mouse Embryonic Stem Cells.

Hyun Ah SHIN ; Eun Young KIM ; Young Jae LEE ; Keum Sil LEE ; Eun Mi PARK ; Hoon Taek LEE ; Kil Saeng CHUNG ; Sepill PARK ; Jin Ho LIM

Korean Journal of Fertility and Sterility.2002;29(2):139-147.

OBJECTIVE: This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. MATERIALS AND METHODS: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to defect cardiomyocytes (anti-sarcomeric alpha-actinin Ab, 1: 100; anti-cardiac troponin I Ab, 1: 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. RESULTS: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3% at 13 days and 69.8% at 15 days, respectively. Also the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and mES02,4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric alpha-actinin Ab and cardiac specific anti-cardiac troponin I Ab. CONCLUSION: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.
Actinin ; Animals ; Antibodies ; Cells, Cultured ; Dimethyl Sulfoxide ; Embryonic Stem Cells* ; Fertilization in Vitro ; Fluorescein-5-isothiocyanate ; Glass ; Mice* ; Microscopy, Fluorescence ; Myocytes, Cardiac* ; Troponin I

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Parthenogenetic Mouse Embryonic Stem Cells have Similar Characteristics to In Vitro Fertilization mES Cells.

Se Pill PARK ; Eun Young KIM ; Keum Sil LEE ; Young Jae LEE ; Hyun Ah SHIN ; Hyun Jung MIN ; Hoon Taek LEE ; Kil Saeng CHUNG ; Jin Ho LIM

Korean Journal of Fertility and Sterility.2002;29(2):129-138.

OBJECTIVE: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. MATERIAL AND METHODS: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and 5microgram/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal perm of hybrid F1 male mice(1x106/ml). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, blastocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identity ES cells, the surface markers alkaline phosphatase, SSEA-1, 3, 4 and Oct4 staining were examined in replated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. RESULTS: Although the cleavage rate (> or =2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic blastocysts (9.6+/-3.1, 35.1+/-5.2) were significantly lower than those of IVF blastocysts (19.5+/-4.7, 63.2+/-13.0) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-1 and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac differentiation derived from mES or P-mES cells was confirmed. CONCLUSION: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.
Alkaline Phosphatase ; Animals ; Antigens, CD15 ; Bisbenzimidazole ; Blastocyst ; Cell Count ; Cell Line ; Embryonic Stem Cells* ; Embryonic Structures ; Ethanol ; Female ; Fertilization in Vitro* ; Humans ; Male ; Mice* ; Oocytes ; Propidium

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In Vitro Neural Cell Differentiation Derived from Human Embryonic Stem Cells: Effects of PDGF-bb and BDNF on the Generation of Functional Neurons.

Hyun Jung CHO ; Eun Young KIM ; Young Jae LEE ; Kyoung Hee CHOI ; So Yeon AHN ; Se Pill PARK ; Jin Ho LIM

Korean Journal of Fertility and Sterility.2002;29(2):117-127.

OBJECTIVE: This study was to investigate the generation of the functional neuron derived from human embryonic stem(hES,MB03) cells on in vitro neural cell differentiation system. METHODS: For neural progenitor cell formation derived from hES cells, we produced embryoid bodies (EB: for 5 days, without mitogen) from hES cells and then neurospheres (for 7~10 days, 20 ng/ml of bFGF added N2 medium) from EB. And then finally for the differentiation into mature neuron, neural progenitor cells were cultured in i) N2 medium only (without bFGF),ii) N2 supplemented with 20 ng/ml platelet derived growth factor-bb (PDGF-bb) or iii) N2 supplemented with 5 ng/ml brain derived neurotrophic factor (BDNF) for 2 weeks. Identification of neural cell differentiation was carried out by immunocytochemistry using betaIII-tubulin (1:100) and GFAP (1:500). Also, generation of functional neuron was identified using anti-glutamate (Sigma, 1:1000), anti-GABA (Sigma, 1:1000), anti-serotonin (Sigma, 1:1000) and anti-tyrosine hydroxylase (Sigma, 1:1000). RESULTS: In vitro neural cell differentiation, neurotrophic factors (PDGF and BDNF) treated cell groups were high expressed MAP-2 and GFAP than non-treated cell group. The highest expression pattern of MAP-2 and betaIII-tubulin was indicated in BDNF treated group. Also, in the presence of PDGF-bb or BDNF, mostof the neural cells derived from hES cells were differentiated into gultamate and GABA neuron in vitro. Furthermore, we confirmed that there were a few serotonin and tyrosine hydroxylase positive neuron in the same culture environment. CONCLUSION: This results suggested that the generation of functional neuron derived from hES cells was increased by addition of neurotrophic factors such as PDGF-bb or BDNF in b-FGF induced neural cell differentiation system and especially gultamate and GABA neurons were mainly produced in the system.
Blood Platelets ; Brain-Derived Neurotrophic Factor* ; Cell Differentiation* ; Embryoid Bodies ; Embryonic Stem Cells* ; gamma-Aminobutyric Acid ; Humans* ; Immunohistochemistry ; Nerve Growth Factors ; Neurons* ; Serotonin ; Stem Cells ; Tyrosine 3-Monooxygenase

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Effects of Reactive Oxygen Species on Sperm Function, Lipid Peroxidation and DNA Fragmentation in Bovine Spermatozoa.

