Human oocyte maturation is dependent on LH-stimulated accumulation of the epidermal growth factor-like growth factor, amphiregulin

Abstract

Background: The LH surge promotes ovulation via activation of multiple signaling networks in the ovarian follicle. Studies in animal models have shown the importance of LH-induced activation of the epidermal growth factor (EGF)signaling network in critical peri-ovulatory events. We investigated the biological significance of regulatory mechanisms mediated by EGF-like growth factors during LH stimulation in humans.

Methods: We characterized the EGF signaling network in mature human ovarian follicles using in vivo and in vitro approaches. Amphiregulin (AREG) levels were measured in 119 follicular fluid (FF) samples from IVF/ICSI patients. Biological activity of human FF was assessed using in vitro oocyte maturation, cumulus expansion and cell mitogenic assays.

Results: AREG is the most abundant EGF-like growth factor accumulating in the FF of mature follicles of hCG-stimulated patients. No AREG was detected before the LH surge or before hCG stimulation of granulosa cells in vitro, demonstrating that the accumulation of AREG requires gonadotrophin stimulation. Epiregulin and betacellulin mRNA were detected in both human mural and cumulus granulosa cells, although at significantly lower levels than AREG. FF from stimulated follicles causes cumulus expansion and oocyte maturation in a reconstitution assay. Immunodepletion of AREG abolishes the ability of FF to stimulate expansion (P < 0.0001) and oocyte maturation (P < 0.05), confirming the biological activity of AREG. Conversely, mitogenic activity of FF remained after depletion of AREG, indicating that other mitogens accumulate in FF. FF from follicles yielding an immature germinal vesicle oocyte or from an oocyte that develops into an aberrant embryo contains lower AREG levels than that from follicles yielding a healthy oocyte (P = 0.008).

Conclusions: EGF-like growth factors play a role in critical peri-ovulatory events in humans, and AREG accumulation is a useful marker of gonadotrophin stimulation and oocyte competence.

Figure 1

Expression of EGF-like growth factors and cognate receptors in the human ovarian follicle. Mural (mGC) and cumulus (CC) granulosa cells were collected 36 h after hCG administration. Total RNA was then isolated and RT–PCR performed. Results are reported as the mean ± SEM of three different samples for EGF-like growth factor expression and three to four different samples for EGF receptor (Erbb) subtype expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

(A) Distribution of AREG protein levels within individual human pre-ovulatory follicles. Individual follicular aspirates were collected 36 h after hCG, and AREG levels determined by ELISA. Each patient contributed a single FF specimen for the analysis. EREG was assayed by ELISA in 20 individual samples and was not detectable in 19 of these, and only present at a low level in one sample. BTC was not detectable in either individual or pooled fluid samples. (B) Follicular AREG is positively correlated with follicular hCG levels. Within individual follicles, AREG and hCG levels were determined and the results plotted. Follicular AREG and follicular hCG levels are positively correlated (P = 0.016).

Figure 3

(A, B) AREG production and secretion is specifically mediated by LH signaling. Primary cell cultures were established from luteinized human granulosa cells obtained at the time of OR. Cells were then stimulated by either recombinant human FSH, hCG or vehicle only for up to 60 h. Cultured medium was assayed for AREG (Fig. 3A) or progesterone (Fig. 3B). Results for AREG production are mean ± SEM values from three different primary cell cultures taken from different patients. Statistical significance is in comparison with the no gonadotrophin stimulation at 24 h. Progesterone results for the primary granulosa cell culture are from an individual patient.

Figure 4

(A) Biologically active AREG in human FF causes CC expansion. Mouse COCs were incubated with 10% human FF (post-hCG) which was immunodepleted (AB) of various EGF-like growth factors. AREG depletion specifically blocks normal CC expansion in this assay. Recombinant AREG (rAREG) is included as a control. Magnification: ×10. (B) Biologically active AREG in human FF causes CC expansion. A histogram showing the mean ± SEM of nine or more COCs analyzed in three separate experiments. Statistical significance determined relative to the mock immunoprecipitated 10% FF. (C) Human FF sampled before hCG administration (pre-hCG FF) does not promote COC expansion. (D) AREG in human FF induces oocyte nuclear maturation. Mouse COCs were incubated with 10% human FF (post-hCG) and assessed for nuclear maturation by light microscopy. Results are mean ± SEM of 15 or more COCs analyzed in three separate experiments. AREG IP, AREG removed by immunoprecipitation.

Figure 5

(A) Mitogenic activity of ovarian FF is not abolished by AREG immunodepletion. [³H] Thymidine incorporation using murine Balb 3T3 cells stimulated with either fetal calf serum (FCS), various FF specimens (post-hCG) or rAREG. Results are expressed as pmol [³H] Thymidine over unstimulated controls. All samples were assayed in duplicate, and all values are mean ± SEM, with minimum n = 3. IP, immunoprecipitation. (B) Two major forms of AREG are distinguishable by ion exchange chromatography. Pooled human FF obtained 36 h after hCG was fractionated under the conditions described in Materials and Methods. Fractions were assayed for total protein by OD280 and BCA, and AREG levels by ELISA.

Figure 6

Levels of AREG in human ovarian follicles yielding normal oocytes or oocytes with compromised developmental potential. Human FF samples were assayed as described in the Materials and Methods. Each point represents a single FF sample with the bars showing the mean. Only a single FF sample was used from a given patient. Where possible, age-matched samples were used, with no significant differences in ages among samples in each of the groups. Data were stratified according to oocyte fertilization after ICSI, as assessed by an embryologist. Abnormal (Abn) embryos are defined as anything other than fertilization of two pronuclei. The GV group is a subgroup where individual follicles produced an immature oocyte.