Follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of pre- and early antral dog follicles

Abstract

Understanding stage-specific requirements of mammalian folliculogenesis is limited in the domestic dog. The present study examined the effects of two potential regulators of dog follicle growth and survival in vitro, namely the original stage of the follicle (i.e. preantral (≤230µm diameter) vs early antral (diameter from >230 to ≤330µm) and FSH and/or LH concentrations. After isolation and alginate encapsulation, follicles were cultured in 0, 1, 10 or 100µgmL FSH and 0, 1 or 10ngmL LH for 20 days. Regardless of stage, FSH promoted growth, but LH did the same only in the absence of FSH. Production of 17β-oestradiol and progesterone was detectable, indicating theca cell activity. The greatest growth occurred in preantral (mean (± s.d.) 61.4±25.9%) versus antral (42.6±20.3%) follicles, but neither developmental stage nor gonadotropin affected survival. Antrum detection was minimal due, in part, to antral collapse, and oocytes exhibited an increasingly pale appearance and chromatin degeneration over time. The results demonstrate that pre- and early antral stage dog follicles encapsulated in alginate grow significantly in vitro. However, because FSH and LH alone or in combination fail to promote antrum development, the next step is identifying factors that enhance antral expansion.

Activin promotes growth and antral cavity expansion in the dog ovarian follicle

“…First, ~85% of our isolated follicles already had begun the cavitation process (i.e., met early antral or antral criteria; [47]. Second, we had determined in a previous study [45] that dog follicles incubated in 0.5% hydrogel developed antral cavities that subsequently collapsed. Therefore, our aim here was to both maintain antrum integrity and expand this cavity's size.…”

“…For both sizing approaches, the widest apparent diameter was measured followed by determining perpendicular width. A mean for these two values was calculated and reported as diameter [45]. We used the same technique on Day 0 to determine oocyte diameter (excluding the zona pellucida).…”

Endocrine and paracrine controls of canine follicular development and function

“…Recently, development of in vitro follicle culture systems for dog ovarian cortices and isolated follicles have allowed close examination of mechanisms regulating ovarian folliculogenesis and oogenesis (Nagashima, Wildt, Travis, & Songsasen, ; Serafim et al., , ; Songsasen, Woodruff, & Wildt, ). Overall, in vitro studies have confirmed that FSH is essential for dog follicle growth (Nagashima et al., ; Serafim et al., ; Songsasen et al., ), although FSH dosages used in these studies were higher than plasma concentrations reported for intact bitches (98 ± 48 ng/ml, Olson, Mulnix & Nett, ; 9.3 ± 0.8 U/L, Kooistra et al, ). Interestingly, sequential exposure of follicles to increasing FSH dosage at 6 days intervals (100, 500 and 1000 ng/ml) to mimic in vivo environment is beneficial, as follicles cultured in this condition grow larger than those incubated in a fixed gonadotropin concentration (100 ng/ml).…”

“…Interestingly, sequential exposure of follicles to increasing FSH dosage at 6 days intervals (100, 500 and 1000 ng/ml) to mimic in vivo environment is beneficial, as follicles cultured in this condition grow larger than those incubated in a fixed gonadotropin concentration (100 ng/ml). Furthermore, LH exerts no beneficial effect on pre‐antral and early antral follicle growth (Nagashima et al., ).…”

Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model

“…This system has provided encouraging results for advancing follicle development in the human [8–10], rhesus monkey [11], baboon [12], goat [13], and dog [14, 15], although with limited or no success at producing fertilizable oocytes. The lack of direct application from achievements in the mouse to other species likely is due to physical, anatomical and physiological differences [15, 16]. For example, the size of a mature cow, sheep, or human follicle is five- to 20-fold larger than for the same stage mouse follicle [16].…”