Endocrine Activity of Extraembryonic Membranes Extends beyond Placental Amniotes

BACKGROUND. During development, all amniotes (mammals, reptiles, and birds) form extraembryonic membranes, which regulate gas and water exchange, remove metabolic wastes, provide shock absorption, and transfer maternally derived nutrients. In viviparous (live-bearing) amniotes, both extraembryonic membranes and maternal uterine tissues contribute to the placenta, an endocrine organ that synthesizes, transports, and metabolizes hormones essential for development. Historically, endocrine properties of the placenta have been viewed as an innovation of placental amniotes. However, an endocrine role of extraembryonic membranes has not been investigated in oviparous (egg-laying) amniotes despite similarities in their basic structure, function, and shared evolutionary ancestry. In this study, we ask whether the oviparous chorioallantoic membrane (CAM) of chicken (Gallus gallus) has the capability to synthesize and receive signaling of progesterone, a major placental steroid hormone. METHODOLOGY/PRINCIPAL FINDINGS. We quantified mRNA expression of key steroidogenic enzymes involved in progesterone synthesis and found that 3β-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone exhibited a 464 fold increase in the CAM from day 8 to day 18 of embryonic development (F5, 68=89.282, p<0.0001). To further investigate progesterone synthesis, we performed explant culture and found that the CAM synthesizes progesterone in vitro in the presence of a steroid precursor. Finally, we quantified mRNA expression and performed protein immunolocalization of the progesterone receptor in the CAM. CONCLUSIONS/SIGNIFICANCE. Collectively, our data indicate that the chick CAM is steroidogenic and has the capability to both synthesize progesterone and receive progesterone signaling. These findings represent a paradigm shift in evolutionary reproductive biology by suggesting that endocrine activity of extraembryonic membranes is not a novel characteristic of placental amniotes. Rather, we hypothesize that these membranes may share an additional unifying characteristic, steroidogenesis, across amniotes at large.Sigma Xi (G20073141634396861); National Science Foundation (2008059161); UF-Howard Hughes G.A.T.O.R. Program; Howard Hughes Medical Institute Professorshi

Steroid biosynthesis pathway.

<p>A simplified version of steroid biosynthesis highlighting the specific steroidogenic enzymes investigated in this study. Filled boxes highlight the steroidogenic enzymes examined by RT-qPCR. Progesterone (P4) is highlighted as the focus of this study. First, the transport protein, steroidogenic acute regulatory protein (StAR) is needed to facilitate the movement of cholesterol from the outer to inner mitochondrial membrane. Cholesterol is then converted to pregnenolone by the action of cytochrome side-chain cleaving enzyme (P450scc). Pregnenolone can then be converted to either 17α-hydroxypregnenolone by 17α-hydroxylase (P45017α) or to P4 by 3β-hydroxysteroid dehydrogenase (3β-HSD). P4 can either be a final product in this pathway or serve as a precursor in the synthesis of glucocorticoids, androgens, or estrogens. 17β-hydroxysteroid dehydrogenase (17β-HSD) functions in the conversion of weaker and stronger androgens and estrogens and was included in this study as a marker of upstream steroid enzyme activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005452#pone.0005452-Norris1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005452#pone.0005452-Payne1" target="_blank">[16]</a>.</p

Relative mRNA expression of steroidogenic enzymes in the chick CAM.

<p>RT-qPCR analysis of mRNA coding for StAR, P450scc, P45017α, 3β-HSD and 17β-HSD on chick embryonic days 8 (n = 10), 10 (n = 17), 12 (n = 14), 14 (n = 10), 16 (n = 14), and 18 (n = 12). Data are reported as relative mRNA expression and represent mean normalized mRNA transcript number in copies/µL±SEM. 3β-HSD increased (F_{5, 68} = 89.282, p<0.0001) and 17β-HSD decreased (F_{5, 68} = 16.027, p<0.0001) significantly between embryonic day 8 and day 18.</p

Progesterone synthesis in the chick CAM.

<p>CAM sections were incubated in culture media for 2, 4, or 8 hours either with (circles) or without (squares) cholesterol (plus cAMP) as a precursor. Concentration of P4 in the culture media is represented as pg/ml of P4 per g of CAM tissue (pg/ml/g). Addition of precursor significantly increased the concentration of P4 in the culture media (F_{1, 58} = 46.917 p<0.0001) with a significant interaction between time of incubation and addition of precursor (F_{2, 58} = 3.709, p = 0.0305). To determine background and cross-reactivity of the P4 assay, controls consisting of only cholesterol and cAMP were incubated for 8 hours with an average P4 concentration of 0.337 pg/ml/g±0.423 SEM (not shown).</p

PR-ab and ERα mRNA expression and PR-ab immunolocalization.

<p>(A) RT-qPCR analysis of mRNA coding for PR-ab and ERα on chick embryonic days 8 (n = 10), 10 (n = 17), 12 (n = 14), 14 (n = 10), 16 (n = 14), and 18 (n = 12). Data are reported as relative mRNA expression and represent mean normalized mRNA transcript number in copies/µL±SEM. PR-ab (F_{5, 68} = 15.897, p<0.0001) and ERα (F_{5, 66} = 14.432, p<0.0001) increased significantly between embryonic day 8 and day 18. (B, C), PR-ab immunohistochemistry of embryonic day 18 CAM. (B) PR-ab positive section. Nuclear staining of PR-ab is localized predominately to the chorionic epithelium (c), and epithelial cells of blood vessels (bv). Positive nuclear staining is also present in allantoic epithelium (a), and mesenchyme (m). (C) Negative control of corresponding CAM section incubated without primary PR-ab antibody does not show specific nuclear staining. Scale bar represents 10 microns.</p