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In vivo and in vitro activation of dormant primordial follicles by EGF treatment in mouse and human. Zhang J et al. (2020) In the mammalian ovaries, dormant primordial follicles represent the reproductive reserve of individual females. Recently, stimulating the activation of primordial follicles in vitro has been practiced, making the utilization of those dormant follicles to treat female infertility possible. However, there are still lacks of effective upstream molecule and strategy to elevate follicle activation in vivo. In the current study, we revealed that growth factor EGF improved a transiently primordial follicle activation in mice by elevating the CDC42-PI3K signaling activity, and EGF treatment also improved the activation and development of human follicles in ovarian cortical pieces. Using a liquid-solid phase transition bio-gel as a carrier, an efficient in vivo activation system was established by ovarian topical EGF administration to living mice. We found that EGF treatment led to an increase of primordial follicle activation in short time but had no effect on long-term fertility in females. By establishing an inducible premature ovarian insufficiency (POI) mouse model through selectively ablating growing follicles in Zp3-Cre;iDTR mice, we further revealed that our in vivo EGF treatment system improved primordial follicle activation and ovulation of POI ovaries significantly. Taken together, our results revealed that in situ ovarian EGF administration could improve the activation of primordial follicles in living animals, and manipulating activation and development of primordial follicles in vivo might be an efficient approach to improve reproductive health in women.//////////////////
Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes. Sugimura S et al. (2015) Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to amphiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2'-O-dibutyryladenosine 3':5' cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2, TNFIPA6 and PTGS2. Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence.//////////////////
Epidermal Growth Factor (EGF) Sustains In Vitro Primordial Follicle Viability by Enhancing Stromal Cell Proliferation via MAPK and PI3K Pathways in the Prepubertal, but Not Adult Cat Ovary. Fujihara M 2014 et al.
This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (<6 mo) versus adult (>8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P < 0.05) follicle viability in prepubertal donors in a dose-dependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by co-incubation with an inhibitor of EGF receptor (AG1478), MAPK (U0126), or PI3K (LY294002). EGF enhanced (P < 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P < 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying age influence in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females.
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118 effects of epidermal growth factor on in vitro survival and antrum formation of isolated ovine preantral follicles. da Paz Santos L 2013 et al.
The aim of this study was to verify the beneficial effects of different concentrations of epidermal growth factor (EGF) on survival and antrum formation of isolated ovine preantral follicles cultured in vitro. Ovine ovaries (n=50) were collected from a local slaughterhouse and secondary follicles (150-200?m in diameter), without antral cavities, were mechanically isolated by microdissection using 26-gauge needles. After selection, the follicles were individually cultured in 100-?L droplets of culture medium at 39?C and 5% CO2 in air for 18 days. The basic control medium consisted of a-minimal essential medium (a-MEM) supplemented with BSA; insulin, transferring and selenium; glutamine; hypoxanthine; and ascorbic acid and then referred to as a-MEM(+). For the experimental conditions, follicles were cultured in a-MEM(+) alone (control) or in different concentrations of EGF (1, 10, or 50ngmL(-1)). Every other day, 60?L of the culture media was replaced with fresh media. The morphological aspects of all ovine follicles were assessed every 6 days using a precalibrated ocular micrometer in a stereomicroscope at 100?magnification. Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. The rupture of the basement membrane was also observed and characterised as follicle extrusion. In addition, antral cavity formation was defined as the emergence of a visible translucent cavity within the granulosa cell layers. Data from morphologically normal follicles, extruded follicles, and antrum formation rate during in vitro culture were expressed as percentages and compared by the chi-squared test, and differences were considered significant when P<0.05. The results showed that the percentage of morphologically normal follicles decreased significantly throughout the culture periods in all the treatments, except in the 50ngmL(-1) EGF group, which maintained the percentage of normal follicles from Day 0 to 6. Considering the same culture period, 50ngmL(-1) EGF treatment significantly increased the percentage of morphologically normal follicles at Day 18 compared with the control group. Moreover, the addition of EGF to the culture medium, at 50ngmL(-1), significantly reduced the precocious extrusion of oocytes and increased the percentage of antrum formation compared with the control and 1ngmL(-1) EGF after 18 days of culture. Notably, there were no significant differences between 10ngmL(-1) EGF, control medium, and 1ngmL(-1) EGF treatments. In conclusion, this study demonstrated that the addition of EGF to the in vitro culture medium, at 50ngmL(-1), increased the proportion of morphologically normal follicles and antrum formation rate of isolated ovine preantral follicles.
