The purpose of this study is to investigate the pharmacokinetics and tissue distribution of recombinant bovine somatotropin (rBST) after subcutaneous adminstration of $^{125}I-rBST$ in male and female rats. A solid state conjugation (Iodo-bead$^{(R)}$) method was confirmed useful for producing $^{125}I-rBST$ because the administration of the conjugated form enabled enough to determine time- concentration relationships of rBST in rats. Subcuatenous administrations showed sex differences that female ($t_{1/2,kc}$, 2.87 h) revealed rapid elimination as compared to male ($t_{1/2,ke}$, 4.81 h), with the absorption ($t_{1/2,ka}$ in male being 0.3 h and that in female 0.75 h) in the reverse order. For subcutaneous administration of rBST in male rats, the liver was the highest in amount, followed by kidney, testes, muscle, and stomach, at the slaughtering tame of 1, 6, 12 and 24 h. But the testes was the highest at the 48 h slaughtered animals, followed by liver, kidney, stomach, and muscle. In slaughtered females at 1, 6, and 12 h after the administration of rBST, the liver was the highest, followed by ovary, kidney, small intestine, and stomach. At 24 and 48 h slaughtered female rats, the ovary was the highest, the liver the second, and the kidney the third.
In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.
Limb bud (LB) and central nerve system (CNS) cells were prepared from 12.5 day old pregnant female Crj:CD (SD) rats and treated with olaquindox and vitamin A. Cytotoxicity and inhibition on differentiation were measured in each cell. Three doses of olaquindox (4, 21 and 100 mgkg), and 0.2 and 75 mg/kg of vitamin A were administered to pregnant rat for 11 days from $6^{th}$ to $16^{th}$ of pregnancy. $IC_{50}$ values of olaquindox for proliferation and differentiation in CNS cell were 22.74 and $28.32\;{\mu}g/ml$ and 79.34 and $23.29\;{\mu}g/ml$ in LB cell and those values of vitamin A were 8.13 and $5.94\;{\mu}g/ml$ in CNS cell and 0.81 and $0.05\;{\mu}g/ml$ in LB cell, respectively. Mean body weights of pregnant rats were decreased at high dose of olaquindox (110 mg/kg) but relative ovary weight, number of corpus lutea, and number of implantation were not changed. Resorption and dead fetus were increased at high dose of olaquindox, and relative ovary weight, the number of corpus lutea and implantation, and sex ratio of male to female were not significantly changed in all dose of olaquindox. Mean fetal and placenta weights were significantly (p < 0.01) decreased in rats of high group. Seven fetuses out of 103 showed external anomaly like bent tail, and 10 out of 114 fetuses showed visceral anomalies at high group. The ossification of sternebrae and metacarpals were significantly (p < 0.01) increased by low and middle dose of olaquindox but it was significantly (p < 0.01) prohibited by high dose of olaquindox. In rats treated with vitamin A, the resorption and dead fetus were increased by high dose. Mean fetal weights were significantly (p < 0.01) increased by low dose but significantly (p < 0.01) decreased by high dose. Thirty four fetuses out of 52 showed external anomaly; bent tail (1), cranioarchschisis (14), exencephaly (14), dome shaped head (22), anophthalmia (15), brcahynathia (10) and others (19). Forty five fetuses out of 52 showed soft tissue anomaly; cleft palate (42/52) and anophthalmia (22/52) by high dose of vitamin A. Sixty one fetuses out of 61 (85.2%) showed skull anomaly; defect of frontal, partial and occipital bone (21/61), defect of palatine bone (52/61) and others (50/61). In summary, we support that vitamin A is strong teratogen based on our micromass and in vivo data, and olaquindox has a weak teratogenic potential in LB cell but not in CNS cell. We provide the in vivo evidence that a high dose of olaquindox could have weak embryotoxic potential in rats.
