This study is the first to demonstrate that prenatal T excess from days 60-90 of gestation impairs final preovulatory follicular growth (ovulatory size: ≥6 mm; [
28]) and reduces intrafollicular concentrations of T, A4, and P4 in preovulatory sized follicles of sheep undergoing controlled ovarian stimulation, a finding consistent with the lower fertility rates reported in these sheep [
19]. The rescue of final growth by postnatal androgen antagonist, but not insulin sensitizer, administration suggests the impairment of prevoulatory follicle growth is mediated via androgenic action. This, in concert with the partial rescue of the intrafollicular steroid milieu with insulin sensitizer, suggests that both androgens and insulin contribute to reproductive disruption [
29‐
31] and reduced fecundity [
17] in adult prenatal T-treated sheep.
Prenatal T programming of follicular dynamics
The GnRHa treatment regimen used was effective in blocking follicular growth beyond the 3 mm stage in both the control and prenatal T-treated sheep, as was the case with T30-90 females exposed to a shorter GnRHa treatment [
32]. The efficacy of pFSH to recruit follicular growth in controls was comparable to that achieved with oFSH or pFSH stimulation in other sheep breeds [
25,
33]. pFSH used in this study was also as effective in stimulating follicular growth in the 3 treatment groups (T, T + F, and T + R) as the combined oFSH- LH regimen used with T30-90 females [
32]. These findings indicate responsiveness to exogenous FSH, and hence recruitment, was not impaired by prenatal T excess or postnatal treatment with androgen antagonist or insulin sensitizer. The finding that recruitment and growth of follicles up to 4 mm size was similar across groups is not surprising, because androgens increase FSH activity [
34] and are not detrimental to follicular survival and growth of preantral and early antral follicles [
35].
The reduced number of follicles ≥6 mm in T females suggests the final maturation of the preovulatory follicle is impaired. Given that
i) the suppression was evident in number of follicles ≥6 mm, but not in the number up to 4 mm, and
ii) in sheep, follicles up to 4 mm size are considered FSH dependent and those beyond 4 mm as LH dependent [
28], the shift from FSH to LH dependency appears to be impaired. One possibility is that this reduction in preovulatory sized follicles might be a function of advancement in LH dependency and hence a requirement for LH. Because LH was not co-administered with FSH, the final transition to preovulatory size might be compromised. This premise is supported by the lack of reduction in preovulatory sized follicles in the T30-90 females [
32], when follicles were stimulated concomitantly with LH and FSH. Findings from both studies (this study and Steckler et al. [
32]) suggest the compromised preovulatory follicular development might be rescued with exogenous LH supplementation. It is unclear if this impairment is a function of reduced number of LH receptors or altered LH signaling, both are aspects yet to be studied in this model but implicated in women with PCOS [
36‐
38].
Importantly, the fact that postnatal androgen antagonist, not insulin sensitizer, treatment was able to rescue the number of preovulatory-sized follicles supports the notion that
i) compromised androgen receptor expression/function in growing follicles or surrounding ovarian stroma is detrimental to progression beyond a critical size (4 mm in this case) and
ii) that blockade of androgen action with an androgen antagonist would help overcome follicular growth arrest. In previous studies, we found prenatal T treatment from days 30-90 of gestation increases granulosa cell androgen receptor expression in antral follicles and is supportive of functional ovarian hyperandrogenism [
15]. Although insulin plays a role in follicular development [
39‐
41], failure of insulin sensitizer treatment to rescue this follicular growth defect suggests this dysfunction is driven primarily by the androgen signaling imbalance within the growing follicle.
Prenatal T programming of follicular steroid milieu
The opposing follicular size-related changes in intrafollicular T and estrogens in control females were similar to previous findings [
20,
42]. A higher androgenic environment prevails in small follicles, with a shift towards a highly estrogenic milieu in preovulatory follicles; this is consistent with increased aromatase activity as follicles mature [
42]. The transition from low E
2 in smaller follicles to high E
2 in larger follicles was the most striking change (4-fold increase) in the control group. Conversely, the E
2 increase was of much lower magnitude (< 1-fold) in the T group. Because androgens are the main substrate for estrogen production, the reduced magnitude of E
2 increase in T females may be driven by a reduced androgenic environment (T and A4) at earlier follicular stages. Androgen and estrogen receptors [
17] and steroidogenic enzymes [
43] are dysregulated in granulosa and theca cells of T30-90 sheep, and these disruptions remain to be determined in T60-90 females. It is also unclear whether the lower magnitude of E
2 increase in T females contributes to the delayed onset of LH surge that was reported earlier in these females [
29].
