• Applied Microscopy
• /
• 제40권4호
• /
• pp.211-218
• /
• 2010

포유동물의 난소는 호르몬과 감각신경 및 교감신경의 지배를 받아 난소 내 혈류의 조절, 스테로이드 호르몬 생성 및 난포의 발달과 관계된 난소의 고유기능이 조절되고 있다. 본 실험에서는 통상적인 추적자인 CT-HRP와 Pseudorabies바이러스(PRV)의 Bartha strain을 신경추적자로 이용하여 뇌하수체 후엽 및 난소와 연결된 중추신경부위를 밝히고자 하였다. 또 oxytocin을 난소 지배 신경축속에서 동정함으로써 신경축내 oxytocinergic neuron들의 존재를 확인하고, 이들이 배란을 중심으로 한 난소생식주기에 따라 보이는 중추 내 oxytocin신경세포의 변화를 조사하고자 하였다. Sprague-Dawley 흰쥐를 대상으로 난소 내에 PRV를 주사하고 48시간 후 실험동물들은 4% paraformaldehyde-lysine periodae로 고정하였으며, 뇌를 적출하여 $30{\mu}m$ 두께의 관상연속 절편을 만들어 CTHRP, PRV 및 oxytocin에 대한 삼중염색을 시행하였다. 본 실험 결과 후뇌에서부터 전뇌에 이르기까지 PRV에 양성반응을 보인 신경핵들이 관찰되어 난소를 지배하는 신경축을 구성할 수 있었다. 또 시상하부의 뇌실옆핵에서 oxytocin, PRV와 CT-HRP에 삼중으로 염색된 세포가 관찰됨으로써 신경내분비축과 자율신경축이 공동으로 기원하고 있다는 것을 형태학적으로 보여주었다. 따라서 oxytocin은 이 두 계통 내에서 호르몬의 역할과 신경전달물질의 역할을 겸할 것이라는 것을 추측할 수 있었다.

• 한국동물생명공학회지
• /
• 제36권1호
• /
• pp.9-16
• /
• 2021

Under the stressed condition, a complex feedback mechanism for stress is activated to maintain homeostasis of the body and secretes several stress hormones. But these stress hormones impair synthesis and secretion of the reproductive hormones, followed by suppression of ovarian function. Cytochrome P450 1A2 (CYP1A2) plays a major role in metabolizing exogenous substances and endogenous hormones, and its expression is recently identified at not only the liver but also several organs with respect to the pancreas, lung and ovary. Although the expression of CYP1A2 can be also affected by several factors, understanding for the changed pattern of the ovarian CYP1A2 expression upon stress induction is still limited. Therefore, CYP1A2 expression in the ovaries from immobilization stress-induced rats were assessed in the present study. The stress-induced rats in the present study exhibited the physiological changes in terms of increased stress hormone level and decreased body weight gains. Under immunohistological observation, the ovarian CYP1A2 expression in both control and the stressed ovary was localized in the antral to pre-ovulatory follicles. However, its expression level was significantly (p < 0.01) higher in the stress-induced group than control group. In addition, stress-induced group presented more abundant CYP1A2-positive follicles (%) than control group. Since expression of the ovarian CYP1A2 was highly related with follicle atresia, increased expression of CYP1A2 in the stressed ovary might be associated with changes of the ovarian follicular dynamics due to stress induction. We hope that these findings have important implications in the fields of the reproductive biology.

