Impact of anisosmotic conditions on structural and functional integrity of cumulus–oocyte complexes at the germinal vesicle stage in the domestic cat

Abstract

During cryopreservation, the immature oocyte is subjected to anisosmotic conditions potentially impairing subsequent nuclear and cytoplasmic maturation in vitro. In preparation for cryopreservation protocols and to characterize osmotic tolerance, cat cumulus-oocyte complexes (COC) at the germinal vesicle (GV) stage were exposed for 15 min to sucrose solutions ranging from 100 to 2,000 mOsm and then examined for structural integrity and developmental competence in vitro. Osmolarities ≥200 and ≤750 mOsm had no effect on incidence of oocyte nuclear maturation, fertilization success, and blastocyst formation compared to control COC (exposed to 290 mOsm). This relatively high osmotic tolerance of the immature cat oocyte appeared to arise from a remarkable stability of the GV chromatin structure as well as plasticity in mitochondrial distribution, membrane integrity, and ability to maintain cumulus-oocyte communications. Osmolarities < 200 mOsm only damaged cumulus cell membrane integrity, which contributed to poor nuclear maturation but ultimately had no adverse effect on blastocyst formation in vitro. Osmolarities >750 mOsm compromised nuclear maturation and blastocyst formation in vitro via disruption of cumulus-oocyte communications, an effect that could be mitigated through 1,500 mOsm by adding cytochalasin B to the hyperosmotic solutions. These results (1) demonstrate, for the first time, the expansive osmotic tolerance of the immature cat oocyte, (2) characterize the fundamental role of cumulus-oocyte communications when tolerance limits are exceeded, and (3) reveal an interesting hyperosmotic tolerance of the immature oocyte that can be increased two-fold by supplementation with cytochalasin B.

Resilience of Oocyte Germinal Vesicles to Microwave-Assisted Drying in the Domestic Cat Model

“…4 Focus on the GV is also timely, given strong evidence that the nucleus of the immature oocyte is more tolerant to certain preservation manipulations than its metaphase II (MII) counterpart. 5 Specifically, abnormalities may occur in MII oocytes because the metaphase spindle and chromosomal plate are highly sensitive to nonphysiological conditions. 1,6 More specifically, the GV is the organelle surrounded by a nuclear envelope that is present within all immature oocytes and which contains (1) de-condensed chromatin arrested at prophase I of meiosis, and (2) nucleoplasmic factors that mix with the cytoplasm during meiotic resumption, both of which are essential for subsequent fertilization.…”

Felis catus ovary as a model to study follicle biology in vitro

“…As we mentioned, the two concentrations studied were (1) the concentration normally used for IVM in IVF clinics (low) and (2) a physiological dose (high) equivalent for the reported surge in cats. In literature, however, gonadotropins of porcine or equine origin, plus higher concentrations, were more commonly used for feline studies [7,19,[24][25][26][27][28][29][30][31]. This was presumably due to experience from in vivo stimulation protocols, where porcine FSH and eCG were used to initiate folliculogenesis in felines (for more information please read review: [36]).…”

“…Cats serve as good models for addressing infertility syndromes in women, such as asynchronous oocyte cytoplasmic and nuclear maturation, ovarian hypersensitivity, and luteal dysfunction after gonadotropin therapy [6]. Interestingly, cat oocytes share several characteristics with human oocytes [7,8]: (1) the diameter of the oocyte proper and the germinal vesicle is equivalent (110 and 45 μm, respectively) in both species; (2) oocytes reach the metaphase II (MII) stage of meiosis after 24 h in culture; and (3) both species have a similar nuclear configuration with a small nucleolus and a fibrillar chromatin. In contrast, these morphological features are distinct or lacking in the typical laboratory mouse model.…”

“…Regarding the domestic cat, efforts to date have focused on oocytes [7,[24][25][26][27][28][29][30] or preantral follicle cultures [19,31]. Therefore, studies were designed to characterize and evaluate the response of individual intact antral follicles from adult female domestic cats to a stimulus of recombinant human LH in vitro by assessing cumulus-oocyte expansion (C-OE) and steroid production (E2 and P4).…”

Maturation outcomes are improved following Cryoleaf vitrification of immature human oocytes when compared to choline-based slow-freezing

“…It may be that the oocyte should be surrounded by an intact cumulus oophorous when vitrifying immature oocytes but studies to date point to the challenges and cellular disruptions resulting from cryopreserving intact cumulusoocyte-complexes [11,[49][50][51][52][53][54][55][56]]. Yet, promising or improved results with the cryopreservation of cumulus-free feline [57] and bovine [58,59] immature oocytes (obtained without hormonal stimulation) warrant further comparison of outcomes when freezing intact versus denuded cumulusoocyte-complexes.…”