Biosynthesis and secretion of anterior pituitary hormones are under the control of specific hypothalamic stimulatory and inhibitory factors. Among them, Growth Hormone Releasing Hormone (GHRH) is the major stimulator of pituitary somatotrophs activating GH gene expression and secretion. Human GHRH is a polypeptide of 44 amino acids initially isolated from pancreatic tumors, and the gene for the hypothalamic form of GHRH is organized into 5 exons spanning over 10 kilobases (kb) on genomic DNA and encodes a messenger RNA of 700-750 nucleotides. Several neuropeptides classically associated with the hypothalamus have been found in the extrahypothalamic regions, suggesting the existence of novel sources, targets and functions. GHRH-like immunoreactivity has been found in several peripheral sites, including placenta, testis, and ovary, indicating that GHRH may also have regulatory roles in peripheral reproductive organs. Furthermore, higher molecular weight forms of the GHRH transcripts were identified from these organs (1.75 kb in testis; 1.75 and >3 kb in ovary). These tissue-specific expression of GHRH gene suggest the existence of unique regulatory mechanism of GHRH expression and function in these organs. In fact, placenta-specific and testis-specific promoters for GHRH transcripts which are located in about 10 kb upstream region of hypothalamic promoter were reported. The use of unique promoters in extrahypothalamic sites could be refered in a different control of GHRH gene and different functions of the translated products in these tissues. Somatotrophs and lactotrophs have been thought to be derived from a common bipotential progenitor, the somatolactotrophs, which give origins to either phenotypes. Although the precise mechanism responsible for the lactotroph differentiation in the anterior pituitary gland has not been yet clalified, there are several candidators for the generation of lactotrophs. In human, the presence of GHRH peptides with different size from authentic hypothalamic form in the normal anterior pituitary and several types of adenoma were demonstrated. Recently our group found the existence of immunoreactive GHRH and its transcript from the normal rat anterior pituitary (gonadotroph> somatotroph> lactotroph), and the GHRH treatment evoked the increased proliferation rate of anterior pituitary cells in vitro. The transgenic mouse models clearly shown that GHRH or NGF overexpression by anterior pituitary cells induced development of pituitary hyperplasia and adenomas particularly GH-oma and prolactinoma. Taken together, we hypothesize that the pituitary GHRH could serve not only as a modulator of hormone secretion but as a paracrine or autocrine regulator of anterior pituitary cell proliferation and differentiation. Interestingly enough, the expression of Pit-1 homeobox gene (the POU class transcription factor) was confined to somatotrophs, lactotrophs and somatolactotrophs in which GHRH receptors are expressed commonly. Concerning the mechanism of somatolactotroph and lactotroph differentiation in the anterior pituitary, we have focused following two possibilities; (1) changes in the relative levels or interactions of both hypothalamic and intrapituitary factors such as dopamine, VIP, somatostatin, NGF and GHRH; (2) alterations of GHRH-GHRH receptor signaling and Pit-1 activity may be the cause of lactotroph differentiation or pituitary hyperplasia and adenoma formation. Extensive further studies will be necessary to solve these complicated questions.
연구방법: 미성숙 백서 난소의 과배란 유도를 위해 PMSG를 주사하고, 배란을 위해서 hCG를 주입하였다. RGS-2의 유전자 발현양상을 조사하기 위하여는 Northern blot 분석과 in situ hybridization 분석을 시행하였다. 결 과: 미성숙 백서에 성선자극호르몬인 PMSG를 복강내 주사했을 때 RGS-2 mRNA 발현에 영향을 미치지 않음을 Northern blot analysis로 확인할 수 있었으나, hCG를 주입했을 때는 1시간에서 3시간 내에 발현이 증가됨을 알 수 있었다. In situ hybridization으로 살펴본 RGS-2 mRNA의 발현세포는 난포의 크기에 관계없이 난자였으나, hCG로 처리한 후에는 배란 전 난포와 성장중인 난포의 과립막 세포이었다. 그러나, RGS-2 단백의 발현은 hCG 처치와 관계없이 난포막 세포이었다. 상기 생체 실험과 마찬가지로 시험관에서도 배란 전 난포의 과립막 세포에 대한 LH 처리는 RGS-2 유전자 발현을 1시간 내에 촉진하였다. 또한, 성선자극호르몬 분비호르몬 2 길항제도 이러한 LH의 촉진작용을 증진시켰다. 결 론: 본 연구로 배란 전 과립막 세포에서 성선자극호르몬인 LH/hCG와 성선자극호르몬 분비호르몬 길항제에 의해 RGS-2의 발현이 증진되는 양상으로 보아 RGS-2가 배란과정 동안에 Gq protein 신호전달을 조절할 것으로 추정된다.
