• Clinical and Experimental Reproductive Medicine
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• 제40권4호
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• pp.155-162
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• 2013

Objective: Stress is known to be an inhibitor of the reproductive hypothalamic-pituitary-gonadal (HPG) axis. However, the neural and molecular connections between stress and reproduction are not yet understood. It is well established that in both humans and rodents, kisspeptin (encoded by the kiss1 gene) is a strong stimulator of the HPG axis. In the present study we hypothesized that endocannabinoids, an important neuromodulatory system in the brain, can act on the HPG axis at the level of kiss1 expression to inhibit reproductive function under stress. Methods: Adult male Wistar rats were unilaterally implanted with an intracerebroventricular cannula. Afterwards, the animals were exposed to immobilization stress, with or without the presence of the cannabinoid CB1 receptor antagonist AM251 (1 ${\mu}g/rat$). Blood samples were collected through a retro-orbital plexus puncture before and after stress. Five hours after the stress, brain tissue was collected for reverse transcriptase-quantitative polymerase chain reaction measurements of kiss1 mRNA. Results: Immobilization stress (1 hour) resulted in a decrease in the serum luteinizing hormone concentration. Additionally, kiss1 gene expression was decreased in key hypothalamic nuclei that regulate gonadotrophin secretion, the medial preoptic area (mPOA), and to some extent the arcuate nucleus (ARC). A single central administration of AM251 was effective in blocking these inhibitory responses. Conclusion: These findings suggest that endocannabinoids mediate, at least in part, immobilization stress-induced inhibition of the reproductive system. Our data suggest that the connection between immobilization stress and the HPG axis is kiss1 expression in the mPOA rather than the ARC.

• Asian-Australasian Journal of Animal Sciences
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• 제14권2호
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• pp.174-183
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• 2001

During the rapid phase of gonadal development of the freshwater teleost, the catfish (Silurus asotus), the influence of hCG upon the inducement of final oocyte maturation and spawning was investigated electrophoretically and ultrastructurally. The electrophoretic patterns obtained were different in the presence and absence of some of the major or minor zones, because of the hormone level in catfish. The vitellogenin of hormone-treated fish was stained more intensively than that of sham-treated fish. These proteins showed some minor or main bands of egg extracts which migrated at positions corresponding to molecular weights of approximately 90,000. However, the thickness of electrophoretic band in molecular weight for hCG-treated fish was slightly lower than that for saline control. It seemed the plasma protein with molecular weight of approximately 45,000 in hCG-treated fish disappeared. In contrast to the control fish, the ovaries in the catfish treated with hCG shows a marked ultrastructural change under the electron microscope. No dilated profiles were seen in the granulosa cells of the mature oocyte before ovulation. After germinal vesicle breakdown (GVBD), the zona radiata interna (ZRI) becomes more compact, and there is a loss of all the processes from the pore canals. There is a wide space between the vitelline membrane and zona radiata. Also, during final maturation, the microvillar processes from the oocyte are seen no longer to penetrate deeply into the extracellular spaces of the overlying granulosa cells, and the reticulate patterns of the zona radiata interna becomes occluded, giving the zona radiata a more solid appearance. It has been possible to initiate 100% oocyte maturation in yolk granules and follicles in vivo by treatment with hCG and a high water temperature ($27^{\circ}C$). In hCG-treated fish, the percentages of successful artificial fertilization and hatching were maximal at 15 h after a single injection. It seems clear that a long acting preparation containing hCG can be successfully used in prespawning fish to advance the final events of gonadal maturation and initiate spawning. Further studies are necessary to evaluate the potential of hCG to either stimulate or inhibit the reproductive development of fish at other stages of the seasonal reproductive cycle.

