ABSTRACT
OBJECTIVES: To investigate the effect of LH receptor in folliculogenesis, we confirm the expression level of LH receptor (LH-R) mRNA in human granulosa cells (GCs) and its expression levels were analyzed by comparison to embryo developmental rate and pregnancy rate. MATERIALS AND METHODS: GCs were obtained at the time of oocyte retrieval from the patients undergoing IVF-ET program. The patients were divided into two groups: Group I (n=20) is poor responder (retrieved oocyte(s)3ea). After the extraction of total RNA, semiquantitative RT-PCR was performed and the expression level of LH-R mRNA was normalized by beta-actin. Statistical analysis was performed by using Chi(2) test, Student's t-test and Pearson correlation. RESULTS: In Group II, the relative values of LH-R mRNA (0.680 vs. 0.463, p<0.005) and pregnancy rate (54.7% vs. 23.1%, p<0.05) were significantly higher than in Group I. Number of retrieved oocyte(s) was gradually increased when the expression of LH-R mRNA was increased (p<0.05). But the quality of retrieved oocyte and transferred embryo were not related with the expression of LH-R mRNA. When the pregnancy rate was compared with FSH only group and FSH combined with hMG group in the ovarian stimulation protocol, FSH combined with hMG group was significantly higher than FSH only group in Group I (37.5% vs. 0%), and the expression of LH-R mRNA was significantly higher in hMG combined group than FSH only group (p<0.05). CONCLUSION: Expression level of LH-R mRNA has important role in ovarian function related with the response to gonadotrophin in human folliculogenesis. Furthermore these data might provide the evidence that additional use of hMG is helpful to poor responders.
Subject(s)
Female , Humans , Pregnancy , Actins , Embryonic Development , Embryonic Structures , Granulosa Cells , Oocyte Retrieval , Oocytes , Ovulation Induction , Pregnancy Rate , Receptors, LH , RNA , RNA, MessengerABSTRACT
No abstract available.
Subject(s)
Animals , Mice , Estrogens , Immunohistochemistry , UterusABSTRACT
No abstract available.
Subject(s)
Animals , Mice , DNA, Complementary , Estrogens , Laser Capture Microdissection , Oligonucleotide Array Sequence Analysis , UterusABSTRACT
No abstract available.
Subject(s)
Animals , Mice , DNA, Complementary , Estrogens , Laser Capture Microdissection , Oligonucleotide Array Sequence Analysis , UterusABSTRACT
OBJECTIVES: The purpose of this study was to evaluate effects of heparin-binding epidermal growth factor (HB-EGF) on the rate of blastocyst formation and hatching in the mouse embryos and the expression of matrix metalloproteinase-9 (MMP-9) and ATPase g-subunit mRNA. METHODS: Late 2-cell mouse embryos was cultured for 72 hours in HTF medium containing with 1, 10, and 100 ng/ml HB-EGF. The mRNA expression level of MMP-9 and ATPase gamma-subunit was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The rate of hatching was significantly higher (p<0.05) in group containing with 1 ng/ml HB-EGF than other groups. Also, the rate of hatched blastocyst was significantly higher (p<0.05) in 10 ng/ml. The mRNA expression level of MMP-9 mRNA was not shown any difference among groups, but ATPase g-subunit was higher than other groups. CONCLUSIONS: Taken together these results suggest that HB-EGF has the positive effect to promote the blastocyst formation and hatching process and influences the blastocoel expansion by promoting the ATPase mRNA expression in the mouse embryos.