Park, Wan-Sung;Lee, Sung-Ho;Kim, Hyun-Sup;Cho, Sa-Sun;Young Namkung;Yoon, Yong-Dal;Paik, Sang-Ho;Cho, Wan-Kyoo;Kim, Kyungjin
한국동물학회지
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제33권4호
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pp.435-445
/
1990
시상하부에서 합성, 분비되는 gonadotropin releasing horrnone (GnRH)의 면역반응성이 생식소를 비롯한 여러 부위에서도 검출됨이 알려졌으나, 이 펩타이트가 과연 생식소에서 국부적으로 합성되는 지에 관해서는 아직 밝혀지지 않았다. 본 연구에서는 흰쥐 생식소에서 GnRH유전자발현을 연구하기 위하여 GnRH-like mRNA와 GnRH펩타이트의 발현과 세포내 분포 양상을 조사하였다. GnRH 방사면역측정법과 GnRH를 크로마토그라피 방법으로 분리한 결과,시상하부에서 합성되는 GnRH와 유사한 GnRH 면역반은이 흰쥐 생식소 추출물에서 상당량 검출되었다. GnRH-면역반응이 흰쥐 난소의 다양한 세포군에서 나타냄에 반하여, GnRH-like mRNA는 granulsa,theca 그리고 luteal 세포에서만 주로 발현되었다. 또한 흰쥐 정소에서 GnRH면역반응성은 원시정세포, Sertoli,Leydig 세포에서만 검출된 반면에, GnRH-like mRNA는 정세관내의 Seertoli세포에서만 발현되었다. 따라서 이 연구는 생식소에 존재하는 GnRH는 생식소 내에서 국부적으로 합성, 발현되는 결과라고 사료되며, 생식소 내에서 생성된 GnRH는 생식소내 세포군간의 정보교환의 매개자로서 역활을 수행하고 있다고 추정된다.
Cytochrome P450 1A2 (CYP1A2) is a member of the cytochrome P450 superfamily enzymes in mammals and plays a major role in metabolizing endogenous hormones in the liver. In recent days, CYP1A2 expression has been found in not only the liver but also other tissues including the pancreas and lung. However, little information is available regarding the expression of CYP1A2 in the ovary, in spite of the facts that the ovarian follicle growth and atresia are tightly associated with controls of endocrine hormonal networks. Therefore, the expression of CYP1A2 in the ovaries of prepubertal and pubertal rats was investigated to assess its expression pattern and puberty-related alteration. It was demonstrated that the expression level of CYP1A2 was significantly (p < 0.01) higher in the pubertal ovaries than prepubertal counterparts. At the ovarian follicle level in both groups, whereas CYP1A2 expression was less detectable in the primordial, primary and secondary follicles, the strongly positive expression of CYP1A2 was localized in the granulosa cell layers in the antral and pre-ovulatory follicles. However, the ratio of CYP1A2-positive ovarian follicle was significantly (p < 0.01) higher in the ovary of pubertal group (73.1 ± 3.1%) than prepubertal one (41.0 ± 10.5%). During the Immunofluorescence, expression of CYP1A2 was mainly localized in Fas-positive follicles, indicating the atretic follicles. In conclusion, these results suggested that CYP1A2 expression was mainly localized at the atretic follicular cells and affected by the onset of puberty. Further study is still necessary but we hypothesize that CYP1A2 expresses in the atretic follicles to metabolize residue of the reproductive hormones. These findings may have important implications for the fields of reproductive biology of animals.
