Structural and endocrine aspects of equine oocyte maturation in vivo

Abstract

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17 beta (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I- to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high.

Relationships of Follicle Versus Oocyte Maturity to Ultrasound Morphology, Blood Flow, and Hormone Concentrations of the Preovulatory Follicle in Mares1

“…The changes in percentage of dark clusters in the ooplasm apparently involve the rearrangement of vesicles and lipid droplets from an even distribution to a more polarized appearance in mares [16]. The positive association between the percentage of ooplasm with dark granules and the oocyte maturation rate in the present in vivo study with equine oocytes is consistent with an in vitro study in cattle that indicated that oocytes with dark clusters of granules had a higher potential for maturation [31].…”

“…In women, both E 2 and P 4 concentrations were greater in follicular fluid of follicles with mature versus immature oocytes [40,41]. In a previous study in mares, E 2 remained high throughout the oocyte maturation process, whereas P 4 was higher during metaphase II than during earlier stages of nuclear maturation [16]. The present study provided temporal indications that intrafollicular E 2 and P 4 were not associated with follicle maturity but were associated with oocyte maturity or immaturity.…”

“…Using a similar hCG-treatment protocol, the proportion of follicles with low E 2 concentrations increased in association with nonrecovery of an oocyte [9]. Nuclear and cytoplasmic changes during final maturation of equine oocytes were associated with an increase in P 4 in the follicular fluid, whereas E 2 remained consistently high [16]. However, in another study with preovulatory follicles (.30 mm), no relationship was found between E 2 , P 4 , and testosterone concentrations and oocyte recovery or nuclear maturation [17].…”

Comparative Evaluation of Nuclear Morphology of Equine Oocytes Aspirated in vivo and Stained with Hoechst and Orcein

“…In the present study, a spot of spherical condensed orcein-stained chromatin was observed in the vicinity of partly filamentous chromatin (configuration B) in a total of 20 oocytes characterized as GV, GVBD or dCh. This structure closely resembles the descriptions of the nucleolus in equine oocytes as visualized by light microscopy [Fulka and Okolski, 1981] and electron microscopy [Grøndahl et al, 1995]. However, after Hoechst staining, a fluorescent ring of perinucleolar chromatin, as observed in GV stages of other mammalian oocytes [Mattson and Albertini, 1990;Debey et al, 1993;Schramm et al, 1993], was not visible.…”

Effect of thein vitromaturation medium on equine oocytes: Comparison of follicular fluid and oestrous mare serum

“…In equine follicles larger than 30 mm, the mean concentration of this hormone is 1575 ng/ml (Goudet et al, 1997), while the level of progesterone is 900 ng/ml. Indeed, the final stage in the maturation of equine oocytes in vivo is accompanied by a significant increase in follicular fluid progesterone (Grondahl et al, 1995). Furthermore, when horse oocytes are matured in vitro in a medium supplemented with 250 ng/ml progesterone, the cumulus cells undergo a greater degree of expansion (Schmid et al, 1998).…”