Slit/Robo pathway participates in luteal cells apoptosis / 生理学报

This study was aimed to examine the expression and function of Slit/Robo family members in mouse ovary. Real-time PCR was used to assess the mRNA expression levels of Slit/Robo family members, and immunohistochemistry was used to examine the location of Slit2 and Robo1 in the ovary. The mRNA and protein expression levels of Slit2 and Robo1 in early-, middle- and late-phase corpus luteum (CL) were examined by real-time PCR and immunohistochemistry, respectively. Blocking agent ROBO1/Fc chimera was used in the luteal cells in vitro to examine the function of Slit/Robo signaling pathway in mouse CL. The results showed that, among the Slit/Robo family members, the expression levels of ligand Slit2 and receptor Robo1 were the highest in mouse ovarian tissue. Moreover, both of them were specifically expressed in mouse luteal cells. Compared with proestrus ovaries, the expression levels of Slit2 and Robo1 mRNA in the ovaries during diestrus were significantly up-regulated (P < 0.01, P < 0.001). The mRNA expression levels of Slit2 and Robo1 in late-phase CL were significantly increased when compared with pregnant CL. Furthermore, blocking Slit/Robo signaling pathway with ROBO1/Fc chimera in the luteal cells in vitro significantly decreased the apoptotic rate of late luteal cells. These results suggest that Slit/Robo family members are mainly expressed in the late-phase CL of ovary and participate in luteal cells apoptosis.

Megalocitose de células luteínicas grandes de vacas prenhes com seneciose crônica / Large luteal cell megalocytosis in pregnant cows with chronic seneciosis

Diversas espécies de Senecio estão amplamente difundidas nas pastagens de propriedades rurais do Sul do Brasil. Criadores dessa região relatam quedas nos índices reprodutivos dos rebanhos bovinos, muitas vezes de causas não determinadas. Várias plantas tóxicas são capazes de causar alterações reprodutivas diretas e indiretas em bovinos em diversos países, incluindo o Brasil, no entanto seus mecanismos patogenéticos ainda são pouco compreendidos. O objetivo desse trabalho é descrever lesões ovarianas em vacas com seneciose crônica proveniente de propriedades rurais da mesorregião Sudoeste Rio-grandense. Foram estudados 21 casos positivos de seneciose crônica diagnosticados entre 2011 e 2014. O estudo revelou que a seneciose crônica é a principal causa de morte de bovinos adultos na região. Quatro vacas prenhes apresentaram lesões hepáticas clássicas da intoxicação por Senecio spp. Essas vacas tiveram seus ovários avaliados histologicamente e células luteínicas grandes (CLG) desses ovários apresentavam megalocitose e pseudoinclusões nucleares. Algumas CLG apresentaram núcleos com até 23,69μm de diâmetro e o aumento no tamanho desses núcleos foi significativamente maior que os de vacas controle. Conclui-se que a intoxicação por Senecio spp. causa alterações ovarianas em vacas e é possível que a intoxicação cause perdas reprodutivas nos rebanhos bovinas da região...

Several species of Senecio are widely distributed on pasture lands in Southern Brazil. Farmers from this region are known to complain about declines in reproductive rates in cattl from undetermined causes. Several poisonous plants can cause direct and indirect reproductive disorders in cattle in several countries, including Brazil. However, their pathogenetic mechanisms are still poorly understood. The aim of this study is to describe ovarian lesions in cows with chronic seneciosis, from farms located in the Southwest Mesoregion of Rio Grande do Sul, a southern state in Brazil. Twenty-one cases of bovine chronic seneciosis diagnosed between 2011 and 2014 were analyzed. The study showed that chronic seneciosis is the major cause of death in adult cattle in the region. Four pregnant cows showing classical necropsy large luteal cells (LLG) from the ovaries of these four cows had marked megalocytosis and nuclear pseudo-inclusions. Some LLG showed nuclei with up to 23.69μm in diameter and the increase in size of these nuclei was significantly greater than measured those of control cows. It is concluded that the ingestion Senecio spp. induces ovarian changes in cows and the intoxication should be considered as a possible cause of reproductive failure in cattle herds from this region...

