Cell Line, Tumor ; Choriocarcinoma ; metabolism ; Chorionic Gonadotropin ; pharmacology ; Female ; Humans ; Tissue Inhibitor of Metalloproteinases ; metabolism

Rabbit follicular oocytes were cultured in a medium supplemented with various elements such as bovine serum(RS), bovine serum albumin(BSA), amino acids and chorionic gonadotrophic hormone(HCG) in order to find which factors among them were most effective for oocyte maturation. The presence of BSA in the basic medium (modified Krebs-Ringer bicarbonate) did not elevate the proportion of oocyte maturation. When BS alone was added to the medium, only a few oocytes could reach to metaphase I and most of them were in degeneration. This implies that BS may act as an inhibitory or a toxic agent to the rabbit oocytes. It was found that the medium supplemented with 0.4% BSA and amino acids together raised the proportion of the oocyte maturation (54-62%). Especially the presence of proline, or of both proline and glutamine, gave a more favourable condition for the initiation of meiotic division than other amino acids. Addition of HCG to the medium did not promote the proportion of the oocyte maturation. As a consequence, it is apparent that amino acids in the medium are the most essential factors in inducing oocyte meiotic division.
Amino Acids/pharmacology* ; Animal ; Chorionic Gonadotropin/pharmacology ; Culture Media* ; Female ; Growth ; Oocytes/physiology* ; Ovum/physiology* ; Rabbits ; Serum Albumin, Bovine/pharmacology

OBJECTIVETo study the effect of mifepristone on the expression of chorionic gonadotropin beta subunit (beta-CG) and collagen type IV in female Rhesus monkey decidua and villus during the first trimester of pregnancy.

METHODSEighteen sexually mature female Rhesus monkeys were allowed to cohabitate with their male counterparts and diagnosed as being pregnant by B-ultrasound at days 38-45 of the menstrual cycle. They were randomly divided into mifepristone treatment group (15 mg/ml.kg.day, 3 days), control group(no treatment) and CMC-Na treatment group(0.5% CMC-Na, 1 ml/kg.day, 3 days). The content and distribution of chorionic gonadotropin beta subunit and collagen type IV in the female Rhesus monkeys were examined by immunohistochemical technique.

RESULTSCompared with the control group, the immunohistochemical reaction intensity of chorionic gonadotropin beta subunit in the plasm of stroma cells, glandular epithelial cells and decidual cells of decidua, and synctial trophoblast cells of villus significantly decreased in the mifepristone treatment group. The positive staining of collagen type IV surrounding the epithelial glandular basal membrane, decidual cells and blood vessels in decidua and surrounding the synctial trophoblast cells in villus were significantly reduced.

CONCLUSIONMifepristone may have a strong effect in decreasing the bioactivity of beta-CG in decidua and the synthesis of beta-CG in villus, and in accelerating the degradation of collagen type IV in decidua and villus.

Animals ; Chorionic Gonadotropin, beta Subunit, Human ; analysis ; Chorionic Villi ; chemistry ; Collagen Type IV ; analysis ; Decidua ; chemistry ; Female ; Immunohistochemistry ; Macaca mulatta ; Mifepristone ; pharmacology ; Pregnancy

BACKGROUNDIt is believed that estrogen plays pivotal roles in the regulation of follicle/oocyte maturation and oocyte fertilizability. It is also involved in the functional preparation of the fallopian tubes for subsequent gamete interaction, in early embryonic development occurring in the tubal microenvironment, and in the preparation of the uterus for implantation. This study was designed to determine whether estrogen is required for follicular and embryonic development.

METHODSThe biosynthesis of estrogen was blocked by a daily injection of the aromatase inhibitor, Arimidex, at a dose of 100 micro g/d, using 3 - 4 week old C57B6 F1 female mice. Injections were continued for 3 days in experiment 1 (n = 10) and for 5 days in experiment 2 (n = 23). Mice in the control group (n = 27) were given the same amount of saline. Exogenous gonadotrophin [7.5 IU pregnant mare serum gonadotrophin (PMSG)] was administered to induce follicular growth and development on the second day. In experiment 1, we tested estrogen and progesterone levels and examined ovary morphology two days later. In experiment 2, 47 hours after PMSG injection, 5 IU human chorionic gonadotropin (hCG) was given and two female mice were then caged with a male mouse overnight. Two days later, we measured estrogen and progesterone levels. We then removed the embryos, cultured them, and examined embryonic development every 24 hours for 3 days.

RESULTSBefore hCG injection, estrogen levels in mice from the Arimidex group were suppressed by 94%, and progesterone levels were suppressed by 75%. There was no difference between the two groups in mean number of total follicles found per animal (30.4 follicles/animal in the control group and 27 follicles/animal in the Arimidex group). Two days after hCG injection, estrogen levels in the Arimidex group were significantly lower than that in the control group (P < 0.01), while progesterone levels were not significantly lower (P > 0.05). The rate of development of embryos, morulae, blastocysts, and hatching blastocysts was not significantly different between the two groups (P = 0.20, 0.10, 0.44, and 0.38, respectively).