Buom Yong RYU ; Yung Chai CHUNG ; Chang Keun KIM ; Hyun A SHIN ; Jung Ho HAN ; Myung Geol PANG ; Sun Kyung OH ; Seok Hyun KIM ; Shin Yong MOON

Korean Journal of Fertility and Sterility.2002;29(2):105-115.

OBJECTIVE: To evaluate the effects of the reactive oxygen species (ROS) generated with a xanthine(X) and xanthine oxidase (XO) system on sperm function, the change of sperm characteristics, lipid peroxidation, and DNA fragmentation in bovine spermatozoa. MATERIALS AND METHODS: ROS were produced using a combination of 100 micrometer X and 50 mU/ml XO. The ROS scavengers: superoxide dismutase (SOD)(200mu/ml) and catalase (500mu/ml) were also tested. Spermatozoa were incubated for 2 hours in BWW medium with a combination of X-XO supplemented with or without ROS scavengers at 37degrees C under 5% CO2 incubator. Sperm movement characteristics by CASA (computer-aided sperm analysis), HOST (hypoosmotic swelling test), Ca-ionophore induced acrosome reaction, malondialdehyde formation for the analysis of lipid peroxidation, the percentage of DNA fragmentation using the method of TdT-mediated nick end labelling (TUNEL) by flow cytometry were determined after 2 hours incubation. RESULTS: The action of ROS on bovine spermatozoa resulted in a decreased in capacity for sperm motility, Ca-ionophore induced acrosome reaction and membrane integrity, an increased in malondialdehyde formation and the percentage of sperm with DNA fragmentation. In the effects of antioxidant, catalase completely alleviated the toxic effects induced by the ROS in terms of sperm function and characteristics, however SOD exhibited no capacity to reduce the toxic effects. CONCLUSION: The ROS can induce significant damages to sperm functions and characteristics. The useful ROS scavengers can minimized the defects of sperm function and various damages of spermatozoa.
Acrosome Reaction ; Catalase ; DNA Fragmentation* ; DNA* ; Flow Cytometry ; Incubators ; Lipid Peroxidation* ; Malondialdehyde ; Membranes ; Reactive Oxygen Species* ; Sperm Motility ; Spermatozoa* ; Superoxide Dismutase ; Xanthine Oxidase

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The Clinical Efficiency of Clomiphene Citrate vs Clomiphene Citrate/GnRH Antagonist on Infertile Women with Normal Ovulatory Cycles.

Woo Seok LEE ; Jae Hong SANG ; Jae Joon KIM ; Gwang June KIM ; Dong Ho KIM ; Sang Hun LEE

Korean Journal of Fertility and Sterility.2006;33(3):149-157.

OBJECTIVE: This study was to investigate the clinical efficiency of clomiphene citrate/GnRH antagonist protocol comparing with the clomiphene citrate only protocol in infertile women with normal ovulatory cycles. METHOD: Among 116 patients, 43 were received assisted reproductive technologies using natural ovulatory cycle, 38 and 35 were received clomiphene citrate only protocol and clomiphene citrate/GnRH antagonist combined protocol, respectively, and the clinical results were compared and analyzed. RESULTS: In each group, basal levels of LH, FSH, E2 and FSH, E2 on hCG day injected were not different, but LH level and endometrial thickness on hCG injected day were decreased significantly and the pregnancy rate was increased significantly in clomiphene citrate/GnRH antagonist group. CONCLUSION: The pregnancy rate was increased significantly in clomiphene citrate/GnRH antagonist group compared with natural ovulatory cycle and clomiphene citrate only group.
Clomiphene* ; Female ; Humans ; Pregnancy Rate ; Reproductive Techniques, Assisted

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Lessons from Mouse Models to Understand Uterine Receptivity for Embryo Implantation.

Haengseok SONG

Korean Journal of Fertility and Sterility.2006;33(3):139-148.

No abstract available.
Animals ; Embryo Implantation* ; Embryonic Structures* ; Female ; Mice* ; Pregnancy

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The Preventive Effect of Systemic Treatment with Interferon-alpha2B for Infertility of Mumps Orchitis.

Ja Hyeon KU ; Yong Soon YIM ; Chang Ho LEE ; Young Ho KIM ; Yun Seob SONG ; Youn Soo JEON ; Min Eui KIM ; Nam Kyu LEE ; Young Ho PARK

Korean Journal of Fertility and Sterility.1999;26(1):111-116.