Follicular fluid levels of prostaglandin E2 and the effect of prostaglandin E2 on steroidogenesis in granulosa-lutein cells in women with moderate and severe endometriosis undergoing in vitro fertilization and embryo transfer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The mechanisms of endometriosis with infertility have not been fully studied. The present study aimed to assess the follicular fluid (FF) levels of prostaglandin E2 (PGE2), which plays a critical role within the ovary, and to investigate the effect of PGE2 on steroidogenesis in granulosa-lutein cells (GLCs) from women with and without endometriosis.</p><p><b>METHODS</b>Thirty-three women with laparoscopically documented endometriosis and 40 controls undergoing in vitro fertilization (IVF) were studied. We assayed the concentrations of PGE2 in FF, the production of E2 and progesterone in FF and in culture medium, and the expression of steroidogenic acute regulatory protein (StAR) and CYP19A1 in GLCs with the intervention of PGE2.</p><p><b>RESULTS</b>PGE2 and progesterone concentrations were increased and displayed positive correlation in endometriotic FF. PGE2 induced the expression of StAR and the production of progesterone in GLCs from women with endometriosis, and the expression of StAR and the production of progesterone were increased in GLCs from women with endometriosis. However, there were no significant effects of PGE2 on promoting the production of E2 or the expression of CYP19A1 in GLCs. Moreover, the production of E2 and the expression of CYP19A1 in GLCs from women with endometriosis were significantly decreased compared to the controls.</p><p><b>CONCLUSIONS</b>PGE2 concentrations are increased in endometriotic FF, along with concomitant increases in progesterone and StAR. In contrast, the E2 and CYP19A1 are decreased in GLCs, which may delay the development of the follicles and cause an imbalance in the follicular steroid hormone levels. These changes may have close relationship with endometriosis-associated infertility.</p>

Sclerosing peritonitis associated with luteinized thecoma and elevated serum CA 125 levels: case report / Peritonite esclerosante associada com tecoma tuteinizante e elevação do CA 125: relato de caso

CONTEXT: Thecomas are benign tumors that account for less than 1 percent of all ovarian neoplasms. The association of ovarian thecoma with sclerosing peritonitis is rare. CASE REPORT: We report the case of a 33-year-old woman, with a two-month history of increasing abdominal volume. Ultrasound showed a complex pelvic lesion and laboratory analysis detected elevated serum CA 125 levels. The patient underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy and peritoneal biopsy. Histopathological analysis revealed the presence of luteinized thecoma of both ovaries associated with sclerosing peritonitis.

CONTEXTO: Os tecomas são tumores benignos e representam menos de 1 por cento das neoplasias ovarianas. A associação de tecoma de ovário com peritonite esclerosante é rara e caracteriza-se pela presença de múltiplos espessamentos nodulares fibróticos no peritôneo. RELATO DE CASO: Relatamos um caso de uma paciente de 33 anos de idade, com aumento de volume abdominal há dois meses. A ultra-sonografia mostrou uma lesão pélvica complexa e a dosagem de CA 125 mostrava-se elevada. A paciente submeteu-se a histerectomia abdominal total com ooforectomia bilateral e biópsia peritoneal. O exame histopatológico revelou a presença de tecoma luteinizado de ovário bilateral associado a peritonite esclerosante.

Role of platelet-activating factor in progesterone synthesis and vascular endothelial growth factor expression in rat luteal cells / 生理学报

