• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.163-168
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• 2005

The aim of this study was to investigate the changes in insulin-like growth factor-I (IGF-I) and growth hormone (GH) in serum, the quantitation of spermato-genesis and the comparable relationships among these measurements during pubertal period in New Zealand White male rabbits. To investigate the age-related testicular changes in DNA contents of spermatogenic cells, the fine-needle testicular biopsies from males aged 10 to 28 wks were evaluated by flow cytometry(FCM). Body weight increased significantly between the ages of 12 and 20 wks (P<0.05) and reached 3.4 kg at 28 wks of age. The highest serum IGF-I level (451.3ng/mL) was observed at 20wks of age (P<0.05) and thereafter remained stable at low levels. Serum GH level at 18 wks of age was 183.3 pg/mL which was significantly higher compared to the other ages (P<0.05), and the rising time in serum GH tend to be somewhat earlier than that of IGF-I. The relative percentage of It-cells in testicular cell compartments was $48.2\%$ at the age of 18 wks which significantly increased than those of 16-wk-old (P<0.05) and thereafter increased with the advance of age to $68\%$. The percentage of 2C-cells in testis was $26.8\%$ at 18 wks of age which was significantly lower than $54.3\%$ at 16 wks old (P<0.05). The percentage of 4C-cells was constantly maintained $2\~6\%$ except the $9.9\%$ at 18 wks of age. In conclusion, the results suggest that the puberty onset occurred at about the 18 wks of age and that the IGF-I and GH in serum during the pubertal period showed the age/growth-specific changes and these changes might be related to the spermatogenesis. The DNA FCM combined with fine-needle testicular biopsy could offer a very sensitive method to monitor the quantitative spermatogenic events related to the puberty onset.

• Development and Reproduction
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• v.2 no.2
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• pp.213-221
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• 1998

Intracytoplasmic sperm injection (ICSI) has been widely used to treat couples with infertility due to severely impaired sperm charateristics and for whom conventional in-vitro fertilization (IVF) had failed. The extent to which the morphology of the oocyte at the light microscopy level is related to the results of ICSI vis controversial. In this study, oocytes from 44 patients were reviewed. The ICSI procedure was recorded through CCD camera. The oocytes were divided into five groups according to the presence of cytoplasmic inclusions, the width of perivitelline space (PVS), the presence of cell debris in PVS, the status of first polar body and the flexibility of oolemma. The results showed that the fertilization rate and embryonic development were not associated with the morphological criteria of oocyte. The degeneraton rate of oocytes after ICSI was significantly higher (P<0.001) in the oocytes whose membranes were broken at the moment of insertion (17.7%) than the oocytes whose membranes were broken by aspiration of cytoplasm (1.6%). More oocytes with cytoplasmic inclusions (48.4% vs. 25.1%, p<0.001), wide PVS (35.2% vs. 19.0%, p<0.001), or cell debris in PVS (53.3% vs. 38.4%, p<0.05) were observed in patients with female factor infertility compared to patients with male factor infertility. These results .suggest that the fertilization rate and embryonic development after ICSI are not correlated with oocyte morphology based on the presence of cytoplasmic inclusions, size of PVS, the presence of cell debris in PVS and the status of polar body. And the degeneration rate of oocytes after ICSI was associated with the flexibility of oolemma.

• Development and Reproduction
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• v.4 no.1
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• pp.115-123
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• 2000