CONCLUSIONSIn the present study, by depriving mice of normal estrogen support, we have been able to rule out the absolute need for rising levels of estrogen for the completion of the follicular maturation process and the development of embryos in vitro.

Animals ; Chorionic Gonadotropin ; pharmacology ; Embryonic and Fetal Development ; Estrogens ; physiology ; Female ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Nitriles ; pharmacology ; Oocytes ; physiology ; Ovarian Follicle ; physiology ; Pregnancy ; Triazoles ; pharmacology

The present study was carried out to investigate the effect of antisense c-myb oligodeoxynucleotides (ODN) on hCG-induced testosterone secretion in isolated rat Leydig cells. The effects of cAMP, Ca(2+) and cycloheximide (CYX) on c-Myb protein expression and testosterone secretion were also observed. The results showed that antisense c-myb ODN inhibited hCG-induced testosterone secretion of isolated rat Leydig cells in a dose-dependent manner. At the same time, integral optical density immunostaining of Myb in Leydig cells was also remarkably reduced. Nonsense tat ODN had no effect on Leydig cells. Further experiments showed that dbcAMP (100 micromol/L) obviously increased hCG-induced testosterone secretion and integral optical density (IOD) immunostaining of Myb in Leydig cells. Verapamil (10 micromol/L), a Ca(2+) channel blocker, and cycloheximide (50 microg/ml), a protein synthesis inhibitor, reduced the immunostaining of c-Myb, and also lowered hCG-induced testosterone secretion in isolated rat Leydig cells. The results indicate that c-myb closely correlates with hCG-induced testosterone secretion, and that cAMP and Ca(2+)-dependent pathway participates in the expression of protooncogene.
Animals ; Cell Separation ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Leydig Cells ; secretion ; Male ; Oligodeoxyribonucleotides, Antisense ; physiology ; Proto-Oncogene Proteins c-myb ; physiology ; Rats ; Rats, Sprague-Dawley ; Testosterone ; secretion

OBJECTIVETo obtain a simple and effective method to isolate and purify adult Leydig cells.

METHODSThe testes of human adults were digested and then the density gradient centrifugation of the cells was performed with four different Percoll concentrations (60%, 34%, 26%, 21%) to isolate Leydig cells, whose characteristics were identified by cytological observation staining, 3beta-HSD staining and detection of hCG and testosterone secretion.

RESULTSHigh-concentration (> 90%) purified Leydig cells were acquired, and many identification experiments demonstrated the adequate testosterone secretory function of the isolated and purified Leydig cells.

CONCLUSIONThis method is easy and efficient for the isolation and purification of adult Leydig cells.

Adult ; Cell Separation ; methods ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Humans ; Leydig Cells ; cytology ; drug effects ; secretion ; Male ; Povidone ; Silicon Dioxide ; Testosterone ; secretion

OBJECTIVETo explore the feasibility of inducing the differentiation of human adipose tissue-derived stem cells (ADSCs) into Leydig cells in vitro.

METHODSWe isolated ADSCs by digestion with Collagenase I from the subcutaneous adipose tissue, cultured them in the DMEM/F12 medium with 10% fetal bovine serum, and detected the expression of vimentin by immunohistochemistry. We exposed the ADSCs to different concentrations of human chorionic gonadotropin (HCG) for different times, determined the expression of StAR mRNA by real-time PCR, and measured the HCG-induced proliferation of ADSCs by MTT. After a week of induction by HCG and DMSO, we conducted 3beta-HSD immunohistochemistry, and detected the testosterone level in the supernatant and lysis of the cells by radioimmunoassay.

RESULTSThe ADSCs grew well with a positive expression of vimentin. The expression of the StAR gene was positively correlated with the increased concentration of HCG, reaching the peak at HCG 10 U/ml in 1 week culture. The proliferation of ADSCs was significantly increased by HCG induction. A positive expression of 3beta-HSD was observed after 1 week induction with HCG 10 U/ml and DMSO 3.2 x 10(-6)mol/L.

CONCLUSIONHCG enhances the expression of the StAR gene and the proliferation of ADSCs. Induced by HCG 10 U/ml and DMSO 3.2 x 10(-6) mol/L, ADSCs tend to differentiate into Leydig cells.

