• Development and Reproduction
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• v.17 no.4
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• pp.469-475
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• 2013

In mammals, puberty is a process of acquiring reproductive competence, triggering by activation of hypothalamic kisspeptin (KiSS)-gonadotropin releasing hormone (GnRH) neuronal circuit. During peripubertal period, not only the external genitalia but the internal reproductive organs have to be matured in response to the hormonal signals from hypothalamic-pituitary-gonadal (H-P-G) axis. In the present study, we evaluated the maturation of male rat accessory sex organs during the peripubertal period using tissue weight measurement, histological analysis and RT-PCR assay. Male rats were sacrificed at 25, 30, 35, 40, 45, 50, and 70 postnatal days (PND). The rat accessory sex organs exhibited differential growth patterns compared to those of non-reproductive organs. The growth rate of the accessory sex organs were much higher than the those of non-reproductive organs. Also, the growth spurts occurred differentially even among the accessory sex organs; the order of prepubertal organ growth spurts is testis = epididymis > seminal vesicle = prostate. Histological study revealed that the presence of sperms in seminiferous tubules and epididymal ducts at day 50, indicating the puberty onset. The number of duct and the volume of duct in epididymis and prostate were inversely correlated during the experimental period. Our RT-PCR revealed that the levels of hypothalamic GnRH transcript were increased significantly on PND 40, suggesting the activation of hypothalamic GnRH pulse-generator before puberty onset. Studies on the peripubertal male accessory sex organs will provide useful references on the growth regulation mechanism which is differentially regulated during the period in androgen-sensitive organs. The detailed references will render easier development of endocrine disruption assay.

• Development and Reproduction
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• v.17 no.4
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• pp.435-440
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• 2013

Previous studies, including our own, have demonstrated that the intratesticular injection of hypertonic saline (20%) decreased serum testosterone level which was similar to the surgical castration in the rat, showing the state of chemical castration. In the present study, we further verify the efficacy of this less invasive method as an alternative of surgical orchidectomy in the andrological field. Sterilized 20% saline was directly injected into the adult male rats (750 ${\mu}l$ per testis). The tested rats were divided into 3 groups including intact group (intact), orchidectomy group (ORX) and saline injection group (SAL) after bilateral orchidectomy was performed at the same day of injection. All rats were sacrificed at 4 weeks after injection. The reproductive organs (testes, epididymis, seminal vesicles and prostates) were collected and used for DNA and protein pattern analyses. Also, patho-histological studies on the testes were performed. In contrast to the intact group, similar DNA damages of testis and seminal vesicle were appeared in ORX group and SAL group. The DNA degradations seemed to be the results of necrosis rather than apoptosis. In the protein pattern analysis, all the testing tissues exerted similar patterns in the ORX group and the SAL group compared to the those of intact group. Patho-histological studies revealed that severe degenerative changes in testicular seminiferous tubules and massive infiltration of immune cells in SAL group. The present study confirmed that direct injection of hypertonic saline into the testis caused the equivalent biochemical changes in the accessory sex organs as shown in the orchidectomized animals. These results suggest that hypertonic saline injection model could be a useful castration model which can substitute for surgical castration when its safety is secured through further study in the future.

• Development and Reproduction
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• v.15 no.3
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• pp.265-272
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• 2011

