• Development and Reproduction
• /
• v.15 no.2
• /
• pp.159-166
• /
• 2011

Simazine (6-chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine) is a triazine herbicide that has been applied worldwide including Korea for agricultural purposes. Simazine is the second most commonly detected pesticide in surfaceand ground-water in the United States, Europe and Australia. It has been shown that simazine is a potent endocrine disruptor in wildlife and laboratory animals. Although many endocrine disruptors can induce apoptosis in various types of cells, the effects of simazine on apoptosis and on the expression of Bcl-2 family genes are not known. Also it is unknown the effect of simazine on the expression of steroidogenesis-regulating genes in testicular cells. In this study, we investigated the effect of simazine on the expression levels of apoptosis- and steroidogenesis-regulating genes in testicular cells. We found that a low concentration of simazine can alter the mRNA expression levels of steroidogenesis-related genes and Bcl-2 family genes in mouse Sertoli cells and rat Leydig cells. Thus, our results suggest that simazine can disturb normal testicular development and reproductive function by altering the expression of genes that are critical for the regulation of apoptosis and steroidogenesis.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.2
• /
• pp.225-230
• /
• 1999

In mouse, the heat shock protein 70-2 (hsp70-2) is found to have special function in spermatogenesis. Based on the observation, the hypothesis that human hspA2 (human gene; 98.2% amino acid homology with hsp70-2) might have important function in spermatogenesis in human testes was proposed. To test the hypothesis, we examined the expression of hspA2 in human tissues. Expression vector pDMC4 for expression of the human hspA2 protein using pTricHisB (invitrogen, USA) was constructed and the expressed hspA2 protein was cross-reacted with antiserum 2A raised against mouse hsp70-2 protein. Based on the cross-reactivity, we determined the expression level of hspA2 protein in human tissues by western blot analysis using the antiserum 2A. We demonstrated that antiserum 2A antibodies detected human hspA2 protein with specificity which was produced in the E.coli expression system. On Western blot analyses, significant hspA2 expression was observed in testes with normal spermatogenesis, whereas a low level of hspA2 was expressed in testis with Sertoli-cell only syndrome. Also, a small amount of hspA2 was detected in breast, stomach, prostate, colon, liver, ovary, and epididymis. These results demonstrate that the hspA2 protein is highly expressed in male specific germ cells, which in turn suggests that hspA2 protein might playa specific role during meiosis in human testes as suggested in the murine model. However, further studies should be attempted to determine the function of hspA2 protein in human spermatogenesis.

• Reproductive and Developmental Biology
• /
• v.33 no.3
• /
• pp.171-177
• /
• 2009

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from $10{\sim}14$-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and $O_2$ tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, $37^{\circ}C$ temperature, and $O_2$ 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

• Development and Reproduction
• /
• v.4 no.2
• /
• pp.215-220
• /
• 2000

Most of endocrine disrupters (EDs) have been reported to exhibit estrogenic or anti-androgenic activity and thereby may disrupt reproductive development in human or wildlife. This study was performed to investigate the effects of estrogen (E$_2$), bisphenol (BP) and octylphenol (OP) on the mouse Leydig cell line (TM3). TM3 originated from testis of 11~13-daly-old BALB/c nu/＋ mice was cultured in DMEM supplemented with 10％ FBS alone or medium with estrogen (E$_2$), bisphenol (BP) and octylphenol (OP; 1 pM, 1 nM, 1 $\mu$M, 1 mM, respectively) for 48 hours. After culture, total cell number and viability were assessed by heamocyto-meter and trypan blue stain. Expression of cytochrome P450scc (CYPscc) mRNA whose product is involved in steroid hormone biosynthesis and estrogen receptor $\alpha$(ER $\alpha$) mRNA were detected by RT-PCR. As a result, treatment of TM3 with E$_2$, BP and OP(1 mM, respectively) significantly decreased the viability but not all of groups as high as 1 $\mu$M. Exposure of TM3 to OP significantly reduced the total cell number but not E$_2$ or BP. The expression of CYPscc mRNA was slightly reduced in BP (1 nM, 1 $\mu$M) and significantly decreased in OP (1 nM, 1 $\mu$M) treated TM3, except E$_2$ group. But the expression of ER $\alpha$ mRNA was sightly increased in all treated groups. In conclusion, BP and OP (high concentration) might inhibit steroidogenesis by decreasing the CYPscc mRNA expression in the mouse testis. These results suggest that BP and OP might impair spermatogenesis and subsequently disturb testicular function.