• 한국수산과학회지
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• 제38권5호
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• pp.309-315
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• 2005

Changes of sex steroid hormones in the plasma of yellowfin goby, Acanthogobius flavimanus were investigated in relation to the gonadosomatic index (GSI), the hepatosomatic index (HSI) and gonadal development. The GSI in females rose rapidly in November and remained high from December to May $(7.26\pm0.89­6.62\pm0.02)$. The Male's GSI also increased gradually from November and was highest in May $(0.16\pm0.08)$. The HSI in both sexes was in reverse correlation with the GSI, and the HSI was low during the spawning season (February-May). In females, the $estradiol-17{\beta}\;(E_2)$ level increased during vitellogenesis (November and December) and reached its maximum $(8.13\pm2.87 ng/mL)$ at the maturing period, in January. $17{\alpha},\;20{\beta}$-dihydroxy-4-pregnen-3-one$(17{\alpha}20{\beta}OHP)$ gradually increased from October $(0.063{\pm}0.02ng/mL)$ to March $(0.16{\pm}0.02ng/mL)$ and increased rapidly in May. The level of testosterone (T) showed a similar tendency of $E_2$. In males, T increased gradually during spermatogenesis from September to December $(0.14{\pm}0.06­0.26{\pm}0.10ng/mL)$ and peaked in January $(0.36{\pm}0.29 ng/mL)$ when the spermatozoa filled the testis. 11-KT also rose markedly in January and then decreased. On the other hand, $17{\alpha}29{\beta}OHP$ in males did not show any clear tendencies.

• 한국발생생물학회지:발생과생식
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• 제5권1호
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• pp.1-8
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• 2001

골든 햄스터의 생식활동은 광주기(하루 중 조명 시간)에 의해 결정된다. 광주기는 일년 동안 예측할 수 있는 환경요인이다. 주간 길이가 야간 길이보다 긴 여름에 햄스터의 생식 활동은 왕성하다. 생식 기능을 유지시키는 조명 시간은 하루에 적어도 12.5시간이다. 송과선을 제거시키면 광주기의 정보가 억제되기 때문에, 광주기의 정보는 송과선을 통하여 중재된다. 송과선을 제거 당한 핼스터는 생식 활동이 유지되고, 생식소 기능을 억제하는 상황에서도 생식 기능을 촉진시킨다. 송과선은 멜라토닌을 분비하고 멜라토닌은 광주기 정보를 반영 한다. 멜라토닌을 적절히 투입하면 송과선과 무관하게 생식소 퇴화가 유도된다. 생식체계를 기능적으로 통합하는 신경내분비 신호로 환경 정보가 전환하는 기전을 멜라토닌이 조절함을 시사한다. 광범위한 연구에도 불구하고, 멜라토닌의 작용부위는 알려지지 않았다. 이는 멜라토닌이 생식 호르몬의 분비에 미치는 즉각적인 효과가 없기 때문이다. 그러나, 성적으로 퇴화된 동물들은 생식 호르몬 수준이 낮고 시상하부 내 GnRH 양이 증가한다. 광주기 혹은 멜라토닌 처리가 생식 기능을 억제하는 효과는 GnRH 신경계에 의해 중재됨을 의미한다. 멜라토닌이 GnRH 신경에 미치는 작용 기전이 조사되어야 한다. 멜라토닌 수용체가 클로닝되어, 목적 조직에 미치는 멜라토닌의 작용 기전과 해부학적 위치를 통하여 멜라토닌의 다양하고 잠재적인 능력을 분자수준에서 연구하는데 공헌할 것이다.

• 한국동물학회지
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• 제39권4호
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• pp.337-351
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• 1996