It is well known that adipose tissue or body fat has been proved as a crucial component of brain-peripheral axis which can modulate the activities of reproductive hormonal axis in female mammals including rodents and human. Concerning the male reproduction, however, the role of adipose tissue has not been thoroughly studied. The present study was carried out to elucidate the effect of a high-fat (HF) diet on the reproductive system of postpubertal male rats. The HF diet (45% energy from fat, HF group) was applied to male rats from week 8 after birth for 4 weeks. The blood glucose levels, body and tissue weights were measured. Histological studies were performed to assess the structural alterations in the reproductive tissues. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus and pituitary, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Body weights (p<0.01) and blood glucose levels (p<0.01) of HF group were significantly higher than those of control animals. Similarly, the weights of epididymis (p<0.05), prostate (p<0.01), seminal vesicle (p<0.01) in HF group were higher than control levels. The weights of testis were not changed. The weights of kidney (p<0.001) and spleen (p<0.01) were significantly higher than control levels while the adrenal and pancreas weights were not changed. There were only slight alterations in the microstructures of accessory sex organs; the shape of luminal epithelial cells in epididymis from HF group were relatively thicker and bigger than those from control animals. In the semi-quantitative RT-PCR studies, the mRNA levels of hypothalamic GnRH (p<0.05) in HF group were significantly higher than those from the control animals. The mRNA levels of kisspeptin in HF group tend to be higher than control levels, the difference was not significant. Unlike the hypothalamic GnRH expression, the mRNA levels of pituitary $LH{\beta}$ and $FSH{\beta}$ were significantly decreased in HF group (p<0.05). The present study indicated that the 4-weeks feeding HF diet during the postpubertal period can alter the hypothalamus-pituitary (H-P) neuroendocrine reproductive system These results suggest that the increased body fat and the altered leptin input might disturb the H-P reproductive hormonal activities in male rats, and the changed activities seem to be responsible for the changes of tissue weights in accessory sex organs.

• 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.

• Fisheries and Aquatic Sciences
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• v.24 no.10
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• pp.330-339
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• 2021

Oyster (Crassostrea gigas) is a marine bivalve mollusk widely distributed in coastal areas, and have been long widely used in industrial resources. Several studies demonstrated that fermented oyster (FO) extract attribute to bone health, but whether administration of FO play as an endocrine disruptor has not been studied. Therefore, in the present study, we investigated the effect of FO on the endocrine system in vitro and in vivo. As the results of the competitive estrogen receptor (ER) and androgen receptor (AR) binding affinities, FO was not combined with ER-α, ER-β, and AR. However, 17β-estradiol and testosterone, used as positive control, were interacted with ER and AR, respectively. Meanwhile, oral administration of 100 mg/kg and 200 mg/kg of FO doesn't have any harmful effect on the body weight, androgen-dependent sex accessory organs, estrogen-dependent-sex accessory organs, kidney, and liver in immature rats. In addition, FO supplementation has no effect on the serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and 17β-estradiol. However, the relative weight of androgen- and estrogen-dependent organs were significantly increased by subcutaneously injection of 4.0 mg/kg of testosterone propionate (TP) and by orally administration of 1.0 ㎍ of 17α-ethynyl estradiol (EE) in immature male and female rats, respectively. Furthermore, TP and EE administration markedly decreased the serum LH and FSH levels, which are similar those of mature Sprague-Dawley (SD) rat. Furthermore, the testosterone and 17β-estradiol levels were significantly enhanced in TP and EE-treated immature rats. Taken together, our findings showed that FO does not interact with ER and AR, suggesting consequentially FO does not play as a ligand for ER and AR. Furthermore, oral administration of FO did not act as an endocrine disruptor including androgenic activity, estrogenic activity, and abnormal levels of sex hormone, indicating FO may ensure the safety on endocrine system to develop dietary supplement for bone health.

• Toxicological Research
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• v.16 no.2
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• pp.163-172
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• 2000

