In lactating dairy cattle, the corpus luteum (CL) is a dynamic endocrine tissue vital for pregnancy maintenance, fertility, and cyclicity. Understanding processes underlying luteal physiology is therefore necessary to increase reproductive efficiency in cattle. A common technique for investigating luteal physiology is reverse-transcription quantitative PCR (RT-qPCR), a valuable tool for quantifying gene expression. However, reference-gene-based RT-qPCR quantification methods require utilization of stably expressed genes to accurately assess mRNA expression. Historically, selection of reference genes in cattle has relied on subjective selection of a small pool of reference genes, many of which may have significant expression variation among different tissues or physiologic states. This is particularly concerning in dynamic tissues such as the CL, with its capacity for rapid physiologic changes during luteolysis, and likely in the less characterized period of CL maintenance during pregnancy. Thus, there is a clear need to identify reference genes well suited for the bovine CL over a wide range of physiological states. Whole-transcriptome RNA sequencing stands as an effective method to identify new reference genes by enabling the assessment of the expression profile of the entire pool of mRNA transcripts. We report the identification of 13 novel putative reference genes using RNA sequencing in the bovine CL throughout early pregnancy and luteolysis: RPL4, UQCRFS1, COX4I1, RPS4X, SSR3, CST3, ZNF266, CDC42, CD63, HIF1A, YWHAE, EIF3E, and PPIB. Independent RT-qPCR analyses were conducted confirming expression stability in another set of CL tissues from pregnancy and regression, with analyses performed for 3 groups of samples: (1) all samples, (2) samples from pregnancy alone, and (3) samples throughout the process of CL regression. Seven genes were found to be more stable in all states than 2 traditional reference genes (ACTB and GAPDH): RPS4X, COX4I1, PPIB, SSR3, RPL4, YWHAE, and CDC42. When CL tissues from pregnant animals alone were analyzed, CST3, HIF1A, and CD63 were also identified as more stable than ACTB and GAPDH. Identification of these new reference genes will aid in accurate normalization of RT-qPCR results, contributing to proper interpretation of gene expression relevant to luteal physiology. Furthermore, our analysis sheds light on the effects of luteolysis and pregnancy on the stability of gene expression in the bovine CL.

中文翻译：

---

在妊娠和黄体溶解的泌乳荷斯坦奶牛黄体中稳定基因的鉴定：对选择逆转录定量PCR参考基因的意义。

在泌乳的奶牛中，黄体（CL）是动态的内分泌组织，对维持妊娠，生育能力和周期性至关重要。因此，了解黄体生理基础的过程对于提高牛的繁殖效率是必要的。研究黄体生理的常用技术是逆转录定量PCR（RT-qPCR），这是定量基因表达的一种有价值的工具。但是，基于参考基因的RT-qPCR定量方法需要利用稳定表达的基因来准确评估mRNA表达。从历史上看，牛中参考基因的选择依赖于一小部分参考基因的主观选择，其中许多参考基因可能在不同组织或生理状态之间表达差异很大。这在CL等动态组织中尤为重要，具有在黄体溶解过程中快速生理变化的能力，并且可能在妊娠期CL维持较弱的时期。因此，显然需要鉴定在广泛的生理状态下非常适合牛CL的参考基因。全转录组RNA测序是通过评估整个mRNA转录本表达谱来鉴定新参考基因的有效方法。我们报告了在整个早期妊娠和黄体溶解期间使用牛CL中的RNA测序鉴定13种新的推定参考基因的方法：RPL4，UQCRFS1，COX4I1，RPS4X，SSR3，CST3，ZNF266，CDC42，CD63，HIF1A，YWHAE，EIF3E和PPIB。进行了独立的RT-qPCR分析，以确认从妊娠和消退的另一组CL组织中表达的稳定性，对3组样本进行了分析：（1）所有样本，（2）仅来自妊娠的样本，以及（3）在CL回归过程中的样本。发现7个基因在所有状态下比2个传统参考基因（ACTB和GAPDH）更稳定：RPS4X，COX4I1，PPIB，SSR3，RPL4，YWHAE和CDC42。当仅分析来自怀孕动物的CL组织时，CST3，HIF1A和CD63也被确定比ACTB和GAPDH更稳定。这些新参考基因的鉴定将有助于RT-qPCR结果的准确归一化，有助于正确解释与黄体生理相关的基因表达。此外，我们的分析揭示了黄体溶解和妊娠对牛CL基因表达稳定性的影响。（3）在整个CL回归过程中的样本。发现7个基因在所有状态下比2个传统参考基因（ACTB和GAPDH）更稳定：RPS4X，COX4I1，PPIB，SSR3，RPL4，YWHAE和CDC42。当仅分析来自怀孕动物的CL组织时，CST3，HIF1A和CD63也被确定比ACTB和GAPDH更稳定。这些新参考基因的鉴定将有助于RT-qPCR结果的准确归一化，有助于正确解释与黄体生理相关的基因表达。此外，我们的分析揭示了黄体溶解和妊娠对牛CL基因表达稳定性的影响。（3）在整个CL回归过程中的样本。发现7个基因在所有状态下比2个传统参考基因（ACTB和GAPDH）更稳定：RPS4X，COX4I1，PPIB，SSR3，RPL4，YWHAE和CDC42。当仅分析来自怀孕动物的CL组织时，CST3，HIF1A和CD63也被确定比ACTB和GAPDH更稳定。这些新参考基因的鉴定将有助于RT-qPCR结果的准确归一化，有助于正确解释与黄体生理相关的基因表达。此外，我们的分析揭示了黄体溶解和妊娠对牛CL基因表达稳定性的影响。PPIB，SSR3，RPL4，YWHAE和CDC42。当仅分析来自怀孕动物的CL组织时，CST3，HIF1A和CD63也被确定比ACTB和GAPDH更稳定。这些新参考基因的鉴定将有助于RT-qPCR结果的准确归一化，有助于正确解释与黄体生理相关的基因表达。此外，我们的分析揭示了黄体溶解和妊娠对牛CL基因表达稳定性的影响。PPIB，SSR3，RPL4，YWHAE和CDC42。当仅分析来自怀孕动物的CL组织时，CST3，HIF1A和CD63也被确定比ACTB和GAPDH更稳定。这些新参考基因的鉴定将有助于RT-qPCR结果的准确归一化，有助于正确解释与黄体生理相关的基因表达。此外，我们的分析揭示了黄体溶解和妊娠对牛CL基因表达稳定性的影响。