• 대한수의학회지
• /
• 제39권3호
• /
• pp.455-462
• /
• 1999

This study was designed to investigate the number of the growing and mature follicles in each stage of estrus cycle in mature rats. Eighteen mature rats(Sprague-Dawley, initially 190~230gm) were randomly alloted into 4 groups(proestrus, estrus, metestrus, and diestrus) according to estrus cycles. The uteri and ovaries of rats were collected and then alternative sections of paraffin embedding ovaries were stained with H-E. Numbers of large, middle and small follicles or only large and middle follicles from secondary and tertiary follicles were investigated by LM photography of preparations. Small follicles were defined as secondary follicles with 2~5 cell layers of granulosa cells surrounding the oocyte, and middle follicles were defined as secondary follicles with more than 5 cell layers or with early signs of antral cavity or with more than one small cleft on either side of the oocytes and large follicles were defined as tertiary follicles with a single medium or large antral cavity. The number of follicles in a pair ovary per rat was appeared to be ranged from 207 to 370 and the mean number of these follicles was $270.4{\pm}52.6$ and the mean number of follicles per ovary was $134.9{\pm}32.0$. The mean number of large, middle and small follicles per ovary was appeared to be $16.4{\pm}4.4$($12.2{\pm}3.3%$), $36.2{\pm}8.6$($26.8{\pm}6.4%$), and $82.7{\pm}24.0$($61.3{\pm}17.8%$), respectively. The mean number of large and middle follicles in each stage group of estrus cycle was appeared to be $17.8{\pm}2.1$ and $38.3{\pm}7.4$ at proestrus stage group, $15.7{\pm}5.2$ and $38.0{\pm}10.0$ at estrus stage group, $16.5{\pm}3.5$ and $33.8{\pm}7.0$ at metestrus stage group, $16.7{\pm}5.8$ and $29.7{\pm}5.5$ at diestrus group, respectively. In histological findings of large follicles during each estrus cycle, the large follicles in proestrus group contain single small antrum, thick granulosa cell layers, and were $300{\sim}500{\mu}m$ in diameter and were growing follicles with PCNA-positive cells in the granulosa cell layers, and other luteinizing follicles of proestrus cycle stage were decreased in size and were thicker in wall thickness and more luteinized than those in metestrus and diestrus stage groups. The large follicles in estrus stage group contain thick granulosa cell layers and nonprominent cumulus-oocyte complexes in antrum, and were $400{\sim}700{\mu}m$ in diameter and were growing follicles with PCNA-positive cells in the granulosa cell layers. The large follicles in metestrus and diestrus stage groups contain enlarged antrums, thinner layers of walls and prominent cumulus-oocyte complexes, and were $700-950{\mu}m$ in diameter, and were nongrowing follicles without PCNA-positive cells or another large follicles contain cells with dark stainability and distinct boundary.

• 한국수정란이식학회지
• /
• 제13권2호
• /
• pp.179-190
• /
• 1998

Prolactin (PRL) surge in cycling rats at proestrous afternoon has previously been reported as an inducer of apoptotic cell death of luteal cells. This death-inducing action of PRL seeins unusual, because PRL can he categorized as a cell-survival factor, if other known physiological functions of PRL are taken into account. In this study, the apoptotic action of PRL was assessed in cultured cells prepared from rat luteal tissue and underlying molecular /cellular mechanism of PRL-induced luteolysis was analyzed. The latest crop of corpora lutea (CLs) were enucleated from rat ovaries at 18:00 h on the proestrous day before the next ovulation. Donor rats were pretreated with CB154, a dopamine agonist, in order to he exempted from the endogenous PRL surge. The harvested GLs were dispersed and cultured with or without PRL (2$\mu$g /ml) for 24 or 48 h. An addition of PRL to the culture medium changed the parameters indicative of cell death via apoptosis: a decrease in cell viability (MTT) and an increase in chromatin condensation. Most of the DNA breakdown in nuclei induced by PRL occurred in steroidogenic cells which were identified by 3$\beta$-HSD activity staining, and the number of 3$\beta$-HSD-positivecells were significantly decreased. Interestingly, most of the cells with an apoptotic nucleus adhered to one or more intact and seemingly non-steroidogenic cells. Because the expression of Fas has heen shown to be abundant in murine ovary, and Fas is known to have an exact physiological role in occurrence of apoptotic cell death, the membrane form-Fas ligand (rnFasL) was quantified in the cell lysate. An addition of PRL increased expression of mFasL. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, in place of PRL, enhanced the apoptotic parameters. Cumulatively, the apoptotic PRL action was addressed to cells unknown than steroidogenic lute~ cells. The most prohable candidate for the direct target cells is Tcells in the luteal tissue that can express mFasL in response to PRL.

• Reproductive and Developmental Biology
• /
• 제34권4호
• /
• pp.283-288
• /
• 2010

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked. changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

• 한국수정란이식학회지
• /
• 제26권4호
• /
• pp.229-235
• /
• 2011

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski protein is implicated in proliferation/differentiation in a variety of cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of this study was, by means of immunohistochemical methods, to locate Ski protein in the rat ovaries during ovulation and corpora lutea (CL) formation to predict the possible involvement of Ski in luteinization. In addition, we performed to examine whether the initiation of luteinization with luteinizing hormone (LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vivo models. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). These results indicate that Ski is profoundly expressed in the luteinized granulosa cells and luteal cells of CL during luteinization, and suggest that Ski may play a role in luteinization of granulosa cells.

• Reproductive and Developmental Biology
• /
• 제35권3호
• /
• pp.355-361
• /
• 2011

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinization to predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadotropin to immature female rat, and luteinization was induced by human chorionic gonadotropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally.