Recently, there is a worldwide concern that a great number of man-made chemicals have a hormone-like action both in humans and in animals. DECD is developing screening programs using validated test systems to determine whether certain substances may have an effect in humans. In the present study. the establishment oj repeated-dose toxicity test method was tried. Flutamide. an anti-androgenic agent. was administered by gavage to Sprague-Dawley rats for 28 days at dose levels of 0. 0.5. 3 and 18 mg/kg body weight (10-15 rats/sex/group) to examine the effects on general findings. especially reproductive and endocrine parameters. Clinical signs. body weights, food consumption, and sexual cycle were checked and measured. For the gross and microscopic examinations. 10 rats/sex/group were sacrificed at the end of dosing period and the remaining animals of control and high dose groups (5 each) were sacrificed after 14 days recovery. Examinations for hematology and clinical chemistry were carried out at necropsy. There were no treatment-related changes in clinical signs. body weights, food consumption. gross necropsy. hematology and clinical chemistry at all doses of both sexes. The period and regularity of sexual cycle were not adversely affected at all doses by the test agent. At 18 mg/kg. both decreased weights of prostate, seminal vesicle and epididymis in males and increased weights of spleen and thymus in females were observed. In addition, decreased number of spermatids and sperms. increased serum testosterone concentration and increased incidence (100%) of interstitial cell hyperplasia were seen in males. At 18 mg/kg of the recovery group. decreased prostate weight. reduced sperm count and increased incidence (20%) of interstitial cell hyperplasia in males and increased thymus weight in females were observed. At 3 mg/kg. reduced sperm count was found. There were no adverse effects on parameters examined at 0.5 mg/kg of both sexes. The results suggested that the potential target organs of flutamide may be accessory sexual glands including testes for males and spleen and thymus for females. Taken together. this test method was found to be a useful screening test system for endocrine disrupting chemicals.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.157-162
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• 2013

Methoxychlor (MXC) was developed to be a replacement for the banned pesticide DDT. HPTE [2,2-bis (p-hydroxyphenyl)-1,1,1-trichloroethane], which is an in vivo metabolite of MXC, has strong oestrogenic and anti-androgenic effects. MXC and HPTE are thought to produce potentially adverse effects by acting through oestrogen and androgen receptors. Of the two, HPTE binds to sex-steroid receptors with greater affinity, and it inhibits testosterone biosynthesis in Leydig cells by inhibiting cholesterol side-chain cleavage enzyme activity and cholesterol utilisation. In a previous study, MXC was shown to induce Leydig cell apoptosis by decreasing testosterone concentrations. I focused on the effects of MXC on male mice that resulted from interactions with sex-steroid hormone receptors. Sex-steroid hormones affect other organs including the kidney and liver. Accordingly, I hypothesised that MXC can act through sex-steroid receptors to produce adverse effects on the testis, kidney and liver, and I designed our experiments to confirm the different effects of MXC exposure on the male reproductive system, kidney and liver. In these experiments, I used pre-pubescent ICR mice; the puberty period in ICR mice is from postnatal day (PND) 45 to PND60. I treated the experimental group with 0, 100, 200, 400 mg MXC/kg b.w. delivered by an intra-peritoneal injection with sesame oil used as vehicle for 4 weeks. At the end of the experiment, the mice were sacrificed under anaesthesia. The testes and accessory reproductive organs were collected, weighed and prepared for histological investigation. I performed a chemiluminescence immune assay to observe the serum levels of testosterone, LH and FSH. Blood biochemical determination was also performed to check for other effects. There were no significant differences in our histological observations or relative organ weights. Serum testosterone levels were decreased in a dose-dependent manner; a greater dose resulted in the production of less testosterone. Compared to the control group, testosterone concentrations differed in the 200 and 400 mg/kg dosage groups. In conclusion, I observed markedly negative effects of MXC exposure on testosterone concentrations in pre-pubescent male mice. From our biochemical determinations, I observed some changes that indicate renal and hepatic failure. Together, these data suggest that MXC produces adverse effects on the reproductive system, kidney and liver.

• Development and Reproduction
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• v.19 no.4
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• pp.167-180
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• 2015

Arsenic is a toxic metalloid that exists ubiquitously in the environment, and affects global health problems due to its carcinogenicity. In most populations, the main source of arsenic exposure is the drinking water. In drinking water, chronic exposure to arsenic is associated with increased risks of various cancers including those of skin, lung, bladder, and liver, as well as numerous other non-cancer diseases including gastrointestinal and cardiovascular diseases, diabetes, and neurologic and cognitive problems. Recent emerging evidences suggest that arsenic exposure affects the reproductive and developmental toxicity. Prenatal exposure to inorganic arsenic causes adverse pregnancy outcomes and children's health problems. Some epidemiological studies have reported that arsenic exposure induces premature delivery, spontaneous abortion, and stillbirth. In animal studies, inorganic arsenic also causes fetal malformation, growth retardation, and fetal death. These toxic effects depend on dose, route and gestation periods of arsenic exposure. In males, inorganic arsenic causes reproductive dysfunctions including reductions of the testis weights, accessory sex organs weights, and epididymal sperm counts. In addition, inorganic arsenic exposure also induces alterations of spermatogenesis, reductions of testosterone and gonadotrophins, and disruptions of steroidogenesis. However, the reproductive and developmental problems following arsenic exposure are poorly understood, and the molecular mechanism of arsenic-induced reproductive toxicity remains unclear. Thus, we further investigated several possible mechanisms underlying arsenic-induced reproductive toxicity.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.383-387
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• 1999