The present study aimed to investigate the role of platelet-activating factor (PAF) in progesterone synthesis and vascular endothelial growth factor (VEGF) expression in rat luteal cells. Immature (25-28 days old) female Sprague-Dawley rats were injected subcutaneously with 50 IU pregnant mare serum gonadotrophin (PMSG), and 25 IU human chorionic gonadotrophin (hCG) 48 h later, to induce follicular development and luteum formation. On day 6 after hCG administration (the day of hCG administration was the first day), the rats were killed by guillotine and the ovarian luteal cells were collected. After incubation for 24 h, luteal cells were incubated without or with different doses (0.1 μg/mL, 1 μg/mL, 10 μg/mL) of PAF at 37 °C (5% CO(2)) for 24 h, and then progesterone concentration was evaluated by radioimmunoassay (RIA); apoptotic rate and VEGF mRNA expression in luteal cells were assessed by flow cytometry and RT-PCR, respectively. The results showed that PAF promoted progesterone production, with a maximal effect at 1 μg/mL (P<0.05); PAF increased apoptotic rate but not in a dose-dependent manner, and 10 μg/mL PAF enhanced apoptotic rate significantly (P<0.05); furthermore, PAF stimulated VEGF mRNA expression in luteal cells, especially at 1 μg/mL (P<0.01). It is suggested that PAF regulates progesterone synthesis and VEGF mRNA expression in luteal cells to mediate corpus luteum formation in rat ovary.

Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação / In vitro progesterone production from bovine corpus luteum throughout gestation

O presente trabalho foi desenvolvido para testar a hipótese de que células luteínicas bovinas em cultivo, provenientes dos três terços de gestação, comportam-se da mesma maneira que células in vivo em relação à produção de P4. Foram coletadas amostras de corpos lúteos (CL) de 90 (n=3), 150 (n=3) e 210 (n=3) dias de gestação obtidos em abatedouro. Sob condições assépticas, as células foram mecanicamente dispersas e cultivadas em placas de 96 poços. Após 24 horas de cultivo foram feitas a lavagem dos poços e a adição do precursor pregnenolona. Os tratamentos foram realizados em octuplicata para cada tempo de tratamento (24, 48 e 96 horas) com três repetições de cada período gestacional. As amostras de meio de cultura e as células foram coletadas 24, 48 e 96 horas após adição do precursor e acondicionadas em freezer a -20ºC até o processamento. A progesterona foi dosada através de radioimunoensaio e o conteúdo protéico pelo método de Lowry. Os resultados foram analisados estatisticamente e considerados diferentes quando p<0.05. Foi observada maior produção de P4 aos 90 dias de gestação (35,277±0,075), posterior decréscimo aos 150 dias (28,820±0,231) e novo aumento aos 210 dias (32,777±0,099). A produção de P4 em células cultivadas por 24 horas foi maior (p<0,05) em células oriundas do grupo de 90 dias (2,912±0,047) quando comparado a 150 (2,669±0,137) e 210 dias (2,741±0,088). As 48 e 96 horas de cultivo, células luteínicas bovinas de 90 dias produziram mais P4 que células de 210 dias (2,934±0,029 e 2,976±0,121 respectivamente x 2,760±0,059 e 2,695±0,149, respectivamente; p<0,05), que por sua vez produziram mais do que células de 150 dias (2,334±0,084 para 48 horas e 2,205±0,136 para 96 horas). Aos 150 dias de gestação a produção de progesterona apresentou diminuição gradativa ao longo das 96 horas de cultivo. Essas diferenças podem ser explicadas pela expressão gênica diferencial de enzimas ou também de fatores presentes na cascata esteroidogênica...

The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20ºC until processing. Progesterone was measured by RIA and protein content by Lowry's method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern...

An Immunohistochemical Study on the Macrophages in the Developing Corpora Lutea of the Rat / 대한해부학회지

Within the corpus luteum, macrophages exert luteotropic and luteolytic actions through secretion of TNF-alpha. However, the mechanisms of luteotropic actions on the development and maintenance of pregnant and nonpregnant corpora lutea are thoroughly unknown.In this experiment, TUNEL, macrophage, and TNF-alpha immunohistochemistry on the corpora lutea of pregnant and nonpregnant rats (Sprague-Dawley strain) were carried out to reveal the role of macrophages in the developing corpora lutea. The results were as follows; 1) In the nonpregnant corpora lutea, the number of macrophages was increased significantly, and the degree of ED1-immunoreactivity of macrophages was increased moderately. But lutein cells showed low-degree TNF-alpha-immunoreactivity. 2) In the pregnant corpora lutea, the number of macrophages was decreased significantly, and the degree of ED1- immunoreactivity of macrophages was low. But lutein cells showed moderate-degree TNF-alpha-immunoreactivity. Based on the above results, it was considered that macrophages in the nonpregnant corpora lutea exert phagocytic action mainly, and the macrophages in the pregnant corpora lutea exert TNF-alpha-secreting action to maintain the structure and function of lutein cells.

