The Capacity of Primordial Follicles in Fetal Bovine Ovaries to Initiate Growth In Vitro Develops During Mid-Gestation and Is Associated with Meiotic Arrest of Oocytes1

Abstract

In cattle and other species in which the pool of resting, primordial follicles is formed during fetal life, little is known about the regulation of the early stages of ovarian follicular development. We used histological morphometry and a combination of observations in vivo and experiments in vitro to study the timing and regulation of follicle formation and the acquisition of the capacity of primordial follicles to initiate growth in cattle. In vivo, primordial, primary, and secondary follicles were first observed around Days 90, 140, and 210 of gestation, respectively. The long interval between the first appearance of primordial and primary follicles suggests that primordial follicles are not capable of activating when they are first formed, or they are inhibited from activating. This hypothesis was confirmed by the finding that most primordial follicles in pieces of ovarian cortex obtained from fetal ovaries older than 140 days activated (i.e., initiated growth) after 2 days in vitro, whereas follicles in cortical pieces from 90- to 140-day-old fetal ovaries did not. We tested the hypothesis that the oocytes of newly formed primordial follicles are not in meiotic arrest and found that before Day 141, most oocytes ( approximately 73%) were in prediplotene stages of prophase I, whereas after Day 140, the majority of oocytes ( approximately 85%) had arrested at the diplotene stage. This observation was further confirmed by the finding that levels of mRNA for YBX2, a protein associated with meiotic arrest, were 2.3 times higher in ovarian cortical pieces isolated after versus before Day 141. Primordial follicles in cortical pieces from 90- to 140-day-old fetal ovaries did activate during a longer, 10-day culture, but activation could be inhibited by adding estradiol or progesterone, but not dihydrotestosterone (all at 10(-6) M). Fetal ovaries secreted estradiol in vitro, and secretion by ovaries from 83 to 140-day-old fetuses declined precipitously ( approximately 30-fold) with age, consistent with the hypothesis that estradiol inhibits activation of newly formed primordial follicles in vivo. In summary, the results show that newly formed primordial follicles do not activate in vivo or within 2 days in vitro and that capacity to activate is correlated with achievement of meiotic arrest by the oocyte and can be inhibited by estradiol, which fetal ovaries actively produce around the time of follicle formation.

Damage to fetal bovine ovarian tissue caused by cryoprotectant exposure and vitrification is mitigated during tissue culture

“…Primordial follicles first appear in the fetal bovine ovary around 90 days of gestation, but only acquire the capacity to activate around 140 days of gestation, when they have achieved meiotic arrest [18], as demonstrated by a decrease in the proportion of primordial follicles and an increase in the proportion of primary follicles in ovarian tissue cultured in the presence of insulin [15]. In this study, primordial follicles within fetal bovine tissue exposed to EG and DMSO as permeable CPAs and sucrose as a non-permeable CPA retained the capacity to activate in the presence of insulin; correspondingly, primordial follicles cultured without insulin remained dormant.…”

“…1a) or activated (an oocyte surrounded by one or more layers of cuboidal granulosa cells; Fig. 1b), as previously described [18]. Follicles were further classified as morphologically Bnormal^(nuclei of oocyte and surrounding granulosa cells were structurally intact; Fig.…”

Follicular assembly: mechanisms of action

“…Alternatively, fetal ovaries could produce E 2 and cyst breakdown could begin when levels of fetal E 2 secretion fall. Fetal cow ovaries produce E 2 and levels drop when primordial follicles begin to form (Yang & Fortune 2008, Nilsson & Skinner 2009.…”

“…Like estrogen, there are several possible sources of progesterone. Two studies using the bovine model have demonstrated that progesterone is produced by the fetal ovary (Yang & Fortune 2008, Nilsson & Skinner 2009. One study reported a drop in fetal ovarian progesterone while progesterone in the fetal or maternal circulation did not change (Nilsson & Skinner 2009).…”

“…In cows, while follicles form before the oocytes reach diplotene arrest, they do not develop to the primary follicle stage until they arrest in diplotene again linking follicle development and meiotic stage. Primordial follicle activation can be inhibited by estrogen, which fetal bovine ovaries produce around the time of follicle formation (Yang & Fortune 2008). In the cow, estrogen may block follicle activation by inhibiting meiotic progression.…”