ABSTRACT
Background: The Wingless-type [Wnt]/beta-catenin signaling pathway controls cell homeostasis. Reproductive tissues are dynamic in response to steroidal hormone changes. Steroidal hormones are known to control the Wnt/beta-catenin pathway, but their role in reproductive tissues remains unknown
Objective: The present study aims to investigate the expression patterns of Wnt/beta-catenin target genes in mouse reproductive tissues during the normal estrous cycle
Materials and Methods: In this experimental study, 16 adult NMRI mice were grouped as proestrus, estrus, metestrus, and diestrus according to vaginal smear and histological evaluation of uterine and ovarian tissues. Uterine horns and ovarian tissues were collected. Reverse transcription quantitative polymerase chain reaction was performed to evaluate the expression of Wnt/beta-catenin target genes [Myc2, Ppard, Id2, Birc5, and Ascl2] at different stages of the estrous cycle
Results: The expression levels of Id2, Ascl2, and Pprd in uterine tissue were significantly higher at the proestrus phase than at the other stages. Meanwhile, Birc5 expression in uterine tissue was significantly higher at the metestrus stage than at the other stages. Furthermore, Myc2 expression was significantly higher at the diestrus stage than at the estrus and metestrus stages. In the ovarian tissue, the highest expression of Id2, Ascl2, and Birc5 was detected at the proestrus stage, whereas the highest expression of Myc2 and Ppard was observed at the estrus stage
Conclusion: This study showed that Wnt/beta-catenin target genes profiles are different among estrous cycle. It seems that different hormonal profiles during estrous cycles play a key role in the expression pattern of Wnt/beta-catenin target genes in ovarian and uterine tissue
ABSTRACT
Background: Therapeutic potential of in vitro maturation [IVM] in infertility is growing with great promise. Although significant progress is obtained in recent years, existing IVM protocols are far from favorable results. The first aim of this study was to investi-gate whether two step IVM manner change reactive oxygen species [ROS] and total antioxidant capacity [TAC] levels. The second aim was to find the effect of alpha lipoic acid [ALA] supplementation on oocyte maturation rate and on ROS/TAC levels during IVM
Materials and Methods: In this experimental study, mouse germinal vesicle [GV] oocytes divided into cumulus denuded oocytes [DOs] and cumulus oocyte complexes [COCs] groups. GVs were matured in vitro in the presence or absence of ALA only for 18 hours [control] or with pre-culture of forskolin plus cilostamide for an additional 18 hours. Matured oocytes obtained following 18 and 36 hours based on experimental design. In parallel, the ROS and TAC levels were measured at different time [0, 18 and 36 hours] by 2',7'-dichlorodihydrofluorescein [DCFH] probe and ferric reducing/antioxidant power [FRAP] assay, respectively
Results: Maturation rate of COCs was significantly higher than DOs in control group [P<0.05], while there was no significant difference between COCs and DOs when were pre-cultured with forskolin plus cilostamide. ROS and TAC levels was increased and decreased respectively in DOs after 18 hours while in COCs did not change at 18 hours and showed a significant increase and decrease respectively at 36 hours [P<0.05]. ROS and TAC levels in the presence of ALA were significantly decreased and increased respectively after 36 hours [P<0.05] whereas, maturation rates of COCs and DOs were similar to their corresponding control groups
Conclusion: ALA decreased ROS and increased TAC but could not affect maturation rate of both COCs and DOs in one or two step IVM manner
ABSTRACT
Background: One of the most major obstacles of ovarian tissue vitrification is suboptimal developmental competence of follicles. Matrix metalloproteinases 2 [MMP-2] and 9 [MMP-9] and their tissue inhibitors TIMP-1 and TIMP-2 are involved in the remodeling of the extracellular matrix in the ovaries
Objective: This study aimed to evaluate the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 genes in the preantral follicles derived from vitrified mouse ovaries
Materials and Methods: In this experimental study, the gene expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the isolated preantral follicles derived from fresh and vitrified ovaries of 14-16 days old female mice through real time qRT-PCR was evaluated. Developmental parameters, including survival rate, growth, antrum formation and metaphase II oocytes were also analyzed
Results: The developmental parameters of fresh preantral follicles were significantly higher than vitrified preantral follicles. The TIMP-1 and MMP-9 expression levels showed no differences between fresh and vitrified preantral follicles [p=0.22, p=0.11 respectively]. By contrast, TIMP-2 expression significantly decreased [p=0.00] and MMP-2 expression increased significantly [p=0.00] in vitrified preantral follicles compared with to fresh ones
Conclusion: Changes in expression of MMP-2 and TIMP-2 after ovarian tissues vitrification is partially correlated with decrease in follicle development
ABSTRACT
Background: The uterus is a dynamic tissue responding to hormonal changes during reproductive cycles. As such, uterine stem cells have been studied in recent years. Transcription factors oct4 and sox2 are critical for effective maintenance of pluripotent cell identity
Objective: The present research evaluated the mRNA expression of oct4 and sox2 in the uterine tissues of ovariectomized mice treated with steroid hormones
Materials and Methods: In this experimental study, adult virgin female mice were ovariectomized and treated with estradiol 17 [E2], progesterone [P4], and a combination of E2 and P4 [E2 and P4] for 5 days. Uterine tissues were removed, and immunofluorescent [IF] staining and quantitative real-time PCR of oct4 and sox2 markers were performed