Subject(s)
Animals , Mice , Adenosine Triphosphatases , Blastocyst , Embryonic Structures , Epidermal Growth Factor , Matrix Metalloproteinase 9 , RNA, MessengerABSTRACT
OBJECTIVE: To evaluate the viability and the characteristics of shed endometrial tissues obtained from menstrual fluid during in-vitro culture. METHODS: The menstrual fluids were collected using Wallace catheter from uterine cavity in 10 women with regular menstruation. The menstrual fluids were washed twice, and the pellets, containing blood cells and shed endometrium, were collected and diluted fivefold with Ham's F-10 medium containing 10% fetal bovine serum. The cell suspension was placed on culture dishes, and cultured for 7 days in an incubator. To evaluate the characteristics of the cultured endometrial cells, immunohistochemical (IHC) staining was performed using anti-cytokeratin and anti-vimentin antibody. RESULTS: The mean volume of menstrual fluids and pellets were 0.7ml and 0.3ml, respectively. Only 15% of the shed endometrial tissues were attached and proliferated in culture dishes, which was considered to have viability. Initially, endometrial epithelial cells and fibroblasts were attached and proliferated, and the area of these cells was increased according to prolong the culture time. Stromal cell colonys were located and proliferated on the epithelial cells. IHC staining showed strongly positive for cytokeratin in epithelial cells and for vimentin in stromal cells. In the confocal microscopic observation of 3-dimensional structure of cultured endometrium, cytokeratin-positive cells (epithelial cells) were located in the pheriphery and cytokeratin-negative cells (stromal cells) inside of the structure. CONCLUSION: From our study, shed endometrial tissues in menstrual fluid showed meaningful viability and closed relationship between epithelial cells and stromal cells during in-vitro culture. Thus, we suggest that the in-vitro culture system of shed endometrium is a suitable model for researches of endometriosis.
Subject(s)
Female , Humans , Blood Cells , Catheters , Endometriosis , Endometrium , Epithelial Cells , Fibroblasts , Incubators , Keratins , Menstruation , Stromal Cells , VimentinABSTRACT
No abstract available.
Subject(s)
Animals , Mice , Embryonic Structures , Gene Expression , Interleukin-1ABSTRACT
OBJECTIVE: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. METHODS: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. RESULTS: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. Fpp (25~100 nM) induced a significant increase in the proportion of B-pattern capacitated spermatozoa, and a significant decrease in the proportion of F-pattern uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maintained higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.
Subject(s)
Humans , Male , Acrosome Reaction , Acrosome , Chlortetracycline , Exocytosis , Fertilization , Fluorescence , Semen , SpermatozoaABSTRACT
In the studies on the hatching mechanisms in mammals, many investigators focused on the embryonic intrinsic factor(s) in in vitro culture, but the uterine environment as the extrinsic factor(s) is thought to play an important role in hatching mechanism. Therefore, to evaluate the effect of uterine environment on the hatching event in vivo, the immature(GV) and ovulated(MII) oocytes, and the late 2-cell embryos of mouse were transferred to pseudopregnant foster mother's uterus during peri-implantation period. So it was verified whether there would happen hatching by only uterine environment independently on embryonic stage. The ultrastructural changes of the zona surface of transferred group were compared with those of in vivo and vitro group by SEM. 36 hrs after transfer, the immature and ovulated oocytes almost degenerated, and the late 2-cell embryos developed to various embryonic stages. However, the embryos which didn't develop to blastula stage did not hatch. The ultrastructural network of ZP in transferred group seemed to be smoothed uniformly, which was different from in vitro group. In conclusion, it is suggested that the uterine environment during peri-implantation period enhances the embryo hatching by provoking the structural change of ZP.
Subject(s)
Animals , Humans , Mice , Blastula , Embryonic Structures , Herpes Zoster , Mammals , Oocytes , Research Personnel , Uterus , Zona PellucidaABSTRACT
SUMMARY: Some of the information concerning sexual function in the male diabetes has been focused upon the problems of endocrine or semen parameters. However, the characteristics of acrosome reaction and spermatozoa concentration at the epididymis and vas deferens have scarcely been studied, and the causes of the infertility has not been critically identified. So, we designed to inspect the spermatozoa concentration and the characteristics of acrosome reaction at epididymis and was deferens of diabetic Wistar rat induced by streptozotocin (STZ, 70 mg/kg, ip). Experimental animal was sacrificed at 3 days and 14 days after the STZ injection. In the diabetes-induced rat, the levels of insulin and glucose had a pattern of inverse proportion. The spermatozoa concentrations in caput and corpus epididymis were significantly decreased in all diabetic condition. In cauda epididymis, however, there was significant decrease in sperm concentration at 14 days onward. In diabetic rat, the spontaneous reaction rate of spermatozoa of cauda and was deferens were significantly higher than the control group. The ARIC (acrosome reaction to ionophore challenge) value of caudal sperm was 28.7 at control, 22.1 at 3 days, and 8.3 at 14 days. In the present study the spermatozoa concentration was decreased and the spontaneous reaction rate was increased by diabetes. In ARIC-test, it is revealed that the fertility of spermatozoa of 14 days group was lower than control or 3 days group. Diabetes mellitus may be provoke the decreased fertilization rate and subsequent infertility.