멜라토닌은 대뇌와 소뇌 사이에 위치한 송과선에서 분비되는 호르몬으로 빛이 없는 밤에만 분비된다. 멜라토닌은 분자적 수준에서부터 개체의 행동에 이르기 까지 다양한 기능을 보인다. 특히, 생식에 미치는 영향은 광범위하여, 온대지방에 사는 대부분의 동물은 주위 환경에 적응하여 종족을 유지하는 유일한 계절적 번식을 한다. 햄스터의 생식활동은 여름에 왕성하고 겨울에 정지된다.이는 많은 환경요소중 광주기의 효과가 송과선에 의해 제거하면 광주기의 영향은 사라진다. 즉 생식에 미치는 광주기의 효과가 송과선에 의해 중재 됨을 의미한다. 또한 송과선 호르몬인 멜라토닌의 적절한 처리는 생식활동을 억제한다. 따라서 멜라토닌은 생식에 미치는 광주기의 정보를 생식내분비계로 전달하는 신경전달물질로 사료된다. 시상하부의 특정부위를 절제한 후 광주기나 멜라토닌을 처리하여 멜라토닌의 작용부위에 관한 연구가 되었으나 동물마다 차이점을 보인다. 대부부의 동물에서 공통적인 부위는 suprachiasmatic nuclei와 pars tuberalis이다. 멜라토닌이 생식에 미치는 작용기작은 아직 밝혀지지 않았다. 이는 멜라토닌의 지속적 처리가 멜라토닌의 장기적처리는 이들 호르몬의 분비를 저하시키고, 시상하부에서의 gonadotropin-releasing hormone (GnRH)양을 증가시킨다. 이 결과는 멜라토닌의 지속적인 처리가 시상하부로부터의 GnRH 양을 분비를 감소킴으로써 생식활동을 억제하는 것으로 사료된다.그러나, 멜라토닌에서 GnRH 신경까지의 정보전달은 아직 밝혀지지 않았다. Opioid 신경에 대한 광주기와 멜라토닌의 효과가 동일한 점은 opioid신경의 매개체 역할을 제시하고 있다. 최근에 멜라토닌 수용체가 개구리의 피부와 몇몇 동물의 뇌와 시세포에서 크로닝되었다. 이수용체는 G protein과 관련되고 cAMP 생성을 억제한다. 앞으로 이 멜라토닌 수용체의 존재여부와 분자생물학적 연구는 멜라토닌의 작용부위와 표적세포에서의 작용기작을 설명하는 데 크게 기여할 것으로 기대된다.

• Animal Bioscience
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• 제35권12호
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• pp.1850-1859
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• 2022

Objective: The quantitative reverse transcription polymerase chain reaction (qPCR) is the most accurate and reliable technique for analysis of gene expression. Endogenous reference genes (RGs) have been used to normalize qPCR data, although their expression may vary in different tissues and experimental conditions. Verification of the stability of RGs in selected samples is a prerequisite for reliable results. Therefore, we attempted to identify the most stable RGs in the hypothalamic-pituitary-gonadal (HPG) axis in sows. Methods: The cycle threshold values of nine commonly used RGs (18S, HPRT1, GAPDH, RPL4, PPIA, B2M, YWHAZ, ACTB, and SDHA) from HPG axis-related tissues in the domestic sows in the different stages of estrus cycle were analyzed using two RG-finding programs, geNorm and Normfinder, to rank the stability of the pool of RGs. In addition, the effect of the most and least stable RGs was examined by normalization of the target gene, gonadotropin-releasing hormone (GnRH), in the hypothalamus. Results: PPIA, HPRT1, and YWHAZ were the most stable RGs in the HPG axis-related tissues in sows regardless of the stages of estrus cycle. In contrast, traditional RGs, including 18S and ACTB, were found to be the least stable under these experimental conditions. In particular, in the normalization of GnRH expression in the hypothalamus against several stable RGs, PPIA, HPRT1, and YWHAZ, could generate significant (p<0.05) elevation of GnRH in the preovulatory phase compared to the luteal phase, but the traditional RGs with the least stability (18S and ACTB) did not show a significant difference between groups. Conclusion: These results indicate the importance of verifying RG stability prior to commencing research and may contribute to experimental design in the field of animal reproductive physiology as reference data.

• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.189-202
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• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.