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Regulation of Gap Junctional Communication Between Cumulus Cells During In Vitro Maturation in Swine, a Gap-FRAP Study. Santiquet NW et al. Intercellular gap junctional communication (GJC) plays an important role in ovarian cell physiology. Closure of GJC has been proposed to be involved in oocyte maturation, particularly in the resumption of meiosis, both in vivo and in vitro, by controlling the flow of meiosis inhibitors such as cAMP and cGMP. Understanding how GJC dynamics are regulated during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and oocyte maturation in vitro. Since little is known about the GJC dynamic regulation between cumulus cells, we have developed an assay based on recovery of calcein fluorescence in photo-bleached cumulus cells, a gap-FRAP assay (Fluorecence Recovery After Photobleaching). The GJC profile has been characterized during the first hours of porcine IVM. We showed that equine chorionic gonadotropin (eCG) and epidermal growth factor (EGF) down-regulated GJC effectiveness between cumulus cells. However, human chorionic gonadotropin was not down-regulating GJC effectiveness. We also showed that the GJC network expanded during this period and that this effect was not regulated by gonadotropins. Porcine follicular fluid present in the maturation medium also had an impact on GJC regulation, increasing GJC network establishment and the effectiveness of calcein transfer rate between cumulus cells. These results show that both eCG and EGF are regulating the decrease in GJC effectiveness after 4.5 h of IVM while the network extension is gonadotropin-independent. Regulation of GJC between cumulus cells would then be specifically regulated during in vitro maturation.
Goritz F, et al. found specific binding sites of EGF were present on granulosa cells of secondary and tertiary follicles and on interstitial gland cells. The EGF-binding capacity of granulosa cells of the cumulus oophorus was greater than that of the mural granulosa cells. Granulosa cells of atretic follicles showed a lower or no affinity for staining. In conclusion, we suggest that EGF plays an important role in ovarian folliculogenesis in cats. Makarevich AV, et al.found that EGF stimulated the release of progesterone (1-100 ng/ml), cAMP (at 100 ng/ml), cGMP (1-100 ng/ml). EGF effect on estradiol output was biphasic: at dose 1 ng/ml it inhibited, whilst at 100 ng/ml it strongly increased estradiol secretion.
Epidermal growth factor-receptor tyrosine kinase activity regulates expansion of porcine oocyte-cumulus cell complexes in vitro.
Prochazka R, et al have recently shown that epidermal growth factor (EGF) strongly stimulates expansion of porcine oocyte-cumulus complexes (OCCs) isolated from large follicles (>6 mm) and does not promote expansion of OCCs from small (3-4-mm) follicles. In order to elucidate the role of EGF in OCCs expansion, in the present study, we first examined the presence of EGF receptors (EGFRs) in cumulus cells isolated from follicles of different sizes. Surprisingly, immunoblotting showed that cumulus cells obtained from all follicular size categories contained similar amounts of EGFR protein. On the other hand, we found a dramatic difference in the pattern of protein tyrosine phosphorylation in a comparison of cumulus cells isolated from small and large follicles treated by EGF. Furthermore, tyrosine-phosphorylated EGFR was specifically immunoprecipitated with antiphosphotyrosine antibodies from EGF-treated cumulus cells isolated from the large follicles. This result strongly indicates that only OCCs from the large follicles contain mature EGFRs that are capable of becoming activated by EGF. Remarkably, preincubation of cumulus cells from small follicles (3-4 mm) with FSH strongly increased EGF-stimulated tyrosine phosphorylation to levels comparable with OCCs from large follicles. The FSH-dependent activation of EGFRs was beneficial for expansion of OCCs isolated from the small follicles since OCCs treated sequentially by FSH (3 h) and EGF (1 h) underwent expansion significantly better then OCCs cultured in FSH or EGF alone. We conclude that a FSH-dependent pathway has an important role in the maturation of the EGFR in cumulus cells and that activation of EGFR-dependent signaling is sufficient to induce expansion.