A few enzymeimmunoassay (EfA) for testosterone (T) have been reported but was not suitable for all biological samples. The present study was designed to develop a rapid, ultrasensitive EIA and to apply this technique for study the physiological changes of T in biological samples. Saliva samples were collected at 06:00～09:00 hour during one menstrual cycle from 18 normally menstruating women and on 09:00～10:00 hour from 20 normal men. The present study shows an established EIA for testosterone, using horseradish peroxidase (HRP), which was covalently bonded to testosterone-3-carboxymethyloxime (T-3-CMO). One batch of T-antisera was also covalently linked to microcrystalline cellulose particles by a mixed anhydride method in order to facilitate separation of bound and free steroids. The established EIA was validated in terms of sensitivity, accuracy, specificity, precisions etc., comparing with conventional radioimmunoassay. The sensitivity of the established EIA was less than 25 pg/tube. The correlation coefficients between the expected T-values and observed T-values measured by EIA or RIA were r=0.985 and r=0.941 respectively. The cross reactivity of antiserum in EIA was a little higher than that of RIA, especially by 5 ${\alpha}$-DHT. The intra- and inter-assay precisions of the present EIA were similar to those of RIA. The present study also demonstrates that the normal T-values in saliva of Korean male ＆ female samples are 265.65${\pm}$15.80 pmol／l and 109.74${\pm}$ 12.01 pmol／l, respectively. The present EIA seems to be established and suitable for use in the endocrinological studies. The advantages of this EIA system also might make the present T-EIA an ideal procedure for use in a routine assay of ordinary laboratory with a conventional spectrophotometer.

• Development and Reproduction
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• v.12 no.1
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• pp.97-105
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• 2008

Vinclozolin (VCZ) is a systemic fungicide commonly used in fruits, vegetables and the wine industry. VCZ and its metabolites, butenoic acid (M1) and enanilide (M2) derivatives, act as anti-androgens through actions on the androgen receptor. Although there is growing body of evidence that VCZ's action as an endocrine disrupting chemical (EDC) in male reproductive physiology and pathphysiology, no evidence on the VCZ's EDC action in female is available yet. Previously we found that the prepubertal VCZ exposures could effectively delay the onset of puberty in female rats, suggesting the postponed or weakened activities of hypothalamus-pituitary-ovary (H-P-O) reproductive hormonal axis. The present study was performed to examine whether the VCZ administration affects the transcriptional activities of reproductive hormone-related genes in the same animal model. VCZ (10 mg/kg/day) was administered daily from postnatal day 21 (PND 21) through the day when the first vaginal opening (V.O.) was observed. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus and pituitary, total RNAs were extracted and applied to the semiquantitative reverse transcription polymerase chain reaction (RT-PCR). As a result, treatment with VCZ significantly lowered the transcriptional activity of nitric oxide synthase-2 (NOS-2) which is known to adjust gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus (p<0.01). Similarly, the mRNA levels of KiSS-1, G protein-coupled receptor 54 (GPR54) and GnRH were significantly decreased in hypothalamus (p<0.01) from VCZ-treated group. As expected, the transcriptional activities of luteinizing hormone-${\beta}$ (LH-${\beta}$) and follicle stimulating hormone-${\beta}$ (FSH-${\beta}$) in the anterior pituitary from VCZ-treated group were also significantly lower than those from the control group. The present study indicates that(i) the inhibitory effect of VCZ exposure on the onset of puberty in immature female rats could be derived from the reduced transcriptional activities of gonadotropin subunits and their upstream modulators such as GnRH and KiSS-1 in hypothalamus-pituitary neuroendocrine axis, and (ii) these inhibitory effects could be mediated by NO signaling pathway.

• Development and Reproduction
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• v.12 no.1
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• pp.57-66
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• 2008