• 한국발생생물학회지:발생과생식
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• 제7권1호
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• pp.35-40
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• 2003

성선자극호르몬 분비호르몬은 시상하부에서 분비되며, 포유동물의 생식에 중추적인 역할을 담당한다. 골든 햄스터는 계절적으로 번식하는 포유동물이며, 생식 활동은 광주기(하루 중 낮의 길이)에 의해 결정된다. 장주기(12.5 시간 이상의 빛)는 생식 능력을 유지시키지만, 단주기(12시간 이하의 빛)는 생식 기능을 억제한다. 성선자극호르몬 분비호르몬과 광주기의 상호 관계를 자세히 조사하기 위하여 랫트 성선자극호르몬 분비호르몬 cDNA를 포함하는 벡터를 여러 농도로 골든 햄스터의 수컷에 주입하고 단주기에 유지하여 생식 활동을 개별적으로 조사하였다. 벡터를 고농도로 처리한 그룹의 골든 햄스터는 기타 실험 그룹에 비해 정소 퇴화가 의미있게(P<0.05) 가속되었다. GnRH의 맥동적 분비가 생식 능력을 유지하는 관점에서 보면, GnRH-cDNA를 포함하는 벡터는 GnRH를 일정하게 고농도로 분비했다고 사료된다. 이 결과는 GnRH를 포함하는 벡터가 뇌하수체 전엽을 desensitization시켜 정소 퇴화를 가속시켰음을 암시한다.

• Clinical and Experimental Pediatrics
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• 제54권5호
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• pp.224-227
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• 2011

A sclerosing stromal tumor of the ovary is an extremely rare benign tumor; it usually is found during the second and third decades of life. Patients present with pelvic pain or a palpable abdominal mass. Hormonal effects such as masculinization are uncommon. Here, an 11-year old premenarchal girl presented with deepening of the voice. In addition, clitoromegaly and hirsutism with a male suprapubic hair pattern were observed. The laboratory findings showed that the testosterone level was elevated to 3.67 ng/ml, andostenedione to above 10 ng/ml, dehydroepiandrosterone-sulfate to 346 ${\mu}g$/dl and 17-hydroxy progesterone (17-OHP) to 11.28 ng/ml. The chromosome evaluation revealed a 46,XX female karyotype. An adrenocorticotropic hormone stimulation test was performed. The 17-OHP to cortisol ratio in 30 minutes was 0.045, which suggested a heterozygote for the 21-hydroxylase deficiency. However, the CYP21A2 gene encoding steroid 21-hydroxylase showed normal. The pelvic ultrasound showed a heterogeneous mass consisting of predominantly solid tissue in the pelvic cavity. The pelvic magnetic resonance imaging revealed an $8.9{\times}6.2{\times}6.6$ cm mass of the left ovary. A left oophrectomy was performed and microscopic examination confirmed a sclerosing stromal tumor. Immunohistochemical studies showed that the tumor was positive for smooth muscle actin and vimentin, but negative for S-100 protein and cytokeratin. Following surgery, the hormone levels returned to the normal range and the hirsutism resolved.

• Advances in Traditional Medicine
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• 제10권2호
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• pp.103-110
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• 2010

The seed powder of Abrus precatorius L. has traditionally been used as oral contraceptive agent by the women in some rural areas in Bangladesh. The present study aimed to examine the antifertility activity of A. precatorius seed extracts in experimental female rats. Finely ground seeds were extracted with aqueous acetone followed by successive partitioning with n-hexane, ethyl acetate (EtOAc), methanol (MeOH) and water. Water suspended crude seed powder, organic fractions of acetone extract and a standard contraceptive drug ($Nordette^{(R)}28$) were separately administered orally to the female rats for 30 days. n-Hexane, EtOAc and MeOH solubles at the doses of 2, 4 and 6 mg/rat/day, respectively and crude seed powder at 100 mg/rat/day exhibited 100% antifertility activity with lowest levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and $17{\beta}$-estradiol. Histological study of ovary and uterus of these rats exhibited reduced number of developing follicles and increased number of atretic follicles in the ovary, and fewer uterine glands with shrunken morphology, reduced endometrial height, poor vascularity and compact stroma in uterus. However, the activities of serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase and the body weight of the rats remained almost unaffected in all the seed extract treated rats compared to control. These results suggest that A. precatorius seed extracts reduced the levels of serum FSH, LH and $17{\beta}$-estradiol probably by affecting hypothalamic-pituitary-gonadal axis. The reduced levels of these hormones might have affected the oestrous cycle, follicular development, and subsequently the establishment of pregnancy in treated rats.