The earliest stages of follicular development: follicle formation and activation.

The formation of primordial follicles to establish a reservoir of resting follicles and the gradual depletion of that reservoir to provide a succession of growing follicles are key to female fertility, but little is known about the regulation of these early stages of follicular development. This review summarizes the efforts of our laboratory to elucidate these critical processes in cattle. Primordial follicles first appear in fetal ovaries around the end of the first trimester of pregnancy (Day 90), during a decline in fetal ovarian production of estradiol and progesterone. In ovarian cortical pieces from 90 to 120-day-old fetuses, follicles form in vitro and estradiol or progesterone inhibits follicle formation, whereas the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) does not. Newly formed bovine follicles are not capable of activating within 2 days in vitro, but they can acquire the capacity to activate during a longer culture; estradiol and progesterone inhibit the acquisition of their capacity to activate. When primordial follicles first form in cattle, their oocytes are not yet in meiotic arrest and acquisition of competence to activate is correlated with their progression to meiotic arrest at the diplotene stage of first prophase. After they acquire the competence to activate, bovine primordial follicles can be stimulated to activate in vitro by insulin or kit ligand, whereas anti-Mullerian hormone (AMH) is inhibitory. Although few follicles progress to the secondary stage in vitro, addition of testosterone or vascular endothelial growth factor (VEGF) dramatically increased the incidence of that transition. Regulation of the earliest stages of follicular development is complex and far from understood; better understanding could lead to new interventions to enhance fertility.

In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue.

Achieving full in vitro growth of oocytes of both domestic animals and humans remains a major challenge. The objective of this study was to examine the in vitro development of primary follicles isolated enzymatically from cryopreserved sheep ovarian tissue. In Experiment 1, isolated primary follicles (mean diameter 60.1+/-0.78microm) were cultured in serum-free medium on fibronectin-coated wells for 42 days. Initially follicular structure was lost as granulosa cells plated down, but by Day 7 two distinct morphologies began to emerge. Nineteen out of 36 oocytes were gradually re-surrounded by granulosa cells, forming follicle-like units (reorganized follicles), and the remaining 17 were not (non-reorganized follicles). On Day 2, there was no difference in diameter of oocytes between reorganized and non-reorganized follicles. The diameter (mean+/-S.E.M.) of oocytes of reorganized follicles increased (P<0.05) from 47.1+/-2.2microm to 65.3+/-2.6microm between Day 2 and Day 42, respectively, but that of oocytes of non-reorganized follicles showed no change. In Experiment 2, oocyte growth and granulosa cell differentiation during long-term culture of primary follicles (>42 days) were examined. Oocytes of reorganized follicles reached a maximum diameter of 75.4+/-2.0microm, a size equivalent to that of oocytes of ovine secondary follicles. Using RT-PCR, mRNA for follicle stimulating hormone receptor was detected in granulosa cells of freshly isolated secondary follicles and of long-term cultured reorganized follicles, but not of non-reorganized follicles. In Experiment 3, we tested if the culture conditions could support further oocyte growth in secondary follicles. The oocytes from enzymatically isolated secondary follicles increased in diameter from 77.7+/-1.6microm to 98.8+/-2.1microm (P<0.05) during 28 days in culture. The changes in oocyte size and in gene expression by granulosa cells support the conclusion that isolated ovine primary follicles developed in vitro to reach the secondary follicle stage.

The in vitro growth and maturation of follicles.

The development of technologies to grow oocytes from the most abundant primordial follicles to maturity in vitro holds many attractions for clinical practice, animal production technology and research. The production of fertile oocytes and live offspring has been achieved in mice following the long-term culture of oocytes in primordial follicles from both fresh and cryopreserved ovarian tissue. In contrast, in non-rodent species advances in follicle culture are centred on the growth of isolated preantral follicles. As a functional unit, mammalian preantral follicles are well-suited to culture but primordial and primary follicles do not grow well after isolation from the ovarian stroma. The current challenges for follicle culture are numerous and include: optimisation of culture media and the tailoring of culture environments to match the physiological needs of the cell in vivo; the maintenance of cell-cell communication and signalling during culture; and the evaluation of the epigenetic status, genetic health and fertility of in vitro derived mature oocytes. In large animals and humans, the complete in vitro growth and maturation of oocytes is only likely to be achieved following the development of a multistage strategy that closely mimics the ovary in vivo. In this approach, primordial follicle growth will be initiated in situ by the culture of ovarian cortex. Isolated preantral follicles will then be grown to antral stages before steroidogenic function is induced in the somatic cells. Finally, cytoplasmic and nuclear maturation will be induced in the in vitro derived oocytes with the production of fertile metaphase II gametes.

Evaluation of synchronization of oestrus based on gonadotrophin-releasing hormone and its potential use for fixed-time breeding in Tuli beef cows.

The objective of the experiment was to compare the reproductive post-partum performance of beef cows synchronized for oestrus using prostaglandin F2alpha (PGF2alpha) alone or with a gonadotrophin-releasing hormone (GnRH)-based drug. Fifty-five post-partum lactating Tuli cows were randomly allocated to three groups. Two groups were synchronized using either two injections of PGF2alpha (500 microg Prosolvin per injection) given 11 days apart (group 1), or GnRH (12.5 microg Receptal per injection) followed 6 days later by an injection of 500 microg PGF2alpha (group 2). The cows were bred by artificial insemination 12 h after they were observed in oestrus. Group 3 was synchronized as for group 2, but a second injection of GnRH was given 54 h after the PGF2alpha injection, at which time the cows were bred by artificial insemination (AI) without detection of oestrus. Blood samples were taken from the cows in group 3 and analysed for progesterone concentration to establish which cows were cycling and in oestrus before and at the time of breeding. Detection of oestrus and breeding by AI was done over 60 days. There were no significant differences (p>0.05) among the three groups in the first service and total conception rates. The percentage of cows in oestrus within 10 days of the synchronization treatment was not significantly different (p>0.05) between groups 1 and 2. The progesterone concentrations in the cows in group 3 showed that only those that were cycling at the start of the experiment responded to the synchronization treatment and conceived after fixed-time breeding. These results suggest that combinations of PGF2alpha and GnRH may be of value in synchronizing oestrus and controlling breeding in Tuli cows. However, the benefit might be greater if only cows that are known to be cycling are bred in this way.