Proporção volumétrica dos constituintes do corpo lúteo de Nelore / Volumetric proportions of the corpus luteum constituints of Nelore

Volumetric proportions and nuclear diameter of the small and large luteinic cells of the corpus luteum (CL) were evaluated in ovaries of 17 Nelore cows and heifers, collected in slaughterhouse and classified into two groups: group I (GI, n=7), non-pregnant animals, and group II (GII, n=10), pregnant animals. The CL was reduced to small cuts (less than 3mm), which were fixed in Bouin solution and processed for morphometric analysis. The volumetric proportion analysis showed higher mean in the GI animals for the nuclei and cytoplasm of luteinic cells, while the mean of connective tissue and fibroblasts was higher in the GII animals, while the mean of the capillary endothelial cells and pericytes did not differ between the groups. The average nuclear diameter of the large and small luteinic cells did not differ between the groups.

Effects on secretory function of rat gonad by Erxian decoction and its disassembled prescriptions / 中国中药杂志

<p><b>OBJECTIVE</b>To explain functions, differences and coordination of three divided combinations of the "Erxian decoction", the famous traditional Chinese formula, on the effective sites of gonad gland at the cell level.</p><p><b>METHOD</b>The effects of Erxian decoction and its main disassembled prescriptions, "Kidney Warming", "Yin Nourishing" and "Chong-ren Adjusting", on the level of testosterone (T) progesterone (P) estradiol (E2), respectively secreted by the primary culture Leydig cell, luteal cell and granulosa cell, were measured by radioimmunoassay.</p><p><b>RESULT</b>(1) Erxian decoction could stimulate the T secretion while its three main disassembled prescriptions would seem no individual promoting effect on the secretion of T. (2) Erxian decoction and the "Kidney Warming" had the stimulating effect on P secretion, and the action of the whole formula being better than that of the "Kidney Warming". (3) Erxian decoction and its main disassembled prescriptions had the stimulating effect on E2 secretion, especially the whole formula.</p><p><b>CONCLUSION</b>Erxian decoction can stimulate the secretion of T of the Leydig cell, P of luteal cell and E2 of granulosa cell. It can be seen that the effect of the whole formula is better than that of its main disassembled prescriptions.</p>

Macrophages in the Corpus Luteum of the Rat : Immunohistochemical and Transmission Electron Microscopic Study / 대한해부학회지

Macrophages in the corpus luteum have many important roles during the periods of functional development and luteal regression. Not only phagocyte the apoptotic luteal cells, but also they secrete many cytokines and exert their effects via autocrine/paracrine actions.In this study, we investigated the changes of number and immunoreactivity of macrophages at various developmental periods of the corpus luteum in the rat ovary. The rats (Sprague-Dawley strain, female)at age of 8 weeks (ovulatory period), GD 6 (pregnant period), and postpartum 5 days (postpartum period)were sacrificed under ether anesthesia and obtained both ovaries, one used for macrophages immunohistochemistry and the other used for TEM. The results were as follows; 1.In the corpora lutea of the rat, macrophages were observed all the developmental periods including ovulatory, pregnant and postpartum periods. 2.In the corpora lutea of the rat, number of macrophages was highest in the ovulatory period, and decreased at postpartum period and pregnant period in order. The immunoreactivity of macrophages was high at ovulatory period, moderate at postpartum period, and low at pregnant period. 3.In TEM observations, two types of macrophages were observed: One type was non-phagocytic macrophage and the other type was phagocytic macrophage. Phagocytic macrophages were observed in the corpora lutea at ovulatory and postpartum periods and contained apoptotic bodies, phagocytic vacuoles and many lipid droplets. Non-phagocytic macrophages were observed in the corpora lutea at pregnancy period and showed slender cell body with long cytoplasmic processes and contained no apoptotic bodies. In the rat, the number and the degree of immunoreactivity of macrophages in the corpus luteum varied with the changes of functional state of the corpus luteum. It was suggested that the main function of the macrophages at the ovulatory and postpartum periods was elimination of apoptotic luteal cells and that at pregnancy period was autocrine/paracrine action.Ultrastructurally, two types, phagocytic and nonphagocytic types, of macrophages confirmed. These results will provide valuable informations on the study of the role macrophages during development and regression of corpus luteum.