By the activation of ovary hormone, many morphological changes occur in the epithelial cell lines and muscle cells in rat uterus. These two cells in uterus are important to the implantation of embryo, maintaining pregnancy and starting parturition. One important change associated with the morphological change of these two cells in uterus is the change on prostaglandin(PG) metabolism. Its presence and synthesis in endometriurn and myometrium in uterus affects estrous cycle and the start of embryo implantation in uterus. It also performs as an important modulator in parturition. So the abnormally weak expression of PG causes difficulty during labor and over-expression causes pre-term labor. PG biosynthesis starts from either free or liberated arachidonic acids from membrane phospholipid by phospholipase. Such arachidonic acids are converted into PG catalyzed by Cyclooxygenase. Under normal physiological condition, Cyclooxygenase-1(COX-1) having 602 units of amino acids controls the synthesis of PG. It acts as a local hormone regulating vasomodulation of blood flow, flexible muscle movement, increasing the blood permeability and contributing the protective role in preserving integrity of the stomach lining and Cyclooxygenase-2 (COX-2) is induced by the inflammation, pregnancy and increased its expression until parturition. Lipid metabolite like PG is located in uterine and expression of COX-2 increased with pregnancy. Increased expression of COX proteins in epithelial cells and myometrial cells are told to increase the muscle contractility in uterus but decreased right after the labor in rat. It is a good sign indicating that COX proteins are deeply related to the start of labor. Currently, Several studies report the use of PG and COX-2 inhibitor as medication for controlled abortion or to prevent pre-term labor but they entail various side-effects. Our study proposed to suggest use of acupuncture as an another mediator to control abortion or pre-term labor without causing unnecessary side-effects by those medicines. Two acupuncture sites, LI-4 & SP-6 were selected due to their known efficacy. From the immunohistochemical staining of COX-2, normal expression of COX-2 protein in nonpregnant SD rat's uterus revealed that COX-2 protein was primarily detected in the lumina epithelial lining and in the epithelial cell lining contacting the stromal cells. High resolution optical microscopic scanning revealed distinguishable staining in the myometrial mucosa. LI-4 acupuncture administered nonpregnant rat's uterus showed strong expression for COX-2 in endometrium contacted with lumina epithelial lining of rat uterus and in myometrial mucosa. Stromal cells showed more staining than untreated nonpregnant rat's uterus and stronger staining in stromal cells contacting myometrial layer compared to untreated nonpregnant rat's uterus. SP-6 acupuncture administered nonpregnant rat's uterus showed weak expression for COX-2 in myometrial layers and stromal cells but no staining was visible in lumina epitheliai and glandular epithelial cells. Few stromal cells and myometrial mucosa were positively stained for COX-2. Pregnant SD rat's uterus was also immunostained for COX-2 expression after 18 days of pregnancy. Unlike to untreated nonpregnant rat's uterus, luminal epithelial cells were not positively stained for COX-2 but stronger staining for COX-2 was revealed in stromal cells. LI-4 acupunctured SD rat's uterus had very strong expression of COX-2 in luminal epithelial lining. Few stromal cells showed stronger positive COX-2 staining and myometrial layers also showed more expression than untreated pregnant rat. SP-6 acupuncture administered pregnant SD rat's uterus showed positive expression of COX-2 in epithelial cells of luminal mucosa layer but weaker than that of LI-4 acupuncture treatment's case. However, strong positive staining was revealed in stromal mucosa and myometrial layers. Virgin SD rat's uterus motility index during LI-4 acupuncture was 66.52 % (Prob〉T = 0.0197) compared to its motility before the acupuncture treatment but the motility index was slighdy elevated up to 79.58 % (Prob〉T = 0.1175) after the acupuncture. During the SP-6 acupuncture treatment for 30 minutes, uterus motility index was 90.52 % (Prob〉T = 0.1832) showing lesser decrement but consequently reached similar motility index decreasal to 