Cumulus-oocyte complexes from small antral follicles during the early follicular phase of menstrual cycles in rhesus monkeys yield oocytes that reinitiate meiosis and fertilize in vitro

Abstract

The stage at which follicle-enclosed cumulus-oocyte complexes achieve developmental competence in primates is unknown. Therefore, studies were designed to characterize the ability of oocytes in small antral follicles present during the menstrual cycle to spontaneously resume meiosis, fertilize, and support early embryo development. Ovaries were removed from adult rhesus monkeys (n = 12) during the early follicular phase (Days 3-4) of spontaneous cycles. Small antral follicles were divided into five groups according to their diameter; group I: <0.5 mm; group II: 0.5-0.99 mm; group III: 1.0-1.49 mm; group IV: 1.5-1.99 mm; and group V: 2.0-2.5 mm. The cumulus-oocyte complex from healthy small antral follicles (devoid of dark oocytes or granulosa cells) were extracted (n = 199) and cultured for 48 h under different conditions: in TALP (tyrode, albumin, lactate, pyruvate) medium alone, SAGE medium alone, or plus gonadotropins. At 48 h, oocyte meiotic status and diameter were measured after treatment of cumulus-oocyte complexes with hyaluronidase. Cumulus-oocyte complexes derived from follicles of 0.5- to 2-mm diameter contain oocytes that typically reinitiate meiosis in the absence or presence of gonadotropins and fertilize via in vitro fertilization or intracytoplasmic sperm injection. Moreover, the inseminated oocytes can reach the morula stage but arrest. Thus, the ability of these oocytes to complete maturation, as monitored from subsequent embryonic development after fertilization, is suboptimal. Further studies on primate IVM of oocytes from SAFs are warranted in order for them to be considered as an additional, novel source of gametes for fertility preservation in cancer patients.

FIG. 1.

A and B) Representative pictures of healthy small antral follicles (SAFs) dissected from the ovaries of adult monkeys during the early follicular phase of the menstrual cycle. COCs are easily observed through SAFs of different diameters. Arrows denote the presence of blood vessels containing red blood cells. Size distribution of healthy SAFs dissected from all animals is summarized in C. SAFs were divided into five groups according to their diameter; group I: <0.5 mm; group II: 0.5–0.99 mm; group III: 1.0–1.49 mm; group IV: 1.5–1.99 mm; and group V: 2.0–2.5 mm. Not every animal yielded SAFs in each size group.

FIG. 2.

Representative pictures of monkey oocytes at different stages of nuclear maturation after isolation from SAFs during the early follicular phase of the menstrual cycle and 48 h of culture (GV: B; MI: C; MII: D) as well as degenerating (A). The surrounding cumulus cells were removed by hyaluronidase treatment. Original magnification ×20.

FIG. 3.

Confocal microscopy images of representative oocytes from SAFs after 48 h of culture in media with or without gonadotropins. A) Oocyte matured in SAGE media without supplementation. Oocyte shows first PB adjacent to MII spindles. Spindle shown separately in inset stained with TUBA (green). B) Oocyte matured in TALP without supplementation. The oocyte shows a MII spindle that is somewhat adjacent to the first extruded PB; tzp fragments remain along the oolemma (red). TUBA staining alone (green) is shown in the inset. C and D) Oocytes matured in SAGE media with LH and FSH. MII oocytes (C and D) showing adjacent spindle to PB placement. The oocytes have small MII spindles and small, extruded first PBs. Bright red staining (F-actin) shows remnants of tzp after IVM. Tubulin staining alone (green) is shown in the inset. E and F) oocytes cultured in TALP with LH and FSH. MII oocyte (E) with spindle placement adjacent to extruded polar body. An oocyte (F) with two spindles (green) from incomplete cytokinesis. Numerous transzonal projections (tzps, arrows) seen with F-actin staining (red). Numerous tzps are also seen around the oocyte (red). TUBA staining alone (green) is shown in the inset. Bars = 50 μm and 25 μm (insets).

FIG. 4.

Representative pictures of MII oocytes prior to (A, F, and K) and after insemination using ICSI (Day 1: B, G, and L; Day 3: C, H, and M; Day 5: D, I, and N; Day 7: E, J, and O). After insemination, the presence of two pronuclei (PN) and two polar bodies (PB) was checked to confirm fertilization (B, G, and L). Some embryos arrested at very early stages (e.g., four cell; D and E), whereas others continued cleavaging until the morula stage (J and O), when they arrested. Original magnification ×20.