• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.95-104
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• 1997

The localization of worm antigen of Neodiplostomum seoulere was examined by immuno-electronmicroscopic observation. Not only the immunized serum of mice with crude worm extract of N. seoulene but also serum of infected mouse were reacted to the worm section. Using immunized serum as primary antibody. the gold particles were deposited on the rough endoplasmic reticulum of the cell of tribocytic organ, spermatozoa in the seminal vesicle, microvilli of the caecum and vitelline follicle. Using infected serum, gold particles were deposited only on the vitelline follicle prominently. This finding suggested that the tribocytic organ, seminal vesicle, caeca and vitelline follicles may play a role of antigen to immunized serum with crude worm extract of N. seoulense, whereas the vitelline follicle, to the infected serum.

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.188-194
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• 1989

These experiments were carried out to investigate fertilizable and developmental ability after zona drilling the unfertilized eggs and the eggs not fertilized by the 1st insemination. The results of in vitro fertilization of the mouse eggs treated by using micromanipulation and acid tyrode's solution with capacitated epididymal spermatozoa were as follows. In the case of ovulated unfertilized eggs, according to sperm count(106, 105, 104 and 103/ml) the rates of in vitro fertlilization treated by zona drilling were 86.0%, 82.0%, 70.0% and 54.0%, respectively, and those of control were 58.0%, 52.0%, 12.0% and 8.0%, respectively. The rates of in vitro fertilization of zona drilled eggs were significantly high compared with those of control, and there were no significant difference between two groups. According to the sperm count the zona drilled eggs developed to the blastocysts were 51.4%, 40.5%, 23.3% and 17.4% and those of control were 35.7%, 26.3%, 0% and 0%, respectively. Also, in the eggs not fertilized by 1st insemination, the fertilization rates of oocytes reinseminated after zona drilling was significantly higher(83.5%) than that of control(34.7%), and the rates of polyspermy were similar. The rates of development to the blastocysts was 18.6% in the zona drilling treated eggs, and that of control was 27.3%, there was no significant difference between two groups. These results indicated that oocytes not fertilized by 1st insemination as well as ovulated unfertilized eggs could be fertilized, improve fertilizing rates by zona drilling treatment, and development potential were normal.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.213-218
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• 2015

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.201-207
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• 2002

Objective: To observe the capability of fertilization and embryo development including blastocyst formation of the oocytes in simple media after thawing of the cryopreserved cumulus-free mouse oocytes by vitrification method. Methods: Oocytes were collected from 5 to 6 weeks old ICR female mice, and were denuded from the cumulus cells by 0.1% hyaluronidase. Recovered mature oocytes in study group were cryopreserved by vitrification method using EM grid for $5{\sim}7$ days. In brief, oocytes were exposed in dPBS containing 1.5 M EG and 5.5 M EG+1 M sucrose for 2.5 minutes and 20 seconds each, and then executed vitrification by plunging in LN2 after loading on EM grid. Thawing treated by exposure of 1, 0.5, 0.25 and 0.125 M sucrose solution for 2.5 minutes each in order and used for experiments. Spermatozoa aspirated form the epididymis of 12 weeks old ICR male mice were used for insemination after capacitation. T6 media containing 0.4% BSA were used for fertilization and development. Results: Survival and fertilization rates after thawing were 76.9% and 79.6% respectively. Fertilization rate was lower (p<0.005) than that of control group (92.9%). There was no difference in embryo developmental rates from 2-cell to morula, however, the blastocyst formation rate and mean cell numbers of blastocysts in study group (63.3%, $58.9{\pm}9.2$) were lower compared with those of control group (76.1%, $63.5{\pm}8.9$). Conclusion: Vitrification is an effective method for mouse mature oocyte cryopreservation with high survival and fertilization rate after thawing. And in simple media, fertilization rates and embryo development of frozen-thawed mouse oocytes are satisfactory.