Prenatal T-induced disruptions in intrafollicular steroid milieu were also reported in non-human primates [
11]. Controlled ovarian stimulation studies in rhesus monkeys found 15-35 days of T treatment starting on gestational days 40-44, but not days 100-115, reduced intrafollicular A4 and E
2 concentrations in preovulatory sized follicles [
11]. Although disruptions in intrafollicular A4 and E
2 parallel findings from the current study, timing and duration of T exposure differ between the sheep and monkey study. Our earlier findings of reduced granulosa cell CYP19A1 expression in antral follicles in the T30-90 model [
43] agrees with the reduced intrafollicular E
2 in large antral follicles evidenced in the present study [
43]. Considering the disrupted intrafollicular steroidal milieu of prenatal T-treated monkeys was accompanied by a reduction in oocyte competence [
11], a similar intrafollicular disruption was evidenced in prenatal T-treated sheep is likely to be associated with compromised oocyte health. This, in fact, may explain the reduced fecundity in T60-90 females [
19]. The strength of the present study is that the intrafollicular steroid milieu was identified in different size follicular classes as opposed to only the preovulatory follicular size in monkeys. Another strength is the parallel assessment of follicular growth at different time points during the ovarian stimulation protocol, which was helpful in dissecting out regulation of follicular growth from steroidogenesis.
Relative to interventions, postnatal flutamide treatment rescued follicular growth to where preovulatory follicle size was achieved, but treatment failed to ameliorate the disruptions in intrafollicular steroidal milieu. The steroidal transition from low E
2 to a high E
2 milieu between 3 to 6 mm size antral follicles seen in controls was not evident in T + F females. In addition, the directionality of change in intrafollicular concentrations of estrone and A4 in T + F animals, namely a reduction in both steroids in the 6 mm compared to 3 mm follicles as opposed to the increase in both steroids in the controls, point to an intrafollicular steroidal disruption that persists through antral follicle growth. The increase of T in the smaller 3 mm follicles of the T + F females may be a compensatory response to the blockade of androgen receptor signaling by flutamide treatment, which was present throughout the course of the study. Such a response would be analogous to the masculinizing effects of flutamide seen relative to other variables [
44,
45]. To what extent the intrafollicular steroidal disruptions play a role in oocyte health and ultimately fertility remains unclear. Paradoxically, while flutamide treatment failed to ameliorate intrafollicular steroidal defects, the same treatment prevented pubertal advancement and enhanced preovulatory LH surge amplitude in the T30-90 females [
24]. Considering T is an aromatizable androgen, the differing effects of flutamide in rescuing the various physiologic functions may be a function of whether androgen or estrogen (via aromatization) is the programming agent [
31].
Despite the fact that T60-90 females are insulin resistant [
13], the lack of rescue in the number of follicles that achieved a preovulatory size by rosiglitazone suggests the insulin pathway is not involved in growth of preovulatory follicles. In contrast, the intrafollicular steroid milieu of T + R follicles was more similar to that of the controls and is supportive of a role for insulin coupled with FSH as follicles mature in maintaining intrafollicular steroid balance [
46]. The beneficial effects of insulin sensitizer therapies in enhancing insulin sensitivity and improving ovulatory function in women with PCOS [
47,
48] may relate to normalization of intrafollicular steroidal milieu, as evidenced in the T + R animals.
In interpreting the impact of the interventions, it is important to recognize that GnRH antagonist treatment given prior to FSH stimulation to achieve a homogeneous follicular pool before FSH stimulation (as achieved in this study, Fig.
1) might have played a role in determining the impact of androgen antagonist and insulin sensitizer on follicular dynamics and the intra-follicular hormone milieu. However, considering that GnRH antagonist treatment is the same across treatments, any variability in the starting pool of follicles across treatment would suggest intrinsic ovarian differences originating from the T treatment and interventions respectively. It needs to be recognized that GnRH agonist and antagonist treatments are routinely used in standard IVF practices [
49], and hence the approach taken with this study is consistent with this practice.
The outcomes achieved with the two interventions, namely the androgen antagonist helping rescue follicular growth and the insulin sensitizer partially rescuing intrafollicular steroidal milieu, suggest that both androgens and insulin may synergize in establishing optimal follicular growth and steroidogenesis. A combined intervention involving both may help compensate for any deficiency that one intervention has in order to achieve better success. While the finding in PCOS women is that combined treatment is more efficacious than monotherapies in treating anovulation [
50] is supportive of this possibility, this remains to be tested.