Changes in luteinizing hormone (LH), serum testosterone (T), and salivary T levels with age were examined in Korean men. Serum was obtained from 167 Korean men of different ages ($20{\sim}69\;y$), and the serum LH and T levels were measured. Saliva samples were also obtained, and the salivary T level was determined. The LH levels did not change considerably until 40 y of age (20s, $2.5{\pm}1.0$; 30s, $2.7{\pm}1.5$; and 40s, $2.5{\pm}1.8\;mIU/mL$) but increased significantly around 50 y (50s, $3.7{\pm}1.8$ and 60s, $3.1{\pm}1.7\;mIU/mL$). Further, the serum T levels also did not change until 40 y of age (20s, $5.3{\pm}2.6$, 30s, $4.4{\pm}1.4$, 40s, $4.1{\pm}1.5\;ng/mL$) but decreased significantly at 50 y (50s, $3.4{\pm}1.5$; 60s, $2.6{\pm}0.8\;ng/mL$). The salivary T levels also showed small changes until the age of 40 y ($20s{\sim}40s$, $0.11{\pm}0.015\;ng/mL$) but decreased significantly at 50 y ($0.08{\pm}0.03\;ng/mL$). Thus, the relative ratio of salivary T to serum T levels did not change significantly in all the ages examined ($2.4{\pm}0.9%$). Linear regression analysis predicted that the LH levels increased 1.5%/y while the serum and salivary T levels decreased 1%/y and 0.8%/y, respectively. The serum T/LH ratio did not change considerably until the age of 40 y ($20s{\sim}40s$, $2.27{\pm}0.14$) but decreased significantly ($1.2{\pm}1.0$) at 50 y. Age-related changes in the salivary T/LH ratio were very similar to those in the serum T/LH ratio. These results demonstrated that LH and T levels in serum or saliva did not change considerably until 40 y of age; instead, in Korean men, from 50 y of age, the LH level increased, while the T level decreased. This suggests that primary testicular failure that occurred due to aging (approximately 50 y) and caused this phenomenon. The present study also shows that the salivary T level can be an indicator of the free T level in serum although the salivary T level correlates weakly with the total T level in serum (r=0.53). Thus, information regarding salivary T levels may be useful for studying the age-related changes occurring in male testicular physiology.

• Development and Reproduction
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• v.13 no.2
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• pp.105-114
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• 2009

Ethane 1,2-dimethane sulfonate (EDS) is a well-known alkylating agent used as selective Leydig cell (LC) toxicant to create a testicular dysfunction model. Previous studies including our own clearly demonstrated the dramatic weight loss of the androgen dependent accessory sex organs such as epididymis, seminal vesicle and prostate gland in this 'LC knock-out' rats. The present study was performed to evaluate the effect of EDS administration on histological changes of the epididymis, seminal vesicle and prostate in adult rats. Adult male Sprague-Dawley rats (350$\sim$400 g B.W.) were injected with a single dose of EDS (75 mg/kg, i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. Tissue weights (testis, epididymis, seminal vesicle and prostate gland) were measured. The histological changes of tissue were observed by a light microscopy using hematoxylin & eosin staining. Weights of the reproductive and accessory organs progressively declined after the EDS treatments (weeks 1, 2 and 3). After this, the decrease was stopped, then gradually returned to the normal levels. There was a partial (about 60%) recovery of the epididymis weight during weeks $6{\sim}7$. The cross section of epididymis revealed an increase in thickness of the epithelium during weeks $1{\sim}3$. In contrast, considerable reduction of epithelial thickness in seminal vesicle was observed during same period. Similarly, a reduction in thickness of prostate epithelial layer was found during weeks $1{\sim}3$, then it was back to normal thickness after week 4. Taken together, the present study demonstrated that the temporally induced androgen-deficiency by EDS treatment could result the prominent alterations in histology of the accessory sex organs. Further studies on the physiological and molecular regulation of these androgen-sensitive organs using EDS model will be helpful to understand the normal and pathological development and differentiation mechanism of these organs.

• Development and Reproduction
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• v.14 no.4
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• pp.297-306
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• 2010

