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. 2015 Aug;30(8):1907-17.
doi: 10.1093/humrep/dev119. Epub 2015 Jun 3.

Differential effects of estrogen and progesterone on development of primate secondary follicles in a steroid-depleted milieu in vitro

A Y Ting  1 , J Xu  2 , R L Stouffer  3
Affiliations

Differential effects of estrogen and progesterone on development of primate secondary follicles in a steroid-depleted milieu in vitro

A Y Ting et al. Hum Reprod. 2015 Aug.

Abstract

Study question: What are the direct effects of progesterone (P4) and estradiol (E2) on the development and function of primate follicles in vitro from the pre-antral to early antral stage?

Summary answer: In a steroid-depleted milieu, E2 improved follicle survival, growth, antrum formation and oocyte health, whereas P4 exerted minimal beneficial effects on follicle survival and reduced oocyte health.

What is known already: Effects of P4 and E2 on follicle development have been studied primarily in large antral and pre-ovulatory follicles. Chronic P4 exposure suppresses antral follicle growth, but acute P4 exposure promotes oocyte maturation in pre-ovulatory follicles. Effects of E2 can be stimulatory or inhibitory depending upon species, dose and duration of exposure.

Study design, size, duration: Non-human primate model, randomized, control versus treatment. Macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks.

Participants/materials, setting, methods: Adult rhesus macaque secondary follicles were encapsulated in 0.25% alginate and cultured individually in media containing follicle stimulating hormone plus (i) vehicle, (ii) a steroid-synthesis inhibitor, trilostane (TRL, 250 ng/ml), (iii) TRL + low E2 (100 pg/ml) or progestin (P, 10 ng/ml R5020) and (iv) TRL + high E2 (1 ng/ml E2) or P (100 ng/ml R5020). Follicles reaching the antral stage (≥750 µm) were treated with human chorionic gonadotrophin for 34 h. End-points included follicle survival, antrum formation, growth pattern, plus oocyte health and maturation status, as well as media concentrations of P4, E2 and anti-Müllerian hormone (AMH).

Main results and the role of chance: In a steroid-depleted milieu, low dose, but not high dose, P improved (P < 0.05) follicle survival, but had no effect (P > 0.05) on antrum formation and AMH production. Low-dose P increased (P < 0.05) P4 production in fast-grow follicles, and both doses of P elevated (P < 0.05) E2 production in slow-grow follicles. Additionally, low-dose P increased (P < 0.05) the percentage of no-grow follicles, and high-dose P promoted oocyte degeneration. In contrast, E2, in a steroid-depleted milieu, improved (P < 0.05) follicle survival, growth, antrum formation and oocyte health. E2 had no effect on P4 or E2 production. Follicles exposed to E2 yielded mature oocytes capable of fertilization and early cleavage, at a rate similar to untreated control follicles.

Limitations, reasons for caution: This study is limited to in vitro effects of P and E2 during the interval from the secondary to small antral stage of macaque follicles.

Wider implications of the findings: This study provides novel information on the direct actions of P4 and E2 on primate pre-antral follicle development. Combined with our previous report on the actions of androgens, our findings suggest that androgens appear to be a survival factor but hinder antral follicle differentiation, E2 appears to be a survival and growth factor at the pre-antral and early antral stage, whereas P4 may not be essential during early folliculogenesis in primates.

Study funding/competing interests: NIH P50 HD071836 (NCTRI), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), ONPRC 8P51OD011092. There are no conflicts of interest.

Keywords: estrogen; follicle development; ovary; progesterone.

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Figures

Figure 1
Figure 1
Follicle (n = 72 follicles/treatment group) survival rate after 5 weeks (Wks) of culture in progestin (P; R5020, A) and estradiol (E2, B) replacement experiments. CONT, control; TRL, trilostane; LP, low-dose P; HP, high-dose P; LE, low-dose E2 and HE, high-dose E2. Data are presented as mean ± SEM. Different letters represent significant differences between treatment groups in each experiment.
Figure 2
Figure 2
Growth characteristics of surviving follicles in progestin (P, A) and estradiol (E2, B) replacement experiments. CONT, control, TRL, trilostane, LP, low-dose P, HP, high-dose P, LE, low-dose E2 and HE, high-dose E2. Different capital, lower case and Greek letters represent statistical differences in fast-grow, slow-grow and no-grow follicle populations, respectively, between treatment groups in each experiment.
Figure 3
Figure 3
Antrum formation rate of surviving follicles in progestin (P, A) and estradiol (E2, B) replacement experiments. CONT, control, TRL, trilostane, LP, low-dose P, HP, high-dose P, LE, low-dose E2 and HE, high-dose E2. Data are presented as mean ± SEM. Different letters represent significant differences between treatment groups in each experiment.
Figure 4
Figure 4
Media anti-Müllerian Hormone (AMH) concentrations (ng/ml) in fast-grow and slow-grow follicles in progestin (P, A) and estradiol (E2, B) replacement experiments at Week 3 of culture. CONT, control, TRL, trilostane, LP, low-dose P, HP, high-dose P, LE, low-dose E2 and HE, high-dose E2. Data are presented as mean ± SEM. Different capital and lower case letters represent differences in fast-grow and slow-grow follicle populations, respectively, between treatment groups in each experiment. Asterisk represents differences between fast-grow and slow-grow follicles within the same treatment group.
Figure 5
Figure 5
Media progesterone concentrations (ng/ml) in fast-grow and slow-grow follicles in progestin (P, A) and estradiol (E2, B) replacement experiments at Week 5 of culture. CONT, control, TRL, trilostane, LP, low-dose P, HP, high-dose P, LE, low-dose E2 and HE, high-dose E2. Data are presented as mean ± SEM. Different capital and lower case letters represent differences in fast-grow and slow-grow follicle populations, respectively, between treatment groups in each experiment. Asterisk represents differences between fast-grow and slow-grow follicles within the same treatment group.
Figure 6
Figure 6
Media estradiol (E2) concentrations (pg/ml) in fast-grow and slow-grow follicles in progestin (P, A) and E2 (B) replacement experiments at Week 5 of culture. CONT, control, TRL, trilostane, LP, low-dose P, HP, high-dose P, LE, low-dose E2 and HE, high-dose E2. Data are presented as mean ± SEM. Different capital and lower case letters represent differences in fast-grow and slow-grow follicle populations, respectively, between treatment groups in each experiment. Asterisk represents differences between fast-grow and slow-grow follicles within the same treatment group.
Figure 7
Figure 7
Follicle, oocyte and embryo development in progestin (P, A) and estradiol (E2, B) replacement experiments. Represented photomicrographs from both experiments showing secondary follicles that grew and formed an atrum (∼week [Wk] 3) with diameters that can reach 1.2 mm (not shown) during 5 weeks of culture. Thirty-four hours after human chorionic gonadotrophin treatment, some oocytes matured to the metaphase I (MI) or MII stage and when subjected to in vitro fertilization, a few exhibited two pronuclei (2PN) formation and early cleavage (3–4 cell on Day [D] 2, 6–8 cell on D3 and 10–16 cell on D4).
Figure 8
Figure 8
Immunofluorescent labeling for actin (red), tubulin (green) and DNA (blue) in germinal vesicle (GV)-intact oocytes from in vitro grown follicles. Healthy GV oocytes showed normal chromatin configuration including non-surrounded nucleolus (diffused), partially surrounded nucleolus and surrounded nucleolus. Extensive development of the transzonal projections (TZPs) was also evident in cumulus–oocyte complexes.
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