• Environmental Analysis Health and Toxicology
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• v.19 no.3
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• pp.261-269
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• 2004

Although the biological functions of metallothioneins (MTs) are still being investigated, they have been suggested to be involved in detoxification of heavy metals, scavenging of free radicals, and protection against alkylating agents. MTs have been reported to be induced in most of animal tissues by heavy metals such as zinc, copper, mercury and cadmium, and the proteins have binding affinities to the metals. However, the presence or induction of MTs was reported not to be clear in leydig cells, which produce testosterone for the maturation of spermatozoa in male testes. In this study, we investigated the inducibility of metallothionein isomers by cadmium in cultured mouse leydig cells. Total RNA was extracted from the near confluent grown leydig cells and RT-PCR was Performed using the Primers which were synthesized on the basis of MT-1, 2, 3 and 4 cDNA from GenBank database. As results, MT-1 and MT-2 mRNA were found to be expressed in cadmium non-treated control cells and MT 1 mRNA expression was dose-dependent when leydig cells were treated with cadmium chloride. But MT-3 which is known to be brain specific and MT-4 which is another isoform of metallothionein, were not expressed. Other genes induced or depressed in cadmium treated leydig cells were also identified by microarray techniques.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.86-93
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• 2004

Nitric oxide (NO) has emerged as an important intracellular and intercellular messenger, controlling many physiological processes and participating in the fertilization process via the autocrine and paracrine mechanisms. This study investigated whether nitric oxide synthase (NOS) inhibitior (L-NAME) and L-arginine could regulate in vitro fertilization and early embryonic development in mice. Mouse epididymal spermatozoa, oocytes, and embryos were incubated in mediums of variable conditions with and without L-NAME or L-arginine (0.5, 1, 5 and 10mM). Fertilization rate and early embryonic development were significantly inhibited by treating sperms or oocytes with L-NAME (93.8% vs 66.3%,92.1% vs 60.3%), but not with L-arginine. In contrast, fertilization rate and early embryonic development were conspicuously reduced when L-NAME or L-arginine was added to the culture media for embryos. Early embryonic development was inhibited by microinjection of L-NAME into the fertilized embryosin a dose-dependent manner, but only by high concentrations of L-arginine. These results suggest that a moderate amount of NO production is essential for fertilization and early embryo development in mice.

• Development and Reproduction
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• v.4 no.1
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• pp.53-59
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• 2000

Endocrine disruptors (EDs) are exogenous chemicals which interfere several aspects of natural hormone properties. EDs with estrogenic activity have been recently reported to cause animal reproductive problems. This study was performed to investigate the effects of bisphenol A (BPA) on the mouse spermatogenesis in vivo. Male ICR mice were orally injected on a daily basis with low dose of BPA 20 mg/kg, high dose of BPA 200 mg/kg, or corn oil (vehicle control) for 7 days, and litter size and weights of body, testis, and cauda epididymis were measured. The level of serum testosterone and the expression of TGF- $\beta$$_1$ mRNA were also analyzed using RIA and RT-PCR, respectively. Also, morphological differences of testes after treatments were examined. Sperm concentration and level of serum testosterone showed a decreasing tendency detected as untreated >corn oil >low >high dose BPA treated mice, although there were no significant statistical differences. Interestingly, in mice treated with a high dose of BPA, partial disappearance of spermatozoa in seminiferous tubular lumen and the expression of TGF-$\beta$$_1$ mRNA were observed. Spermatogenesis was disrupted through TGF-$\beta$ system in the seminiferous tubules, resulting in no development of germ cells. Similarly, the litter size treated with a high dose of BPA was significantly different from that of untreated control group. In conclusion, these results that a high dose of BPA (200 mg/kg) acts as an endocrine disruptor during apermatogenesis in male mice md that there are BPA-specific lesions in the adult male reproductive tract might represent a permanently altered responsiveness to testosterone by BPA in the affected target tissue.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.41-49
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• 1996