Objective: There is increasing evidence for the expression of rat in gene in several extrapituitary sites including testis and ovary. We also have demonstrated that the local LH expression in the rat epididymis and uterus, the major accessory sex organs in male and female reproductive system, respectively. Design: The present study was undertaken to elucidate whether the gene for LH receptor is expressed in rat uterus and whether the expressions of uterine LH and its receptor are differentially regulated during estrous cycle. Presence of the transcripts for rat LH receptor in the rat uterine tissue were confirmed by touchdown reverse transcription-polymerase chain reaction (RT-PCR). Results: In $LH{\beta}$ semi-quantitative RT-PCR, the highest expression level was shown in estrus stage. The level of ill receptor transcripts was also fluctuated during estrous cycle. In ovariectomized rats (OVX + Oil), the expressions of both uterine LH and LH-R were markedly reduced when compared to those from normal rats. Supplement with estradiol $17{\beta}$ to the ovariectomized rats (OVX + $E_2$) restored the expression levels of LH and its receptor to the levels in uteri from normal rats. Conclusion: Our findings indicated that 1) LH and its receptor gene are expressed in the rat uterus from cycling rats, 2) the expression of uterine LH and its receptor is mainly, if not all, under the control of ovarian sex steroid(s). These results suggested that the uterine LH may act as a local regulator with auto and/or paracrine manner, though the posibility that the pituitary LH may act directly on the regulation of uterine functions could not be discarded.

• Development and Reproduction
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• v.16 no.1
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• pp.53-58
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• 2012

Circadian rhythmicity (e.g. secretory pattern of hormones) plays an important role in the control of reproductive function. We hypothesized that the alteration of feeding pattern via meal time shift/restriction might disrupt circadian rhythms in energy balance, and induce changes in reproductive activities. To test this hypothesis, we employed simple animal model that not allowing $ad$ $libitum$ feeding but daytime only feeding. The animals of $ad$ $libitum$ feeding group (Control) have free access to food for 4 weeks. The day feeding (=reverse feeding, RF) animals (RF group) have restricted access to food during daytime (0900-1800) for 4 weeks. After completing the feeding schedules, body weights, testis and epididymis weights of animals from both group were not significantly different. However, the weights of seminal vesicle (control : RF group = $0.233{\pm}0.014g$ : $0.188{\pm}0.009g$, $p$<0.01) and prostate (control : RF group = $0.358{\pm}0.015g$ : $0.259{\pm}0.015g$, $p$<0.001) were significantly lower in RF group animals. The mRNA levels of pituitary common alpha subunit ($C{\alpha}$; control : RF group = $1.0{\pm}0.0699$ AU : $0.1923{\pm}0.0270$ AU, $p$<0.001) and $FSH{\beta}$ (control : RF group = $1.0{\pm}0.1489$ AU : $0.5237{\pm}0.1088$ AU, $p$<0.05) were significantly decreased in RF group. The mRNA levels of ACTH were not significantly different. We were unable to find any prominent difference in the microstructures of epididymis, and there were slight alterations in those of seminal vesicles after 4 weeks of reversed feeding when compared to control samples. The present study demonstrates that the shift and/or restriction of feeding time could alter the pituitary gonadotropin expression and the weights of seminal vesicle and prostate in rats. These data suggest the lowered gonadotropin inputs may decrease androgen secretion form testis, and consequently results in poor response of androgen-dependent tissues such as seminal vesicle and prostate.