Tecoma luteinizado de ovario: a propósito de un caso / Luteinizado Tecoma of ovary: with regard to a case

Se describe el caso clínico de una paciente de 32 años, que acudió a Solca de Quito presentando mal estado general, dolor pélvico y baja de peso, en la valoración ginecológica se encuentra una tumoración pélvica por lo que se programa una laparotomía exploratoria y el estudio histopatológico definitivo fue un tecoma luteinizado de ovario. Dentro de los tumores de los cordones sexuales y células del estroma ovárico, se encuentran los tecomas luteinizados. Se presentan en pacientes jóvenes en un 30 por ciento por ser un caso raro y poco frecuente describimos su anatomía patológica y una revisión actualizada del tema.

Effect of L-tyrosine on 3beta-HSD activity of rat luteal cells in vitro / 中国应用生理学杂志

<p><b>AIM</b>To study the effects of L-tyrosine on 3beta-HSD activity of rat luteal cells in vitro.</p><p><b>METHODS</b>Luteal cells were isolated from ovary tissues of female rats pretreated with PMSG and hCG. Luteal cells were cultured with 95% oxygen and 5% carbon dioxide in 37 degrees C. 3beta-HSD activity was measured by radioimmunoassay (RIA).</p><p><b>RESULTS</b>(1) 0.2 mmol x L(-1) and 2.0 mmol x L(-1) L-tyrosine significantly inhibited 3beta-HSD activity. (2) 0.2 mmol x L(-1) L-tyrosine exerted different effects on 3beta-HSD activity at different concentrations of pregnenolone (Ph). It increased 3beta-HSD activity at 0.1 micromol x L(-1) and 1 micromol x L(-1) of Pn concentration. With further increase in the concentration of Pn to 100 micromol x L(-1), the stimulating effect of L-tyrosine was switched to suppression effect. (3) L-tyrosine and L-tyrosine hydrazide both inhibited 3beta-HSD activity induced by hCG.</p><p><b>CONCLUSION</b>L-tyrosine affects 3beta-HSD activity of rat luteal cells in vitro. L-tyrosine and tyrosine hydrazide inhibits hCG induced 3beta-HSD activity.</p>

Apoptosis and Peripheral Benzodiazepin Receptor (PBR) Expression in Human Granulosa-Luteal Cells by GnRH-agonist / 대한불임학회지

OBJECTIVE: To investigate whether GnRH-agonist (GnRH-Ag) using in IVF-ET affects apoptosis of human granulosa-luteal cells and expression of peripheral benzodiazepine receptor (PBR) protein involved in the apoptosis of the cells. METHODS: Granulosa-luteal cells obtained during oocyte retrieval were cultured and treated with 10(-5) M GnRH-Ag. Apoptosis of the cells by the treatment was confirmed using DNA fragmentation analysis 24 h after culture. The presence of PBR protein within the cells was examined by immunofluorescence staining and the expression of the protein was analyzed by Western blotting. In addition, it was measured for progesterone and nitric oxide (NO) produced by granulosa-luteal cells after GnRH-Ag treatment. To evaluate the relationship between NO production and PBR expression, sodium nitroprusside (SNP) as a NO donor was added in media and investigated the expression of PBR protein by Western blotting. RESULTS: Apoptosis increased in the granulosa-luteal cells 24 h after GnRH-Ag treatment, whereas the expression of PBR protein significantly decreased. Furthermore, the production of progesterone and nitric oxide (NO) by the cells significantly fell from 12 h after the treatment. In the results of Western blotting after SNP treatment, the expression of PBR protein increased in the treatment with SNP alone to the granulosa-luteal cells, but was suppressed in the treatment with GnRH-Ag and SNP. Additionally, the staining result of PBR protein in the cells showed the even distribution of it through the cell. CONCLUSION: These results demonstrate that GnRH-Ag treatment induces apoptosis, decreasing expression of PBR protein and NO production in human granulosa-luteal cells. The present study suggests that one of the apoptosis mechanism of human granulosa-luteal cells by GnRH-Ag might be a signal transduction pathway via NO and PBR.