Subject(s)
Animals , Humans , Male , Rats , Acrosome Reaction , Acrosome , Diabetes Mellitus , Epididymis , Fertility , Fertilization , Glucose , Infertility , Insulin , Semen , Spermatozoa , Streptozocin , Vas DeferensABSTRACT
Protein expression patterns of matrix metalloproteinases (MMPs) were examined in mouse reproductive organs during estrous cycle. Estrous cycle was classified into diestrus, proestrus, estrus or metestus and MMP expression was analyzed by zymography using gelatin as a substrate. Uterine fluid (UF) obtained both at diestrus and proestrus exhibited 4 major MMPs including 106kDa, 64kDa, 62kDa and 59kDa gelatinases. However, in UF at estrus, the gelatinolytic activity of 64kDa MMP disappeared and that of 106kDa and 62kDa MMPs dramatically decreased. At metestrus, 64kDa MMP activity reappeared and 106kDa and 62kDa MMP exhibited increased activities such that the band intensity of 106kDa was comparable to that in UF at diestrus. Gelatinolytic activity of 59kDa MMP was not changed throughout the cycle. Both ovarian and oviductal tissue homogenate revealed 4 MMPs which corresponded to the 4 MMPs of UF. However, unlike UF MMPs, gelatinolytic activity of these MMPs did not show distinct changes throughout the cycle. Either an inhibitor of MMP, 1, 10-phenanthroline, or a metal chelator, EDTA, abolished the appearance of the above MMP activities in gelatinated gel whereas a serine proteinase inhibitor, phenylmethylsulfonyl fluoride, failed to inhibit the appearance of MMP activities, proving that gelatinolytic activity of the above reproductive tissues were due to the enzymatic activity of MMP. When gelatinolytic activity of mouse serum was examined, it revealed 5 MMPs (131kDa, 106kDa, 89kDa, 64kDa and 62kDa bands) and one gelatinase (84kDa) band. From these results, it is concluded that the protein expression of MMPs of mouse reproductive organs, particularly uterus, is temporally regulated during estrous cycle and uterine 106kDa, 64kDa and 62kDa MMP,3 are suggested to play an important role in cyclic tissue remodeling of mouse uterus.
Subject(s)
Animals , Female , Mice , Diestrus , Edetic Acid , Estrous Cycle , Estrus , Gelatin , Gelatinases , Matrix Metalloproteinases , Metestrus , Oviducts , Phenylmethylsulfonyl Fluoride , Proestrus , Serine Proteases , UterusABSTRACT
PURPOSE: To verify the expression of the egg activator oscillin in mouse testis and adult organs. MATERIALS AND METHODS: Genomic PCR using primers for oscillin was conducted to confirm that the PCR product was derived from cDNA. Total RNA isolated from developing, immature, and mature testis was subjected to RT-PCR and restriction analysis. In situ hybridization of adult testis was performed to localize the oscillin transcript using cRNA probe. RESULTS: Genomic PCR using the primer for RT-PCR revealed no amplification product, suggesting that the oscillin gene consists of at least two exons. The RT-PCR product of oscillin mRAN was detected in testis beginning 2 weeks after birth. Oscillin mRAN was detected in both germ and somatic cells such as Sertoli and Leydig cells by in situ hybridization. The testis showed al high level of oscillin mRAN compared with other adult organs. CONCLUSION: Oscillin is not a testis-specific transcript and therefore may have another function intracellularly as well ad serving as a trigger for egg activation.