• Clinical and Experimental Reproductive Medicine
• /
• v.34 no.4
• /
• pp.293-303
• /
• 2007

Objective: Previously, we identified differentially expressed genes between GV and MII stage mouse oocytes using ACP technology. When we study one of GV selective genes, Obox family, we found Obox4 mRNA expression in ovaries that has been reported as expressed exclusively in testis. Therefore, this study was conducted for characterization and functional analysis for Obox4. Methods: Expression of Obox4 mRNA was examined in gonads and oocytes by RT-PCR. To determine the role of Obox4 in oocyte maturation, Obox4 dsRNA was microinjected into the cytoplasm of GV oocytes followed by 16 h of incubation in the plain medium or by 24 h of incubation in the medium containing IBMX. After RNAi, phenotypes and maturation rates were observed, change in mRNA expression was evaluated, and chromosomal status was confirmed by orcein staining. Results: Obox4 has minimal expression in the ovary compared to that of the other family members. When oocytes were cultured for 16 h in M16 medium after RNAi, maturation rate was not changed significantly, compared with that of non-injected or buffer-injected control oocytes. Surprisingly, however, when oocytes were cultured for 24 h in M16 containing IBMX, in which oocytes were supposed to arrest at GV stage, Obox4 RNAi oocytes were advanced to MI and MII. Spindle structure was disappeared and the chromosomes were condensed in the oocytes after Obox4 RNAi. Conclusions: This is the first report on the expression of Obox4 in the ovary and oocytes. Results of the study suggest that Obox4 plays a crucial role in spindle formation and chromosome segregation during meiosis in oocytes. In addition, Obox4 may play an important role in cAMP-dependent signal cascades of GV-arrest in mouse oocytes.

• Journal of Animal Science and Technology
• /
• v.46 no.6
• /
• pp.957-966
• /
• 2004

The objective of this study was to investigate the effects of BPA on reproductive characteristic and blood hematological and chemical values in mice. The male mice were intraperitoneally injected BPA in native control(no treatment), positive control(corn oil, 3$m\ell$/kg B.W), 0.05, 0.5 and 5.0mg BPA/kg B.W and female mice were injected BPA in control(corn oil, 3$m\ell$/kg B.W), 0.05, 0.5 and 5.0mg BPA/kg B.W with 5 times at 3 days interval for 14 days. The administration of BPA in male mice didn't affect the body and reproductive organ weight such as testis, epididymis, vesicular gland and coagulating gland. We found that the 5.0mg BPA group was significantly reduced the sperm concentration and increased the sperm abnormality compared to native, positive control and 0.05mg BPA groups(P<0.05), but any other effects were not found in sperm viability and motility in BPA treatment groups. The RBC, HB, HT, MCV, MCH, MCHC, albumin, BUN and total protein of blood hematological and chemical values in male were not different in all experimental groups(P>0.05). However, the value of WBC was slightly lower in BPA treatment groups than that of control groups and PLT was slightly higher in BPA groups than that of control groups, but not significantly defference among the experimental groups(P>0.05). In female mice, the effects of BPA on body weight didn’t affect in all experimental groups, but ovary weights in 0.5 and 5.0mg BPA groups were significantly increased compared to those in control and 0.05mg BPA group(P<0.05). The uterine weight in BPA groups was slightly higher than that of control group, but not significantly different in all experimental groups(P>0.05). The RBC, Hb, HT, MCV, MCH, MCHC, albumin and total protein of blood hematological and chemical values in female were not different in all experimental groups(P>0.05). The values of WBC and PLT in BPA groups were slightly higher than that of control, but not significantly different among the experimental groups. The concentration of BUN was the higher in BPA groups than that of control group. The histological evaluation of testis, ovary and uterus were not different in all experimental groups.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.23 no.3
• /
• pp.502-508
• /
• 1994

A total of 24 specimens of the pufferfish, Fugu xanthopterus, purchased at a fixhmarket in Pusan, korea were examined for toxicity using the assay method of tetrodotoxin (TTX). Also, the toxins isolated from the puffer liver were partially purified and analyzed for their chemical composition by instrumental behaviors. On the whole, when the level of toxicity in each organ was analyzed compared to that of liver, they were 100 % of the lover, 92 % for the intestine, 75% for the skin, 17% for the muscle, 785 for the testis, 87% for the ovary, and 71% for bile. The highest and average scores of toxicity for the liver were 917 and $231{\pm}51MU/g$ liver, respectively. The toxins of the puffer gave four peaks in HPLC whose retention times (10, 20, 22 and 25 min) were close to those of TDA, TTX, 4-epi-TTX, and and -TTX, respectively.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.9
• /
• pp.1319-1326
• /
• 2007