본 연구는 체외수정에 의해 생산된 생쥐 배반포기배를 vitrification 방법으로 동결보존하였을때 높은 생존율을 얻기 위한 적정조건을 검토하고자 실시하였다. 배반포기배를 생산하기 위하여, B6CBA F1 (C57BL/6, (표현불가)${\times}CBA/N$, (표현불가)) 계통의 생쥐 미수정란에 $1{\times}10^6$ spermatozoa/ml 농도의 정자로서 수정을 유도하였으며, 이후 $37^{\circ}C$, 5% $CO_2$배양기내에서 96시간동안 체외배양하였다. 배양 4일째의 배반포기배는 발달상태에 따라 early, middle 그리고 hatching blastocysts로 구분하였다. 본 실험에 사용된 동결보존액은 30% Ficoll과 0.5mol의 sucrose가 첨가된 mDPBS 용액에 40%의 ethylene glycol를 첨가한 EFS 40 (Zhu et al., 1993) 이었고, 수정란은 $25^{\circ}C$의 상온에서 먼저 20% ethylene glycol에 노출된 후 EFS 40 용액으로 옮겨 액체질소에 침지하는 2단계 동결법에 의해 동결보존되었으며, 급속융해하여 다음과 같은 결과를 얻었다. 1. 체외수정율과 배양 4일째 배반포기까지의 배발달율은 각각 89.4%와 86.1%였다. 2. 20% ethylene glycol에서 5분간 평형된 후 EFS 40 용액에 냉동보존된 후 융해된 난자의 생존율은 20% ethylene glycol에 0, 1, 3분간 평형된 난자의 생존율에 비해 유의하게 높았다. 3. 배반포기배를 20% ethylene glycol에서 5분간, EFS 40 용액에 1분간 차례로 노출한 다음 체와배양하였던 바, 배양 24시간째 생존율은 $82.9%{\sim}88.4%$ 였다. 본 연구 결과, 체외수정, 배양된 생쥐 배반포기배는 20% ethylene glycol과 EFS40에 대한 노출만으로는 난자의 생존성에 나쁜 영향을 미치지 않는 것으로 미루어 보아 배반포기배의 초자화 동결이 가능함을 시사하였다. 따라서 동결 융해 후 높은 생존율은 상온에서 난자를 2단계 즉, 20% ethylene glycol에 5분간 평형시킨 후 EFS 40 용액에 노출하여 1분내에 LN2에 직접 침지하는 간편한 동결방법으로 얻을 수 있었다.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.361-368
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• 2002

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilization. After 44h in vitro maturation, spermatozoa was injected into the cytoplasm of oocytes. After injection, all oocytes were transferred to NCSU23 medium and cultured at 39'E under 5% CO2 in air. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8 to 9 h following the injection of porcine sperm, and 6 to 8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte center. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. These results suggested that DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, and the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.1-6
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• 1996

There are a number of problems during the process of culture in vitro on fertilization and embryo development compared to those on in vivo counterparts. And the platelet activating factor (PAF), which is found not only in mammalian spermatozoa but also preembryos, is implicated on reproductive process. To improve the environment of culture on in vitro fertilization and embryo development, coculture using salpingeal epithelial cells has been considered to accept the better result on pregnancy rate. This study was designed to determine if two different culture systems, coculture alone and PAF treated coculture, are positive or negative influence on process of in vitro fertilization and embryo culture in mouse. The cell cleavage rate reached to 2-4 cell stage at 24 hours of culture is 56.81% (50/88) and 48.21%(54/112) respectively, in PAF treated group which is added PAF on coculture and in coculture group. But the rate of cells cleavage was similar in both group after 48 hours of culture. The rate of unfertilization after insemination of oocytes was higher in coculture group(55..53%) than in PAF treated group(42.37%). And in assessment of undeveped embryos, the rate of equalized cell block was similar on both, coculture alone (35.3%)and PAF treated coculture(35.5%). while unequalized cell block was higher rate in PAF treated coculture(19.4%) than coculture alone (11.8%). But the rate of cytoplasmic degeneration of undeveloped embryos was significantly higher in PAF treated coculture than coculture alone. In conclusion, we have observed that PAF treated coculture is superior in the rates of in vitro fertilization and early embryo cell cleavage compared to those in coculture alone, but there is no difference on the rates of embryo develpments, cell degeneration, cell quality in both PAF treated coculture and coculture alone when the embryo cells were continuosly cultured for 48 hours or more.