Subject(s)
Adult , Animals , Humans , Male , Mice , DNA, Complementary , Exons , In Situ Hybridization , Leydig Cells , Ovum , Parturition , Polymerase Chain Reaction , RNA , RNA, Complementary , TestisABSTRACT
It is well known that the bona pellucidae of mouse oocytes become 'hardened' when they are allowed to mature in vitro in the absence of serum components. To see if oocytes already undergone meiotic resumption in vivo exhibit similar zona hardening, hardening of ZP of cumulus-enclosed oocytes(CEOs) was examined after culture in vitro since their release from follicles various hours after hCG injection. When CEOs matured in vivo for 3h or longer were subjected to culture in vitro for 14h with BSA alone, zona hardening was significantly reduced compared to those cultured in vitro from the begining of maturation. However, when CEOs matured in vivo for 5h were freed from cumulus cells and then cultured in vitro with BSA alone, little reduction of zona hardening was observed. Preincubation of CEOs for 5h with fetuin, one of the well known inhibitor of in vitro zone hardening, did not prevent bona hardening during its subsequent culture of CEOs for 14h without fetuin. However, when CEOs precultured with both fetuin and PMSG for 5h and then further cultured with BSA alone for 14h, zona hardening was dramatically reduced. Under these conditions, the expansion of cumulus cell was observed. In addition, CEOs cultured with both BSA and dbcAMP to prevent their meiotic resumption showed a significant increase of zona hardening. Whether the observed zona hardening was correlated with the conversion of ZP2 to ZP2f was examined. Zona pellucida, isolated from CEOs matured for 5h in vivo and then further cultured with BSA alone was subjected to SDS-PAGE. Most of ZP2 molecules from these CEOs did not undergo conversion from ZP2 to ZP2f. From these results, it is concluded that CEOs undergone meiotic resumption in vivo do not exhibit bona hardening when they were subsequently cultured in vitro without serum components. It appears that cumulus cells play an important role in this phenomenon.
Subject(s)
Animals , Mice , Bucladesine , Cumulus Cells , Electrophoresis, Polyacrylamide Gel , Fetuins , Herpes Zoster , Oocytes , Zona PellucidaABSTRACT
This study aimed to evaluate the effect of seminal vesicle fluid (SVF) on the acrosome reaction (AR) occurred spontaneously or induced by Ca2+ ionophore A23187, follicular fluid, and progesterone in mouse epididymal sperm. SVF was divided into high (MW>10 kM)) and low (MW<10 kD) fractions by ultrafiltration. The low MW fraction of SVF decreased the rate of spontaneous AR, however the high MW fraction did not. It suggested that the low MW fraction of SVF might have contained decapacitation factor(s) responsible for prolonging of time need for capacitation. When sperm preincubated for 60 min in the presence of SVF, the rate of AR induced by A23187 was decreased, but prolongation of preincubation time for 120 min significantly potentiated the AR by A23187. It suggested that addition of SVF into sperm preincubation medium imposed the epididymal sperm a condition similar to ejaculation. AR induced by human follicular fluid or progesterone was also inhibited by SVF. It suggested that substance in SVF might have affected AR of mouse sperm by inhibiting the interaction between AR inducing ligands and sperm surface receptors involved in acrosomal exocytosis.
Subject(s)
Animals , Female , Humans , Male , Mice , Acrosome Reaction , Acrosome , Calcimycin , Ejaculation , Exocytosis , Follicular Fluid , Ligands , Progesterone , Seminal Vesicles , Spermatozoa , UltrafiltrationABSTRACT
The present study was designed to define the role of prostaglandin in the development and hatching of mouse embryo. The effects of indomethacin, an inhibitor of prostaglandin synthesis, on the development and hatching of morula and blastocyst were examined. In early morula stage, embryos were degenerated significantly at 100 muM and 200 muM indomethacin. However, :he viability of embryos was not influenced by concentration in any other embryonic stages. In all embryonic stages, the hatching was suppressed with concentration dependent manner, but expansion was not suppressed. Particularly, in 84h embryos post hCG injection, the hatching was suppressed significantly compared with post hCG 72h or 96h embryos. When embryos were treated with 100 muM indomethacin for a specific time (12h) in according to the development stage, the hatching was suppressed all groups. These suppressional effect was decreased as embryonic development stage was progressed. However, the expansion was not affected in all treatment group. This study suggests that hatching-related metabolic substances are synthesized from morula stage and intraembryonic signaling mediated prostaglandin was important for development and hatching of mouse embryo.
Subject(s)
Animals , Female , Mice , Pregnancy , Blastocyst , Embryonic Development , Embryonic Structures , Indomethacin , MorulaABSTRACT
No abstract available.