O-024. Factors regulating in-vitro maturation of oocytes

Reproductive BioMedicine Online, 2011

The purpose of the study was to evaluate whether a lower concentration of FSH or 2-h incubation with FSH would improve the outcome of in-vitro maturation of oocytes. The immature oocytes were obtained from FVB mice, and were allocated to four groups and incubated in the maturation media for 24 h. The maturation media were supplemented with 10 mIU/ml FSH for 24 h (group 1), 10 mIU/ml FSH for 2 h (group 2), 75 mIU/ml FSH for 24 h (group 3) or 75 mIU/ml FSH for 2 h (group 4). In each group, half of the in-vitro-matured oocytes were fertilized and cultured to blastocysts and the remaining matured oocytes were analysed for growth differentiation factor (GDF)-9 and bone morphogenetic protein (BMP)-15 mRNA to assess the oocyte quality. The maturation rates and oocyte BMP-15 mRNA concentrations were similar among the four groups. The GDF-9 mRNA concentrations were similar in group 2 and group 4. The fertilization and blastocyst rates were higher in groups 2 and 4 than in groups 1 and 3. It is concluded that 2-h incubation with FSH is better than 24-h incubation in terms of the fertilization rate and blastocyst development. RBMOnline

Reproduction, 1979

The effect of adding LH (10 \ g=m\ g NIH-LH-B8/ml) to the medium in which oocytes were undergoing maturation in vitro was studied. The fertilizability of the oocytes was evaluated in the sterile oviduct of a unilaterally ovariectomized, mated recipient. Freshly ovulated oocytes, used as a control of the method, were fertilized at a rate of 72%. Only 14% of oocytes matured in culture (without LH) were penetrated by spermatozoa, and 11% were fertilized normally. Addition of LH to the medium increased these proportions to 43 and 33% respectively. Oocytes matured in the presence of LH were able to develop into apparently normal rats. It is concluded that, although oocytes can mature in vitro spontaneously, and that these matured oocytes can be fertilized, addition of LH increases the numbers 3-fold. LH therefore has a direct maturation-promoting action on the rat oocyte\p=n-\cumulus complex in vitro.

Reproduction, 2012

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC w...

Theriogenology, 2007

There is increasing evidence demonstrating that oocyte quality depends on the events that occur before germinal vesicle breakdown (GVBD), suggesting that the oocyte must accumulate the appropriate information for meiotic resumption fertilization and early embryonic development before chromosome condensation. This situation seems to prevail in large mammals and particularly in the bovine where we have more information than in other species. Signaling events at two different levels controls the changes that must take place for follicular growth and attainment of oocyte developmental competence. The first signaling event comes from the proper differentiation of the follicle as it normally occurs in the dominant follicle in preparation for ovulation. The second signaling event occurs as the process of follicle differentiation signals directly to the oocyte, possibly through the cumulus cells, that conditions are suitable for further embryo development. The first signal, follicular differentiation, becomes possible though a rise and fall of FSH in the circulation, while the second signal might be mimicked partially by the same hormone acting on the cumulus cells. Although FSH is likely involved in these two signaling events, the processes involved are quite different and analysis of gene expression in granulosa, cumulus and oocyte is starting to reveal the complexity of this system. The next challenge is to combine these two pathways into a functional signaling cascade. To be successful and obtain meaningful information, these genomic analyses must be developed and performed in precisely defined conditions of follicular growth and differentiation or culture conditions. Functional genomics already started with the study of function of several genes and genes families in the regulation of follicular growth and follicle-oocyte co-differentiation (i.e. IGF and BMP genes families, EGF). #

Animal Reproduction Science, 2004

The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 μM cysteamine and 1 μg/ml estradiol-17β. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1 UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 μg/ml FSH and 5 μg/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6–7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5 min, followed by 20% EG, 20% DMSO and 0.5 M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN2. After warming, the blastocysts were transferred in pairs into synchronized ewes.The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%).In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production.

Reproduction in Domestic Animals, 2008

The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade-A cumulus oocyte complexes, aspirated from slaughterhouse cycling-gilt ovaries, were cultured in vitro in 400 ll of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (FolltropinÒ-V, 0.50 mg/ml) or ovine FSH (Ovagen TM , 0.44 iu/ml), in four-well dishes under mineral oil, at 38.5°C, 5% CO 2 in humidified air. At the end of each 3-h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000·), after fixation (methanol/ acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi-square and t-test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 ± 12.97%, 71.34 ± 9.86%, mean ± SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.

Indonesian Journal of Obstetrics and Gynecology, 2016

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: T...

Small Ruminant Research, 2008

The roles of gonadotrophins in the regulation of primordial ovarian follicular development remains unclear. The aims of the present study were to investigate the effects of LH alone or in combination with FSH on the survival, activation and growth of in vitro caprine primordial follicles, using histology and transmission electron microscopy (TEM). To this end, samples of the caprine ovarian cortex were cultured for 1 or 7 days (39 • C), in an atmosphere of 5% CO 2 , in minimum essential medium (MEM-control medium) supplemented with different concentrations of LH (0, 1, 5, 10, 50 or 100 ng/ml-Experiment 1). In Experiment 2, the control medium was supplemented with FSH (50 ng/ml) and the different concentrations of LH (0, 1, 5, 10 or 50 ng/ml also used). Small samples of non-cultured ovarian tissue, as well as those cultured for 1 or 7 days in a specific medium were processed for classical histological evaluation and TEM to evaluate the follicular integrity and to calculate the percentage of normal follicles. Additionally, the effects of FSH on the oocyte and follicle diameters of the cultured follicles were evaluated. Results showed that after 7 days of culture, the presence of LH (10, 50 or 100 ng/ml) in the culture media significantly increases the percentage of developing follicles (P < 0.05). In addition, fragments cultured in media supplemented with FSH alone or FSH plus LH had a higher percentage of developing follicles after 1 and 7 days of culture, compared to the control (P < 0.05). At the end of culture period, 1 and 5 ng/ml LH increased the follicular diameter, while the addition of 1, 5 or 10 ng/ml LH increased the oocyte diameter (P < 0.05). A combination of FSH with 1 and 5 ng/ml LH increased the follicular diameter. With regard to follicle survival, low doses of LH (1 ng/ml) maintained follicle viability-similar to day 0 and in the control medium after 7 days of culture. However, higher concentrations of LH (5, 10, 50 and 100 ng/ml) induced atresia in the goat follicles. Culture of the ovarian cortex for 7 days in medium supplemented with FSH alone or FSH plus 1 ng/ml LH kept the ultrastructural characteristics of follicles similar to that of day 0. In conclusion, it can be said that, the addition of low concentrations of LH (1 ng/ml) combined with or without FSH maintained the goat follicular ultrastructural integrity, but LH in doses higher than 5 ng/ml induced atresia in in vitro goat preantral follicles.

2024

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