Postpubertal mumps may result in ochitis and permanent testicular atrophy may develop following infection. This present study was initiated to evaluate the preventive effect of interferon-alpha2B on infertilty after mumps orchitis. There were 21 patients with mumps orchitis between May 1990 and June 1997. Patients were randomly distributed into 2 groups: group 1 patients (n=13) maintained therapy with interferon-alpha2B (3x10(6) IU per day) and group 2 were managed by conservatively. All of the patients were evaluated with testis size measurement, mumps virus titer, hormone level, and if possible semen analysis. For group 1 patients symptoms disappeared within 2 to 3 days and the volume of testis returned to normal within 11 days and testis atrophy was not observed in all patients in follow up. But asthenospermia was continued in 4 patients (unilateral 2, bilateral 2). For group 2 patients symptoms disappeared within 5 to 6 days and the volume of testis returned to normal within 10 days and testis atrophy was observed in 3 patients (unilateral 2, bilateral 1) in floow up. Asthenospermia was continued in 4 patients (unilateral 2, bilateral 2). Sperm count and morphology were recovered all the recover in group 1, 4 patients had persistent reduced sperm count and morphology in group 2, respectively. These observations suggest that systemic interferon-alpha2B treatment is highly effective in preventing infertility as well as testicular atrophy after mumps orchitis.
Atrophy ; Follow-Up Studies ; Humans ; Infertility* ; Male ; Mumps virus ; Mumps* ; Orchitis* ; Semen Analysis ; Sperm Count ; Testis

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The Lack of a Direct Effect of Tumor Necrosis Factor-Alpha on Sperm Motility.

Eun Seop SONG ; Young Ku LIM ; Yun Seob SONG

Korean Journal of Fertility and Sterility.1999;26(1):97-101.

Male genital tract inflammatory conditions may be associated with unexplained infertility. The presence of cytokine such as tumor necrosis factor-alpha (TNF-alpha) was reported in the semen of infertile men. However, the effect of these cytokines on human sperm function is still unclear. The purpose of this study was to investigate the in-vitro effects of TNF-alpha on human sperm motility with computer assisted sperm analysis. Washed sperm from 16 normal men were incubated without and with TNF-alpha(0.1, 10, 1000 ng/ml). The changes of parameters of sperm motility were recorded at different time intervals (0, 5, 24 hour). There was no significant change of parameters of sperm motility in the incubation with TNF-alpha. It is suggested that TNF-alpha alone does not interfere with the sperm motility and more studies are needed.
Cytokines ; Humans ; Infertility ; Male ; Semen ; Sperm Motility* ; Spermatozoa* ; Tumor Necrosis Factor-alpha*

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Spermatozoa Characteristics of Streptozotocin-induced Diabetic Wistar Rat: Acrosome Reaction and Spermatozoa Concentration.

Yong Pil CHEON ; Chung Hoon KIM ; Byung Moon KANG ; Yoon Seok CHANG ; Joo Hyun NAM ; Young Soo KIM ; Myung Chan GYE ; Moon Kyoo KIM ; Kil Soo KIM

Korean Journal of Fertility and Sterility.1999;26(1):89-96.

SUMMARY: Some of the information concerning sexual function in the male diabetes has been focused upon the problems of endocrine or semen parameters. However, the characteristics of acrosome reaction and spermatozoa concentration at the epididymis and vas deferens have scarcely been studied, and the causes of the infertility has not been critically identified. So, we designed to inspect the spermatozoa concentration and the characteristics of acrosome reaction at epididymis and was deferens of diabetic Wistar rat induced by streptozotocin (STZ, 70 mg/kg, ip). Experimental animal was sacrificed at 3 days and 14 days after the STZ injection. In the diabetes-induced rat, the levels of insulin and glucose had a pattern of inverse proportion. The spermatozoa concentrations in caput and corpus epididymis were significantly decreased in all diabetic condition. In cauda epididymis, however, there was significant decrease in sperm concentration at 14 days onward. In diabetic rat, the spontaneous reaction rate of spermatozoa of cauda and was deferens were significantly higher than the control group. The ARIC (acrosome reaction to ionophore challenge) value of caudal sperm was 28.7 at control, 22.1 at 3 days, and 8.3 at 14 days. In the present study the spermatozoa concentration was decreased and the spontaneous reaction rate was increased by diabetes. In ARIC-test, it is revealed that the fertility of spermatozoa of 14 days group was lower than control or 3 days group. Diabetes mellitus may be provoke the decreased fertilization rate and subsequent infertility.
Acrosome Reaction* ; Acrosome* ; Animals ; Diabetes Mellitus ; Epididymis ; Fertility ; Fertilization ; Glucose ; Humans ; Infertility ; Insulin ; Male ; Rats* ; Semen ; Spermatozoa* ; Streptozocin ; Vas Deferens

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