GnRH-agonist Induces Apoptosis of Human Granulosa-luteal Cells Via Caspase-3 and -9 and PARP Cleavage / 대한산부인과학회잡지

OBJECTIVE: GnRH-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for IVF-ET has been known to affect directly on apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-3 and -9 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism(s) by which GnRH-Ag induces apoptosis in human granulosa-luteal cells. METHODS: Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10(-6) M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after the treatment. Activity of caspase-3 and -9 in the cells was examined by using a fluorogenic substrate. Caspase-3 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage were analyzed by Western blotting. RESULTS: DNA fragmentation in the cells increased in the higher concentration over 10(-6) M GnRH-Ag. In the result of TUNEL assay, the rate of apoptotic cells in GnRH-Ag treatment increased significantly compared with that of saline treatment (p<0.05). The activity of caspase-3 and -9 investigated by using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, the cells treated with GnRH-Ag revealed an increase in active forms of caspase-3 and -9 compared with those of the saline treatment. In addition, cleavage of PARP also increased in the cells treated with GnRH-Ag. CONCLUSION: These results suggest that activation of caspase-3 and -9 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.

The Effect of Gonadotropins and Cytokines on Human Luteal Cell Apoptosis / 대한산부인과학회잡지

OBJECTIVE: Our object is to evaluate the detailed mechanisms of support and regression of the human corpus luteum. METHODS: To investigate the regulation of luteal function by gonadotropins, cytokines, and prostaglandins, the frequency of apoptosis and expression of Fas, Fas-L, Bcl-2, Bax, p53, caspase-8 were examined in cultured human luteal cells after treatment with various doses of FSH (30, 100, or 300 ng/mL), LH (30, 100, or 300 ng/mL), TGFbeta1 (1, 10, or 100 ng/mL), TNFalpha (1, 10, or 100 ng/mL), or PGF2alpha (1, 10, or 100 ng/mL) for 24 h. Cells were tested for apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling TUNEL) method and cell death detection ELISA. Immunostaning was performed using anti-Fas, Fas ligand, Bcl-2, Bax, and p53 antibodies. RESULTS: Incidence of apoptosis determined by TUNEL method in the group without treatment was 1.7+/-0.5% (0 h), 10.8+/-1.6% (24 h), and 12.9+/-1.2% (48 h), respectively. Spontaneous increase was significant at the latter time points. Significant suppression of incidence of apoptosis was observed with LH and TGFbeta1 (P<0.05). On the other hand, significant induction of incidence of apoptosis was observed with TNFalpha and PGF2alpha (P<0.05). Immunostaining revealed that p53 and Bax expressions after treatment with LH or TGFbeta1 were significantly lower than those without treatment. Bcl-2 and caspase-8 expressions were not significantly affected by any substance addition. Also we found that inductions of apoptosis by TNFalpha and PGF2alpha were not correlated with the expression of Fas, Fas ligand, Bcl-2, Bax, p53 and caspase-8. CONCLUSION: Our results suggest that LH and TGFbeta1 may be involved in the support of luteal function via suppression of apoptosis, and that TNFalpha and PGF2alpha may contribute to luteal regression via its induction in human corpus luteum during early luteal phase. Also, Fas, Fas-L, Bax and p53 may play roles in this apoptosis controlled by LH, and TGFbeta1.