Tributyltin (TBT), an endocrine disrupting chemical, has been reported to decrease testicular function by causing apoptosis in the testis, but this mechanism is not fully understood. Thus, in this study we examined whether TBT induces adipogenesis of the Leydig cells to find out the correlation between adipogenesis and apoptosis in the testis. Three week old SD male rats were orally administrated with sesame oil, 1 mg/kg of TBT, or 10 mg/kg of TBT daily for 1 week and weighed after administration. The testes obtained on day 8 were weighed and stained with BODIPY and TUNEL kit. Using total RNA extracted from the isolated Leydig cells, adipogenesis and apoptosis-related genes were analyzed by real-time PCR. The testicular weights of the rats treated with 10 mg/kg TBT were significantly decreased compared to those in the control rats treated with sesame oil. As a result of BODIPY staining, the number of Leydig cells stained with BODIPY was increased in the rats treated with 10 mg/kg TBT compared with the control rats. Similar to BODIPY staining results, the TUNEL assay showed that the apoptosis of Leydig cells was increased in TBT treated rats. The results of the gene expression analysis in the Leydig cells showed that the expression of adipogenesis-related genes (PPAR${\gamma}$, aP2, Perilipin, CD36) and apoptosis-related genes (TNFRSF1A, TNFSF10) was increased after TBT administration. The present study demonstrates that TBT induces the expression of adipogenesis-related and apoptosis-related genes in the Leydig cells leading to adipogenesis and apoptosis in the testes. These results suggest that the dysfunction of Leydig cells by TBT exposure may cause a loss in testicular function.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.203-207
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• 2004

The transgenic mice carrying human Interleukin-10 (hIL-10) gene in conjunction with bovine (3 -casein promoter express hIL-10 in milk during lactation. In this study, stability of germ line transmission and expression of hIL-10 transgene integrated into host chromosome were monitored up to generation F8 of transgenic mice. When male mouse of generation F8 was crossbred with normal females, approximately half of offspring (50.9±5.8%) were identified as transgenic mice. Generation F9 to F15 mice also showed similar transmission rates (66.0±20.1%, 61.5±16.7%, 41.1±8.4%, 40.7±20.3%, 61.3±10.8%, 49.2±18.8% and 43.8±25.9%, respectively), implying that hIL-10 transgene can be transmitted stably up to long term generation in the transgenic mice. Expression levels of human IL-10 from milk of generation F9 to F14 mice were 3.6± 1.2 mg/ml, 4.2±0.9 mg/ml, 5.7±1.5 mg/ml, 6.3±3.5 mg/ml, 6.8±4.5 mg/ml and 6.8±3.1 mg/ml, respectively, which was showed high-level expression compared with that of generation F1 (1.6 mg/ml) mice. In conclusion, our results suggest that transgenic mice can be continuously passed their transgenes to the progeny through the breeding program with the same productivity of human IL-10 protein in their milk.

• Journal of Sericultural and Entomological Science
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• v.15 no.2
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• pp.15-20
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• 1973

This will be second publication regard with silkworm selection using anesthetic followed by previous report. This treatise investigated for nacorsis recovery concerned with silkworm health through more directions than the previous paper. After the author has found a possibility of silkworm seperation through nacorsis recovery, he has recommended to use Balance Health (BH) value system to evaluate or identify silkworm health and to produce better homogeneous silkworm eggs. Also, the author has continued the observation for the next generation how once nacortized parents affect to their family. The obtained results are summarized as followings. 1. Basic investigation a) Hybrid silkworm variety recovers from nacorsis earlyer than pure line because it is stronger. b) Strong pure line variety recovers from nacorsis earlyer than weak one because it is stronger. c) Male worm recovers from nacorsis earlyer than female worm because it is stronger. 2. Establishment of Balance Health(BH) value system. In order to identify each seperated group by nacorsis and their family, the following Balance Health (BH) value indicating system was established.(Table Omitted) 3. In order to select better silkworm during breeding service, the BH value indicating system is strongly recommended. That is, in case BH value is smaller than four, it would be strong group or in case BH value is larger than four, it would be weak group. 4. Once nacortized parents do not harm any nature for next generation regardless P$_1$ or F$_1$ production. 5. The seperation through nacorsis may delay retrograde phenomenon some years than other case, so, this system should be used for pure line production every year. 6. It is recommended to nacortize silkworm for five minutes on breeding purpose, but two and half minutes on pure lines reproduction at fifth instar. 7. This report has been discussed in relation to practical industry promotion aspects, but there are many subjects to be solved from academical aspect in future