Department of Anatomy, Centre for Neuroendocrinology, University of Otago School of Biomedical Sciences, Dunedin, New Zealand.
Department of Physiology, Centre for Neuroendocrinology, University of Otago School of Biomedical Sciences, Dunedin, New Zealand.
J Neuroendocrinol. 2021 Dec;33(12):e13058. doi: 10.1111/jne.13058. Epub 2021 Nov 8.
Prenatal exposure to excess androgens is associated with the development of polycystic ovary syndrome (PCOS). In prenatally androgenised (PNA) mice, a model of PCOS, progesterone receptor (PR) protein expression is reduced in arcuate nucleus (ARC) GABA neurons. This suggests a mechanism for PCOS-related impaired steroid hormone feedback and implicates androgen excess with respect to inducing transcriptional repression of the PR-encoding gene Pgr in the ARC. However, the androgen sensitivity of ARC neurons and the relative gene expression of PRs over development and following prenatal androgen exposure remain unknown. Here, we used a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) of microdissected ARC to determine the relative androgen receptor (Ar) and progesterone receptor (Pgr) gene expression in PNA and control mice at five developmental timepoints. In a two-way analysis of variance, none of the genes examined showed expression changes with a statistically significant interaction between treatment and age, although PgrA showed a borderline interaction. For all genes, there was a statistically significant main effect of age on expression levels, reflecting a general increase in expression with increasing age, regardless of treatment. For PgrB and Ar, there was a statistically significant main effect of treatment, indicating a change in expression following PNA (increased for PgrB and decreased for Ar), regardless of age. For PgrA, there was a borderline main effect of treatment, suggesting a possible change in expression following PNA, regardless of age. PgrAB gene expression changes showed no significant main effect of treatment. We additionally examined androgen and progesterone responsiveness specifically in P60 ARC GABA neurons using RNAScope® (Advanced Cell Diagnostics, Inc.) in situ hybridization. This analysis revealed that Pgr and Ar were expressed in the majority of ARC GABA neurons in normal adult females. However, our RNAScope® analysis did not show significant changes in Pgr or Ar expression within ARC GABA neurons following PNA. Lastly, because GABA drive to gonadotropin-releasing hormone neurons is increased in PNA, we hypothesised that PNA mice would show increased expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA production. However, the RT-qPCR showed that the expression of GAD encoding genes (Gad1 and Gad2) was unchanged in adult PNA mice compared to controls. Our findings indicate that PNA treatment can impact Pgr and Ar mRNA expression in adulthood. This may reflect altered circulating steroid hormones in PNA mice or PNA-induced epigenetic changes in the regulation of Pgr and Ar gene expression in ARC neurons.
产前暴露于过量雄激素与多囊卵巢综合征（PCOS）的发生有关。在产前雄激素化（PNA）小鼠模型中，孕激素受体（PR）蛋白在弓状核（ARC）GABA 神经元中的表达减少。这表明 PCOS 相关类固醇激素反馈受损的一种机制，并暗示雄激素过多与诱导 ARC 中 PR 编码基因 Pgr 的转录抑制有关。然而，ARC 神经元的雄激素敏感性以及 PR 基因在发育过程中的相对表达以及产前雄激素暴露后的相对表达仍不清楚。在这里，我们使用定量逆转录聚合酶链反应（RT-qPCR）对微分离的 ARC 进行了分析，以确定在五个发育时间点的 PNA 和对照小鼠中 ARC 中的相对雄激素受体（Ar）和孕激素受体（Pgr）基因表达。在双因素方差分析中，尽管 PgrA 显示出边缘相互作用，但没有一个检查的基因显示出具有统计学意义的处理和年龄之间的表达变化相互作用。对于所有基因，年龄对表达水平都有统计学上显著的主要影响，这反映了无论治疗如何，随着年龄的增长表达水平普遍增加。对于 PgrB 和 Ar，治疗有统计学上显著的主要作用，表明 PNA 后表达发生变化（PgrB 增加，Ar 减少），而与年龄无关。对于 PgrA，治疗有边缘主要作用，表明 PNA 后表达可能发生变化，而与年龄无关。PgrAB 基因表达变化没有治疗的主要作用。我们还使用原位杂交技术（Advanced Cell Diagnostics，Inc. 的 RNAScope®）专门检查了 P60 ARC GABA 神经元中的雄激素和孕激素反应性。该分析表明，正常成年雌性动物的大多数 ARC GABA 神经元中均表达 Pgr 和 Ar。然而，我们的 RNAScope®分析并未显示 PNA 后 ARC GABA 神经元中 Pgr 或 Ar 表达的显着变化。最后，由于 PNA 中促性腺激素释放激素神经元的 GABA 驱动增加，我们假设 PNA 小鼠会表现出谷氨酸脱羧酶（GAD）表达增加，GAD 是 GABA 产生的限速酶。然而，RT-qPCR 显示成年 PNA 小鼠与对照组相比，编码 GAD 的基因（Gad1 和 Gad2）的表达不变。我们的研究结果表明，PNA 处理会影响成年期的 Pgr 和 Ar mRNA 表达。这可能反映了 PNA 小鼠中循环类固醇激素的改变，或者 PNA 诱导的 ARC 神经元中 PR 和 Ar 基因表达的表观遗传变化。