Expression of Mouse Homolog of Glucosamine-6-Phosphate Deaminase in Reproductive Organs / 대한남성과학회지

PURPOSE: To verify the expression of the mouse homolog of glucosamine-6-phosphate deaminase (GNPDA) in the testis and reproductive organs. MATERIALS AND METHODS: Expression of GNPDA was examined using a polyclonal antibody raised against a synthetic oliogopeptide of the N-terminus of the protein. RESULTS: In Western blots, an immunoreactive band of Mr 35 kDa was detected in the testis, ovary, and uterus extracts. Expression of GNPDA was greater in the adult than in the immature testis. With immunostaining, a positive signal was found in the cytoplasm of interstitial, Sertoli, and germ cells of adult testis. In the ovary, positive staining was found in the interstitial and luteal cells. Conclusion: The expression of GNPDA in many reproductive tissues suggests that the enzyme plays a housekeeping role in cell physiology and in the differentiation of the seminiferous tubules.

Effects of GnRH Agonist Used for Ovarian Hyperstimulation in Human IVF-ET on the Apoptosis of Preovulatory Follicular Cells / 대한불임학회지

There have been many reports to date regarding the role of GnRH as a local regulatory factor of ovarian function as studies of human and rat ovaries revealed GnRH and its receptor. In recent studies it has been shown that GnRH directly causes apoptosis in the granulosa cells of the rat ovary, and such results leads to the suggestion that the use of GnRH agonist for more stable long term ovarian hyperstimulation in human IVF-ET programs causes granulosa cell apoptosis which may lead to follicular atresia. Therefore this study attempts to determine if granulosa-luteal cell apoptosis occurs in patients during IVF-ET programs in which GnRH agonist is employed for ovarian hyperstimulation. The quality of oocyte-cumulus complexes obtained during ovum pickup procedures were assessed morphologically and then the fertilization rate and developmental rate was determined. Apoptotic cells among the granulosa-luteal cells obtained during the same procedure were observed after staining with Hematoxylin-rosin. The fragmentation degree of DNA extracted from granulosa-luteal cells was determined and comparatively analyzed. There was no difference in the average age of the patients, the number of oocytes retrieved, and fertilization and developmental rates between the FSH/hMG group and GnRH-long group. There was also no difference in the apoptosis rate and pyknosis rate in the granulosa-luteal cells between the two groups. However, when the oocyte-cumulus complexes were morphoogically divided into the healthy group and atretic group without regard for the method of hyperstimulation, the results showed that the number of oocytes obtained averaged 11.09+/-8.75 and 10.33+/-4.53 per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental ,ate (65.96%, 41.51%, respectively, p<0.01) was significantly increased in the healthy group when compared to the atretic group. The degree of apoptosis in the granulosa-luteal cells showed that in the healthy group it was 2.25% which was not significantly different from the atretic group (2.77%), but the pyknosis rate in the atretic group (27.81%) was significantly higher compared to the healthy group (11.35%, p<0.01). The quantity of DNA fragmentation in the FSH/hMG group was 32.22%, while in the GnRH-long group it was 34.27%, showing no significant difference. On the other hand the degree of DNA fragmentation was 39.05% and 11.83% in the healthy group and atretic group, respectively, showing significantly higher increase in the atretic group (p<0.01). The above results suggest that death of granulosa-luteal cells according to the state of the oocyte-cumulus complex is more related to pyknosis rather than apoptosis. Also, the GnRH agonist used in ovarian hyperstimulation does not seem to directly affect the apoptosis of retrieved oocytes and granulosa-luteal cells, and which is thought to be due to the suppression of the apoptogenic effect of GnRH agonist as a result of the high doses of FSH administered.

