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

Abstract

Purpose The current study was designed to evaluate the response of individual intact antral follicles from adult female domestic cats to a luteinizing hormone (LH) stimulus in vitro by assessing cumulus-oocyte expansion (C-OE) and steroid production. Methods C-OE and steroid levels (estradiol [E2] and progesterone [P4]) obtained from individual antral feline follicles (n=366 follicles; n=56 cats) were analyzed after 12 or 24 h of culture in the presence or absence of LH (low [3.4 ng/ml] or high [100 ng/ml]). Results At the end of the culture, the highest percentage of expanded cumulus-oocyte complexes (COCs) was observed in the LH groups at 12 or 24 h in comparison to their controls (p<0.001). There was a significant increase in expanded COCs when comparing LH concentrations (high vs. low) at 12 or 24 h. Higher levels of both E2 and P4 were observed in the media from antral follicles after 12 and 24 h of culture in the presence of LH (both concentration, p<0.05). There was no association between hormone levels and follicle diameter; high variability was observed in the steroid levels produced by antral follicles within all treatment groups.Conclusions These data indicate, for the first time, that feline antral follicles (0.5-2 mm) from different stages of the natural estrous cycle can be cultured and will respond to an LH stimulus, based on an increase in steroid levels as well as C-OE after 12 or 24 h in culture.

Direct role of the C-C motif chemokine receptor 2/monocyte chemoattractant protein 1 system in the feline cumulus oocyte complex†

“…Follicle isolation from the ovary was performed under a dissecting microscope using 30-gauge needles as previously described [16][17][18]. Briefly, isolated antral follicles (n = 133) were individually cultured for 6, 12, 24, and 36 h in the presence or absence of recombinant human LH (75 mIU/ml rhLH, Merck Serono) in a 48-well plate containing 300 μl alpha minimum essential medium (αMEM, Sigma) supplemented with 15 ng/ml recombinant human FSH (equivalent to 205 mIU/ml, Merck Serono), 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium (ITS, Sigma), streptavidin/penicillin (100 IU/ml-100 mg/ml, Sigma), and 0.30% BSA (Fraction V, Natocor), as previously reported [16]. At the end of culture, the follicle walls and the COCs were isolated and stored at -80 • C for subsequent RNA extraction and RT real-time PCR.…”

“…Images of the individual COCs at 6, 12, 24, and 36 h retrieved post-culture were taken using a digital camera attached to a bright-field microscope to assess the CO-E by an "all or none" response to treatments. The substantial expansion/enlargement of the adjacent cumulus cells of the COC under the dissecting microscope can be easily visualized [16]. CO-E is reportedly the most reliable index of oocyte maturation in cats, since polar body extrusion is difficult to identify in feline oocytes due to the dark appearance of their oocytes [19].…”

“…As previously reported [16], even though only healthy antral follicles (devoid of dark follicles/granulosa cells; n = 133) were isolated and used for this study, at the end of the culture a few (less than 10%) of the retrieved COCs contained naked (n = 8) or dead (n = 1) oocytes. Thus, these samples were excluded for the C-OE analysis.…”

“…In addition to the potential to expand knowledge of feline reproduction, the use of this model system has the advantage of providing an excellent surrogate for understanding events involving human COCs that are necessary for fertility. In our laboratory, we recently estab-lished two different feline culture systems, showing that the culture of feline antral follicles was a robust and valuable system to study follicular development, steroidogenesis, and periovulatory events as well as follicle biology in general [16]. Also, we demonstrated that the culture of feline COCs from antral follicles was an appropriate system to study periovulatory events such as CO-E along with oocyte and cumulus cell biology in general [17].…”

Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium

“…In the supplemented medium, 84% of preantral follicles showed more than 75% alive granulosa cells, confirming that the growth factors have a positive influence on the quality of granulosa cells. It has been suggested that they reduce the loss of cell to cell contacts and avoid degeneration and death of preantral follicles (Rojo, Linari, Musse, & Peluffo, ; Wongbandue, Jewgenow, & Chatdarong, ).…”