Results: IF showed oct4 and sox2 expression in the uterine endometrium and myometrium among all groups. The mRNA expression of oct4 [p=0.022] and sox2 [p=0.042] in the E2-treated group significantly were decreased compared to that in the control group. By contrast, the mRNA expression of oct4 and sox2 in the P4 [p=0.641 and 0.489 respectively] and E2 and P4-treated groups [p=0.267 and 0.264 respectively] did not show significant differences compared to the control group
Conclusion: The results indicate ovarian steroid hormones change the expression of oct4 and sox2 in the mice uterine tissues, which suggest the involvement of steroid hormonal regulation in uterine stem cells
ABSTRACT
Since the uterine is a sensitive tissue to steroid hormones, the aim of the present study was to investigate the effects of 17 beta-estradiol [E2] and progesterone [P4] alone or in combination on morphological and morphometrically parameters of ovariectomized mouse uterus
Adult virgin female mice [8-10 weeks old]were ovariectomized and treated with E2, P4, E2 followed by P4 and the oil vehicle alone for 5-days period. Uterine tissue was removed, and processed for histology assessment. The total uterine diameter were significantly higher [P < 0.05] following E2 treatment and Maximum diameter of uterine lumen, myometrium and endometrium were recorded after this treatment regimen. Sequential treatment with oestradiol and then progesterone caused both mitotic activity and cell degeneration. P4 treatment induced signs of active secretion in the endometrium glands and symptoms of degeneration and cell death. Estradiol treatment induced growth of uterine tissue. Subsequent treatment with progesterone stimulated uterine tissues to reach maximum size and maturity which is necessary to modify the uterus in preparation for pregnancy
ABSTRACT
The aim of this study was to investigate the in vitro development of isolated pre-antral follicles derived from vitrified ovaries in the presence of coenzyme Q[10] [CoQ[10]]. Mice pre-antral follicles derived from fresh and vitrified-warmed ovarian tissues were cultured individually in alpha-MEM medium supplemented with or without CoQ[10], followed by adding human Chorionic Gonadotropin [hCG] to induce ovulation. The follicle development parameters and ovulated oocyte maturation were assessed. The developmental parameters of pre-antral follicles and oocyte maturation rates were significantly higher in CoQ[10] pretreatment groups of both vitrified and fresh samples compared to the respective CoQ[10] free groups. CoQ[10] improves the in vitro development of pre-antral follicles derived from fresh and vitrified -warmed ovaries
ABSTRACT
In assisted reproduction techniques [ART] settings, reactive oxygen species [ROS] can be produced from endogenous and exogenous sources during in vitro manipulation. Endogenous sources of ROS include gametes and embryo, whereas exogenous sources are oxygen tension, light exposure, culture media, and the nature of some protocols, such as centrifugation or cryopreservation. Elevated ROS production can result in oxidative stress [OS], which is harmful to gametes and embryos, and reduces the procedure's outcomes. Therefore, addressing various aspects of the adverse effects of oxidative stress and its management is necessary
ABSTRACT
Occurrence of oxidative stress [OS] following in vitro culture is inescapable. Therefore, the aim of the present study was to determine the efficacy of Coenzyme Q[10] [CoQ[10]] supplementation medium on developments and Total Antioxidant Capacity [TAC] of mouse vitrified preantral follicles. Fresh and vitrified-warmed isolated preantral follicles from ovaries of 14-16 day-old mice were cultured in supplemented alpha- MEM medium with or without CoQ[10]. On the twelfth day of culture period, ovulation was induced by adding 1.5 IU/ml Human Chorionic Gonadotropin [hCG]. Rates of survival, antrum formation and developmental stages of released oocytes [GV, MI, MII] were evaluated. Separately, Total Antioxidant Capacity [TAC] levels were measured at initial time, 24, 48, 72 and 96 hours of culture period by Ferric Reducing/Antioxidant Power [FRAP] assay. The results showed that the mean diameter and rates of survival, antrum formation, ovulation and MII oocytes of fresh and vitrified-warmed isolated preantral follicles with pretreatment of CoQ[10] were significantly higher than those of respective groups without pretreatment of CoQ[10]. TAC levels significantly increased up to 96 hours in fresh and vitrified-warmed preantral follicles with pretreatment of CoQ[10] compared with those which were cultured without CoQ[10]. Supplemented culture medium with CoQ[10] promotes TAC levels and development of both fresh and vitrified-warmed isolated preantral follicles
ABSTRACT
Cryopreservation of ovarian tissues and pre-antral follicles is a promising prospect for preservation of women fertility. The aim of this study was to evaluate the in vitro developmental competence of mouse vitrified pre-antral follicles in comparison to isolated pre-antral follicles derived from vitrified ovaries in the presence of alpha lipoic acid [ALA]. Pre-antral follicles derived from fresh, vitrified-warmed ovarian tissues and vitrified-warmed pre-antral follicles were cultured individually with or without ALA, followed by adding hCG to induce ovulation. The follicle growth, oocyte maturation, and embryo development were assessed. The diameter and development of follicles, oocyte maturation and embryo development rates were significantly higher in ALA supplemented groups compared to the respective ALA-free conditions groups. Aforementioned parameters were significantly higher in vitrified-warmed follicles in comparison to follicles derived from vitrified-warmed ovaries. These findings support a superior performance of pre-antral follicles when vitrified rather than when isolated from vitrified ovaries with regard to increasing the rates of developmental parameters. Moreover, ALA improves the in vitro maturation of pre-antral follicles in vitrified and non-vitrified samples.