79.95 % (Prob〉T = 0.0215) after the acupuncture treatment as LI-4 showed. LI-4 acupuncture tend to be a quick treatment to reducing the uterus motility in a virgin rat but eventually both two acupuncture administration created very similar reduction of uterus motility seeing the index after the both acupunctures. The uterus movement monitored during the LI-4 acupuncture administered for 30 minutes, Pregnant SD rat showed decreased motility down to 77.90 % (Prob〉 T = 0.0076) compared to uterus motility before the acupuncture and it continuously decreased down to 71.81 %(Prob〉T = 0.0214) after the removal of needle. The statistical analysis using paired t-test showed significance difference for both two motility indexs at =0.05. SP-6 acupuncture administered to pregnant SD rat also had similar pattern of decreasing uterus motility index down to 74.70 % (Prob〉T = 0.1730) during the initial 30 minutes acupuncture administration and it was continuously lowered to 71.52 % (Prob〉T = 0.0155) after the acupuncture. The paired t-test resuit for SP-6 suggest prompt response of uterus motility index to the SP-6 acupuncture treatment but consequently reached same level of inducing the motility reduction as LI-4 at =0.05 level.
Fowl typhoid is a septicemic disease of turkeys and chickens. The antigen is Salmonella(S) gallinarum, which cross-reacts with S pullorum because of common antigen. The organism is maintained in the ovary of carrier birds and transmitted vertically in the york. It is also transmitted horizontally through feces and broken eggs. The symptoms are weakness, drowsiness, wing drooping, hyperexcitability, paresis and diarrhea. In the many cases, the birds are found dead in the morning before any symptoms have been noticed. this experiment was carried out to investigate the characteristics of S gallanarum and scrutinized about the control systems. 1. Fowl typhoid was came about in the winter than the other seasons. The average of mortality was 46.3%. And it was came about not only adult poultry but also chicks. 2. S gallinarum was isolated from liver and spleen in infected chickens. Total isolated strains were 60. 3. The organisms were all extinct at 60$^{\circ}C$, 30mins. 4. A survival rate was higher in underground water than a secondary distilled water. It was detected in underground water until 30 days. 5. When being exposed to the solar light, it became extinct almost within 24 hours. 6. It was survived in a refrigerator until 12 months. 7. It was extinct among 5 mins in 0.1% phenol and 2% formalin. 8. When the cultured organism fluid was inoculated in SD-rat abdominal cavity, rat died within 24 hours. 9. Pullorum-typhoid related program must disciplines in order to destroy the fowl typhoid, and breeding system must be converted to cage system.
The equine chorionic gonadotropin (eCG) subunits $\alpha$ and ${\beta}$ are transcribed from different genes and associate noncovalently to form the bioactive eCG heterodimer. Dimerization is rate limiting for eCG secretion, and dissociation leads to hormone inactivation. The correct conformation of the heterodimer is alto important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduction. To determine whether ${\alpha}$ and ${\beta}$ subunits can be synthesized as a single polypeptide chain (tethered-eCG) and also display biological activity, the tethered-eCG molecule by fusing the carboxyl terminus of the eCG ${\beta}$-subunit to the amino terminus of the af-subunit was construe-ted and transfected into chinese hamster ovary (CHO-Kl) cells. LH- and FSH-like activities were assayed in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells and granulosa cells, respectively. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG ${\alpha}$/${\beta}$ and native eCG. FSH-like activity of the tethered-eCG was also shown similarly in comparison with the native and wild type eCG ${\alpha}$/${\beta}$. Our data for the first time suggest that the tethered-eCG can be expressed efficiently and the produced product by the CHO-K1 cells is fully LH- and FSH-like activities in rat in vitro bioassay system. Our results also suggest that this molecular can imply particular models of FSH-like activity not LH-like activity in the eCG. Taken together, these data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.
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