Dekel N, Sherizly I 1985 reported that epidermal growth factor induces maturation of rat follicle-enclosed oocytes.
Gonadotropin-induced differentiation of ovarian granulosa cells in culture is inhibited by epidermal growth factor (EGF). The present study was undertaken to test a possible inhibitory effect of EGF on LH-induced maturation of rat follicle-enclosed oocytes. We have found that EGF not only failed to affect LH action but served by itself as an inducer of maturation of follicle-enclosed oocytes. EGF action on the oocytes was dose and time dependent and could be prevented by (Bu)2 cAMP. The response of the oocytes was specific to EGF and could not be elicited by other growth factors such as nerve growth factor and insulin. The response to EGF was not limited to the large antral follicles, as oocytes enclosed by small antral follicles (less than 0.4 mm) were induced to mature by EGF as well. In addition, we have demonstrated that oocytes, induced to mature by EGF, are concomitantly uncoupled from the follicular cells. Based on these results we suggest that EGF may terminate the transfer of a follicular inhibitor to the oocyte. It is also possible, however, that EGF induces oocyte maturation by a mechanism independent of its effect on communication between the cellular components of the follicle.
Epidermal growth factor enhances preimplantation developmental competence of maturing mouse oocytes. De La Fuente R et al. The objective of this study was to determine whether epidermal growth factor (EGF) promotes nuclear and cytoplasmic maturation of mouse oocytes grown in vivo or in vitro. In-vivo-grown oocytes were isolated at the germinal vesicle (GV) stage from gonadotrophin-primed (PR) or -unprimed (UPR) 22-day-old mice before in-vitro maturation (IVM). In-vitro-grown (IVG) oocytes were isolated from preantral follicles of 12-day-old mice and grown in vitro without gonadotrophins for 10 days before maturation (IVG/IVM oocytes). IVM and IVG/IVM oocytes were matured in medium supplemented with either EGF (10 ng/ml), follicle stimulating hormone (FSH) (100 ng/ml), EGF plus FSH, or with neither ligand (control). When oocyte-cumulus cell complexes were isolated from PR and UPR mice, IVM with EGF (10 ng/ml), alone or in combination with FSH (100 ng/ml), increased (P < 0.05) the incidence of nuclear maturation to metaphase II. Cytoplasmic maturation of oocytes from PR females, manifested as increased frequency of cleavage to the 2-cell stage and development to the blastocyst stage, was also enhanced with EGF (P < 0.05). Moreover, EGF increased the number of cells per blastocyst, but only in the absence of FSH (P < 0.01). In contrast, EGF, FSH, or EGF plus FSH did not affect the percentage of oocytes from UPR mice completing preimplantation development, but did increase the number of cells per blastocyst. These ligands also increased the proportion of IVG oocytes reaching metaphase II (53-57%) compared with controls (25%; P < 0.05). EGF alone or in combination with FSH increased (P < 0.05) the frequency of blastocyst formation (23% and 28%, respectively) compared with controls (13%). EGF treatment of maturing IVG oocytes produced blastocysts with more cells than other IVG groups (P < 0.05). It is concluded that gonadotrophins in vivo increase the sensitivity or responsiveness of cumulus cell-enclosed oocytes to EGF, thereby promoting both nuclear and cytoplasmic maturation. However, oocyte-granulosa cell complexes grown in vitro become responsive to EGF without gonadotrophin treatment. Thus, nuclear and cytoplasmic maturation of IVG oocytes is promoted by EGF treatment during meiotic maturation.
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