ABSTRACT
Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species [ROS] and total antioxidant capacity [TAC] levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid [ALA]. Isolated pre-antral follicles [140-150 micro m in diameter] were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and nonvitrified samples through increasing follicular TAC level and decreasing ROS levels
ABSTRACT
In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid [ALA] on in vitro oocyte maturation and fertilization. Mouse germinal vesicle [GV] oocytes were categorized into cumulus denuded oocytes [DOs; n=896] and cumulus oocyte complexes [COCs; n=1077] groups. GV oocytes were matured in vitro with or without ALA; [I] without the meiotic inhibitors; [II] supplemented with cilostamide; [III] supplemented with forskolin and [IV] supplemented with Forskolin plus cilostamide. The obtained metaphase II [MII] oocytes were subjected to in vitro fertilization. Independent-samples t-test and ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner
Subject(s)
Female , Animals, Laboratory , In Vitro Oocyte Maturation Techniques , Cyclic AMP , Mice , Cumulus Cells , OocytesABSTRACT
Oocyte maturation and embryo development are controlled by intra-ovarian factors such as steroid hormones. Progesterone [P4] exists in the follicular fluid that contributes to normal mammalian ovarian function and has several critical functions during embryo development and implantation, including endometrial receptivity, embryonic survival during gestation and transformation of the endometrial stromal cells to decidual cells. It is well known that the physiological effects of P4 during the pre-implantation stages of some mammal's embryos are mediated by P4 receptors and their gene expression is determined. The effects of P4 on oocytes and embryo development have been assessed by some investigations, with contradictory results. P4, a dominant steroid in follicular fluid at approximately 18 hours after the luteinizing hormone [LH] surge may have a critical role in maturation of oocytes at the germinal stage. However, it has been shown that different concentrations of P4 could not improve in vitro maturation rates of germinal vesicles [GV] in cumulus oocyte complexes [COCs] and cumulus denuded oocytes [CDOs]. Culture media supplemented with P4 significantly improved mouse embryo development. In addition, an in vivo experimental design has shown high blastocyst survival and implantation rates in P4-treated mice. In this review we explain some of the findings that pertain to the effects of P4 on oocyte maturation and embryo development both in vitro and in vivo
ABSTRACT
The aim of the present study was to investigate the role of progesterone on the developmental competence of cumulus-oocyte complexes [COCs] and cumulus-denuded oocytes [CDOs] at germinal vesicle [GV] stage. GV oocytes of pregnant mare's serum gonadotropin [PMSG]-primed prepubertal mice were divided into two groups: CDOs and COCs. The oocytes were cultured in TCM199 with different concentrations of progesterone [10, 38, 50 and 100 micro M] and without progesterone [controls]. The number of oocytes at the GV, germinal vesicle breakdown [GVBD] and metaphase II [MII] stages were counted. In vitro fertilization [IVF] of MII oocytes and their development to the blastocyst stage were evaluated. Significantly different MII rates were observed between the COCs [85%] and CDOs [68%] control groups. The MII rates of 83%, 48%, 14% and 0% for COCs and 65%, 53%, 20% and 0% for CDOs were obtained in TCM199 that contained 10, 38, 50 and 100 micro M progesterone concentrations, respectively. These MII rates were lower [p<0.05] in both COCs and CDOs as compared to their respective control groups, except for 10 micro M. The fertilization and blastocyst rates of COCs [83% and 35%, respectively] were higher [p <0.05] than those of the CDOs [51% and 5%, respectively] control groups. The fertilization and blastocyst rates in the presence of 10 micro M [81% and 36%, respectively] and 38 micro M [85% and 30%, respectively] progesterone in COCs and CDOs [52% and 4% for 10 micro M; 56% and 4% for 38 micro M] were similar to their respective control groups. Adding progesterone to the medium could not improve maturation of mouse GV oocytes and their development to the blastocyst stage