Adipocytes ; cytology ; drug effects ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Humans ; Leydig Cells ; cytology ; drug effects ; Male

The objective of this study was to analyze the expression of telomerase in granulosa cells and the influential factors in telomerase expression. TRAP-ELISA (telomeric repeat amplification protocol-enzyme linked immunoadsordent assay) method was used to study the expression and control of telomerase in rat preovulatory ovary. We also used radioimmunoassay (RIA) to determine the expression of estradiol (E(2)) and progesterone (P(0)). Furthermore we used MTT to study the proliferation of ovarian granulosa cells. Telomerase activity was significantly enhanced by human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), verapamil and dbcAMP, and was significantly reduced by antisense-c-myb oligodeoxynucleotide (anti-c-myb ODN) treatment. RIA was used to determine the secretion of P(0) and E(2) in all these cell culture media. We found that the secretion of these two hormones was increased when verapamil and FSH were added; no change after adding HCG and dbcAMP; and reduced when anti-c-myb was added. In MTT assay, we found that the antisense hTERT ODN significantly inhibited the proliferation of ovarian granulosa cells. These results demonstrate that telomerase activity is present in ovary antral granulosa cells and its activity is controlled by FSH, HCG, verapamil and anti-c-myb, and is directly related with the function of proliferation.
Animals ; Cell Proliferation ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; enzymology ; Humans ; Ovary ; enzymology ; physiology ; Rats ; Rats, Sprague-Dawley ; Telomerase ; drug effects ; physiology ; Verapamil ; pharmacology

The present experiment aims to examine the efficiency of estrus synchronization using progesterone and equine chorionic gonadotrophin (eCG) and to look at luteal function. During the non-breeding and breeding season, 5 adult female Korean native goats were injected intramuscularly with 2.5 ml of physiological saline as the control. A progesterone impregnated intravaginal sponge was then kept in the same goats for 10 days followed, after a week, by an intramuscular injection of 500 IU eCG. Five adult female Nubian goats were mated with a fertile buck during the non-breeding season. During the non-breeding season 2 of the 5 goats showed a normal estrous cycle (ranging from 18 to 21 days) and 3 a short estrous cycle (ranging from 3 to 6 days). During the breeding season the equivalent figures were 1 and 2. The major axes of the corpus luteum (CL) were measured by means of calipers built into the ultrasonography system, and the concentrations of plasma progesterone (P(4)) were determined by double antibody radioimmunoassay. The mean major axes of the CL in goats showing the short cycle (6.1 +/- 0.5 mm) was significantly smaller than in those showing the normal cycle (8.9 +/- 0.5 mm; p < 0.01) and also the value of P4 in goats showing the short cycle (4.2 +/- 2.1 ng/ml) was significantly lower than for those showing the normal cycle (10.3 +/- 4.3 ng/ml; p < 0.05) at day 3 following ovulation. Three out of 5 Nubian goats became pregnant but only one goat carried to full term. The present experiment indicated that a combination of progesterone and eCG was effective in inducing estrus, although it resulted in a high incidence of short luteal lifespan. The low kidding rate and high incidence of embryonic loss may be due to the instability of the luteal lifespan.
Animals ; Chorionic Gonadotropin/*pharmacology ; Corpus Luteum/*drug effects/*physiology ; Estrus Synchronization/*drug effects/physiology ; Female ; Fertility/*drug effects ; Fertility Agents, Female/pharmacology ; Goats/*physiology ; Horses ; Pregnancy ; Progesterone/blood/*pharmacology

The objectives of this study were to explore whether ovarian vascular endothelial growth factor (VEGF) expression in mice can be regulated by IL-6 (interleukin-6), angiotensin II, FSH, and hCG; and to test whether the mouse ovarian VEGF expression can result in angiogenesis. The ICR mice were sacrificed, and their ovaries were recovered. Recovered ovaries were treated with IL-6, angiotensin II, FSH, and hCG separately and incubated for 24 hours in alpha-MEM. Expression of mRNA and protein of VEGF were assessed by RT-PCR and immunohistochemistry. The resulting angiogenesis was evaluated through immunohistochemical analysis for CD34. Treatment of mice ovaries with IL-6, FSH, and hCG resulted in a significant increase of VEGF mRNA, and IL-6 was the most potent inducer of VEGF. IL-6 and FSH resulted in increased neovascularization in the follicular phase of mouse ovaries. In contrast, angiotensin II could not increase VEGF expression or neovascularization. We documented an in vitro increase in VEGF expression by IL-6, FSH, and hCG; and reaffirmed that the proliferative response of murine ovarian endothelial cells paralleled an increase of VEGF expression.
Vascular Endothelial Growth Factor A/analysis/*genetics ; RNA, Messenger/analysis ; Ovary/*metabolism ; Mice, Inbred ICR ; Mice ; Interleukin-6/pharmacology ; Immunohistochemistry ; Gene Expression Regulation/*drug effects ; Follicle Stimulating Hormone/pharmacology ; Female ; Chorionic Gonadotropin/pharmacology ; Antigens, CD34/analysis ; Animals