A Case of Benign Ovarian Steroid Cell Tumor with Huge Ascites and Elevated Serum CA125 / 대한부인종양콜포스코피학회잡지

Steroid cell tumor of ovary, first described as lipid cell tumor, is rare lesions composed entirely of cells resembling typical steroid hormone - secreting cells, that is lutein cells, Leydig cells, and adrenal cortical cells. Steroid cell tumors oftcn secret androgen and manifest themselves with symptoms of virilization. Other presentations include abdominal swelling or pain, menstrual dysfunction, postmenopausal bleeding, or rarely ascites. We experienced a case of right ovarian steroid cell tumor, not otherwise specified(NOS), manifested hirsuitism and amenorrhea in 49 - year - old patient. The tumor was about 5 cm in size, and associated with huge ascites (l3,000 ml), both pleural effusion, and elevated serum CA 125. We present a case of Meigs syndrome associated with benign ovarian steroid cell tumor with a brief review of the literature.

Cistos teca-luteínicos na gravidez: relato de caso / Theca-lutein cysts in pregnancy: a case report

Este é o relato de um caso de cistos teca-luteínicos na gravidez, havendo apenas 35 casos previamente relatados na literatura médica mundial (Tabela I). A paciente foi examinada, ecograficamente, na sua 34ª semana de gestaçäo, quando diagnosticamos extensas massas císticas anexiais bilaterais. A única anormalidade clínica observada foi clitoromegalia. Após isto, exames laboratoriais específicos mostraram aumento acentuado de taxas hormonais. Em dois exames ecográficos de controle, durante a gravidez, o aumento anexial bilateral persistiu. Também observamos hidronefrose bilateral no feto. Após o parto, na 39ª semana de gestaçäo, a criança, do sexo masculino, teve bons Apgar e peso. Uma ressecçäo subtotal bilateral dos ovários foi submetida a análise anatomopatológica, que revelou extensa luteinizaçäo e edema, compatíveis com múltiplos cistos teca-luteínicos bilaterais. Durante o controle ecográfico da paciente, após nove semanas, observamos normalizaçäo dos ovários, clitóris e taxas hormonais. Este caso demonstra a importância da visibilizaçäo dos anexos durante todas as semanas da gestaçäo. Entretanto, mesmo com o extenso aumento dos anexos, bilateralmente, e do seu aspecto ecográfico complexo, esta é uma condiçäo benigna autolimitada pela gravidez e nenhum tratamento e indicado

Diagnostic Significance of 3beta-Hydroxysteroid Dehydrogenase Polyclonal in Human Steroidogenic Tumors / 대한해부학회지

The enzymatic complex 3beta-hydroxysteroid dehydrogenase Delta5-Delta4 isomerase(3beta-HSD) catalyzes the obligatory oxidation and isomerization of Delta4-hydroxypregnene and Delta5-hydroxyandrostene steroid precursors into Delta4-ketosteroid necessary for the formation of all classes of steroid hormones such as glucocorticoid, mineralocorticoid, progesterone, androgen and estrogen. The 3beta-HSD enzymatic activity is present at the syncytiotrophoblast, the sebaceous gland of the skin, the Leydig cells of the testis, the thecal cells and granulosa lutein cells ofthe ovary in human. So we thought that 3beta-HSD antibody can be used to diagnose several steroidogenic tumors in human, but there has been few trial for it. In this study, we made polyclonal antibody against human placental 3beta-HSD. And then immunohistochemical studies with the antibody were performed to diagnose several kinds of steroidogenic tumors in human. The polyclonal antibody detected specifically the placental protein of 43 KDa by Western blotting. And the polyclonal antibody reacted on the syncytiotrophoblast, the sebaceous gland of the skin, the Leydig cells of the testis, adrenal cortex, the thecal cells and granulosa lutein cells of the ovary with immunohistochemical staining. In immunohistochemical staining, the polyclonal antibody against 3beta-HSD reacted on the primary and metastatic choriocarcinoma, placental site trophoblastic tumor, sebaceous carcinoma, adrenal cortical adenoma and carcinoma. But it did not react on the squamous cell carcinoma, the basal cell carcinoma and malignant melanoma of the skin, the adenocacinoma and the leiomyosarcoma of the uterus, the metastatic and primary renal cell carcinoma. From these results, the polyclonal antibody against human placental 3beta-HSD demonstrated to have specific reaction on steroidogenic tumors and also it can be used in differential diagnosis of primary and metastatic related to steroid hormone.