Amphiregulin promotes the maturation of oocytes isolated from the small antral follicles of the rhesus macaque

Abstract

Background: In non-primates, the epidermal growth factor (EGF) and EGF-related ligands such as amphiregulin (AREG) serve as critical intermediates between the theca/mural cells and the cumulus-oocyte-complex (COC) following the mid-cycle LH surge. Studies were designed in primates (1) to analyze AREG levels in follicular fluid (follicular fluid) obtained from pre-ovulatory follicles, as well as (2) to assess dose-dependent effects of AREG on oocytes from small antral follicles (SAFs) during culture, including meiotic and cytoplasmic maturation.

Methods: Controlled ovulation protocols were performed on rhesus monkeys (n=12) to determine AREG content within the single, naturally selected dominant follicle after an ovulatory stimulus. Using healthy COCs (n=271) obtained from SAFs during spontaneous cycles (n=27), in vitro maturation (IVM) was performed in the absence or presence of physiological concentrations of AREG (10 or 100 ng/ml) with or without gonadotrophins (FSH, 75 mIU/ml; LH, 75 mIU/ml). At the end of the culture period, oocyte meiotic maturation was evaluated and ICSI was performed (n=111), from which fertilization and early embryo development was followed in vitro.

Results: AREG levels in follicular fluid from pre-ovulatory follicles increased (P<0.05) following an ovulatory bolus of hCG at 12, 24 and 36 h post-treatment. At 12 h post-hCG, AREG levels in follicular fluid ranged from 4.8 to 121.4 ng/ml. Rhesus macaque COCs incubated with 10 ng/ml AREG in the presence of gonadotrophins displayed an increased percentage of oocytes that progressed to the metaphase II (MII) stage of meiosis (82 versus 56%, P<0.05) and a decreased percentage of metaphase I (MI) oocytes (2 versus 23%, P<0.05) relative to controls, respectively. The percentage of either MI or MII oocytes at the end of the culture period was not different between oocytes cultured with 100 ng/ml AREG or in media alone. Fertilization and first cleavage rates obtained by ICSI of all IVM MII oocytes were 93 and 98%, respectively, and did not vary among treatment groups. Of the MII oocytes that fertilized (n=103), 37 were randomly selected and maintained in culture to assess developmental potential. A total of 13 early blastocysts were obtained, with four embryos developing to expanded blastocysts.

Conclusions: These data indicate that AREG levels increase in rhesus macaque pre-ovulatory follicles after an ovulatory stimulus, and a specific concentration of AREG (10 ng/ml) enhances rhesus macaque oocyte nuclear maturation but not cytoplasmic maturation from SAFs obtained during the natural menstrual cycle. However, owing to the small number of samples in some treatment groups, further studies are now required.

Figure 1

AREG levels in the follicular fluid of pre-ovulatory follicles. Follicles were obtained at 0, 12, 24 and 36 h post-hCG from rhesus monkeys undergoing COv protocols. Values are the mean ± SEM, n = 3/time point. Different letters represent significant differences among time points (P< 0.05).

Figure 2

Representative pictures of MII oocytes from rhesus monkeys after 48 h of IVM. (A and C) Images of MII oocytes taken by light microscopy (20×). (B and D) Confocal microscopy (63×) images of the spindles and PBs from the same MII oocytes after immunofluorescence (red = F-actin, green = tubulin, blue = DNA). (B) shows a typical barrel-shaped spindle with aligned chromosomes and a normal PB, whereas (D) shows a normal-shaped spindle but with an abnormal, diffuse PB.

Figure 3

Representative pictures of an early (A) and an expanded (B) blastocyst obtained after ICSI of IVM MII oocytes from rhesus monkeys (20× magnification).