Fructose-1,6-bisphosphatase (FBP1) is a key regulatory enzyme of gluconeogenesis that catalyzes the hydrolysis of fructose-1,6-bisphosphate to generate fructose-6-phosphate and inorganic phosphate. Deficiency of fructose-1, 6-bisphosphatase is associated with fasting hypoglycemia and metabolic acidosis. The enzyme has been shown to occur in bacteria, fungi, plants and animals. The bovine FBP1 gene was cloned and characterized in this study. The full length (1,241 bp) FBP1 mRNA contained an open reading frame (ORF) encoding a protein of 338 amino acids, a 63 bp 5' untranslated region (UTR) and a 131 bp 3' UTR. The bovine FBP1 gene was 89%, 85%, 82%, 82% and 74% identical to the orthologs of pig, human, mouse, rat and zebra fish at mRNA level, and 97%, 96%, 94%, 93% and 91% identical at the protein level, respectively. This gene was broadly expressed in cattle with the highest level in testis, and the lowest level in heart. An intronic single nucleotide polymorphism (SNP) (A/G) was identified in the $5^{th}$ intron of the bovine FBP1 gene. Genotyping of 133 animals from four beef breeds revealed that the average frequency for allele A (A-base) was 0.7897 (0.7069-0.9107), while 0.2103 (0.0893-0.2931) for allele B (G-base). Our preliminary association study indicated that this SNP is significantly associated with traits of Average Daily Feed Intake (ADFI) and Carcass Length (CL) (p<0.01). In addition, the FBP1 gene was assigned on BTA8 by a hybrid radiation (RH) mapping method.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.28 no.1
• /
• pp.31-34
• /
• 1995

Ten specimens (5 males and 5 females) of the pufferfish, fugu stictonotus ('gachilbog'), were collected at a fish market of Pusan, Korea in July 1993, and examined for anatomical distribution of toxicity by mouse assay method. The frequency of toxic specimens was $40\%\;for\;liver,\;60\%$ for ovary, $40\%\;for\;skin\;and\;60\%$ for bile in female puffers. The highest toxicities were 107, 107, 29 and 93MU/g for liver, ovary, skin and bile, respectively; and average toxicity $\pm S.E.\;values\;were\;14\pm11,\;48\pm22.4\pm3\;and\;12\pm9MU/g,$ respectively. The range of total toxicity was shown to be from 0 to 35,316MU. The characteristic pattern of toxin distribution observed on these specimens was exhibited; both muscle and testis were non-toxic, but others were weakly toxic. Also, there was significant difference for toxicity between male and female specimens.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.38 no.12
• /
• pp.1724-1731
• /
• 2009

This study was aimed to investigate the effect of phyto-extract fermented mixture (MP119) on the male sexual functions. The MP119 was evaluated for anti-impotency and anti-hypertensive effects via ACE (angiotensin converting enzyme) or PDE (phosphodiesterase) inhibition assay. $IC_{50}$ values of MP119 against ACE and PDE were 241.3${\pm}$35.5 ppm and 372.2${\pm}$33.8 ppm, respectively. To investigate the effect of testosterone expression by MP119, we performed cell media test using mouse Leydig-derived TM3 cells. Production of testosterone in TM3 cell was increased by MP119. Also, NO (nitric oxide) production of HUVEC (human umbilical vein endothelial cell) was increased when MP119 was added to the cultures. Forty male ICR mice were divided into 4 groups. MP119 was orally intubated for 7 days to group 1 and 3, and same volume of vehicle to group 2 and 4 as controls. After that, group 3 and 4 were intraperitoneally injected cadmium chloride at a single dose of 2 mg/kg. On the 8th experimental day, weights of testis, epididymis and seminal vesicle, number of sperm, concentrations of serum testosterone and cGMP were determined. The number of sperm, the concentrations of testosterone and cGMP were significantly increased in two experimental groups (group 1, 3). These results suggest that MP119 enhanced the sexual function of male mice, and could protect the sexual organs from the cadmium chloride as one of the endocrine disrupters.