• Applied Microscopy
• /
• 제41권2호
• /
• pp.109-116
• /
• 2011

다낭성 난소증후군(polycystic ovary syndrome, PCOS)은 내분비 및 대사장애로 호르몬 불균형 상태를 야기하여 가임기 여성의 불임 및 여러 합병증을 유발한다. 폐경기 여성의 호르몬대체 치료에 사용되는 estradiol valerate (EV)는 과다 투여 시 PCOS를 유발하는 것으로 알려졌다. 신경성장인자(nerve growth factor, NGF)는 발생 중인 신경세포의 생존과 성숙을 조절하는 인자로 난소에서도 합성되고, EV 처리에 의해 유도된 다낭성난소(polycystic ovary, PCO)에서 발현이 크게 증가하는 것으로 보고됐다. 따라서 본 연구에서는 난소유래 세포인 CHO 세포주와 다낭성 난소를 이용해 호르몬 불균형 상태인 PCOS 진단 시 NGF가 생물학적 표지인자로서 사용될 수 있는 가능성을 규명하고자 하였다. CHO 세포에 EV 2 mg과 3 mg 처리는 초기에 세포증식을 억제했으나 1 mg 처리는 영향을 미치지 않음으로써 실험 시 최적농도로 사용하였다. 하지만 농도별 EV 처리에 따른 CHO 세포의 형태학적 차이는 관찰되지 않았다. CHO 세포에 EV 처리 후 NGF mRNA 및 단백질 발현은 대조군에 비해 30분 후 크게 증가하였고, 다수의 난포낭이 관찰된 PCOS 조직에서도 정상군에 비해 크게 증가하였다. 따라서 이상의 결과들을 종합하면 EV 처리 후 난소에서 유도되는 NGF 과발현은 비정상적 난포발달을 유도하는 인자로 작용할 것으로 보이며, 다낭성난소 진단 시 표지인자로서 사용될 수 있을 것으로 생각된다.

• 대한수의학회지
• /
• 제37권1호
• /
• pp.79-86
• /
• 1997

This study was designed to investigate the effects of superovulation on the growing and mature follicles following gonadotrophin treatments in mature rat by immunohistochemical methods. Eighteen mature rats (Sprague-Duwely, 190~230gm) were randomly alloted into 3 groups. One group was control group, another FSH-treated group was injected intramuscularly with 0.5 units of follicular stimulating hormone(FSH) / rat, and third PMS and HCG-treated group was injected intramuscularly with 20~25IU of pregnant mare serum(PMS) / rat and then at the 48 hrs later, with 20~25IU of human chorionic gonadotropin(HCG) / rat. Half the number of rats were administrated intraperitoneally with bromodeoxyuridine(Brdur, 0.2mg/gm BW once) at 2 hours before exanguination and the remainder of rats were sacrified without Brdur administration. The investigation by immunohistochemical methods using paraffin sections of ovaries was performed by using anti-Brdur antibody and PCNA(proliferating cell nuclear antigen) antibody for labeling proliferating cells in follicles. In immunohistochemical findings, follicles squeezed by peripheral corpus luteum or follicles large follicles with loosly and irregularly distributed granulosa cells and although with compacted granulosa cells, middle follicles with dilated round or oval follicular antrum were confirmed as atretic follicles. The proportions of atretic follicles in control group were 29.8%, 21.7% and 14.2% respectivley at large, middle and small follicles and mean proportions of these all 3 grade follicles were 26.7%. The proportions of atretic follicles in FSH-treated group were 35.4%, 24.9% and 10.4% respectively at large, middle and small follicles and mean proportions of these all 3 grade follicles were 28.1%. The proportions of atretic follicles in PMS and HCG-treated group were 44.7%, 24.0% and 12.7% respectively at large, middle and small follicles, and mean proportions of these all 3 grade follicles were 29.7%. The above findings reveal that the group with higher proportion of atretic follicles were ordered as large, middle and small follicles in size, and these proportions were increased in hormone treated two groups with more number of more growing and mature follicles when compared with control group.

• 한국임상수의학회지
• /
• 제24권4호
• /
• pp.563-567
• /
• 2007

This study evaluated the effect of ANTORIN R-10(pFSH), a commercially available follicle stimulating hormone on ovarian morphology, on follicular development and serum estradiol levels in rats. Immature female Wistar S/T rats(27 day old; 80-100 g B.wt) maintained under controlled environmental conditions($22{\pm}2^{\circ}C$; 50% humidity; 12 h light/12 h dark cycle) with free access to standard laboratory feed and tap water were utilized. Animals were allowed to acclimatize to the new environment for at least 2 weeks before being included in the experiment. Rats were randomly allotted to 5 groups(Control, SL 0.1AU, SH 0.2AU, TL 0.1AU and TH 0.2AU). ANTORIN R-10 was subcutaneously injected twice daily for 3 days. Twenty hours after hormone treatment, blood was collected to estimate the serum estradiol $17-\beta$ concentration. Immediately, all rats were sacrificed and the ovarian morphology, ovary weight and number of follicles were recorded. Ovaries were fixed for histomorphological examination. Higher standard and treatment groups were significantly increased on ovary weight and the number of follicles more than 1mm compared with lower standard and treatment. However, no difference revealed between standard and treatment groups. ANTORIN R-10 was similar effects of follicles development and maturation compared with House standard FSH.