Modulation of Apoptotic Mechanisms in Developing Bovine Corpora Lutea May Regulate Susceptibility to Luteolysis in Response to PGF2A

Jillian M. Hughes-Brown¹; Joy L. Pate^1; Camilla H.K. Hughes¹

1.       Department of Animal Science, Pennsylvania State University, State College, USA

Abstract Text:

The corpus luteum (CL) is a transient endocrine gland that maintains pregnancy by producing progesterone. Although the mature CL will regress in response to prostaglandin F2A (PGF2A), the developing CL is resistant to PGF2A-induced regression. This acquisition of luteolytic capacity occurs in cattle on day 5 of the estrous cycle. To understand the mechanisms promoting resistance to luteolysis, we evaluated changes in the transcriptome and proteome of the CL during the acquisition of luteolytic capacity. CL were collected on days 4 and 6 of the estrous cycle and frozen (n=4/group). Total luteal RNA and proteins were extracted, quantified, and tested for differential abundance using RNA sequencing (NextSeq 2000) and proteomics (Q-Exactive HF mass spectrometry; Data Independent Acquisition mode). Among 19539 transcripts, 2679 were more abundant on day 4 and 1344 were more abundant on day 6 (Padj < 0.10). In total, 5179 proteins were identified, with 63 more abundant on day 4 and 394 more abundant on day 6 (Padj < 0.10). Gene ontology analysis revealed changes in mechanisms associated with apoptosis and nuclear transport during acquisition of luteolytic capacity. Specifically, proteins of several members of the apoptosis-regulating BCL-2 gene family were upregulated in day 6 CL, including brain abundant membrane attached signal protein 1 (BASP1; fold change = 3.497), BCL2 antagonist/killer 1 (BAK1; fold change = 2.329) and BCL2-like 13 (BCL2L13; fold change = 2.456).  Nuclear transport is a critical downstream component of apoptosis. Regulators of nuclear import also increased on day 6, including nucleoporin (NUP) 37 (FC = 2.395), NUP43 (FC = 1.772), and NUP93 (FC = 2.994). Surprisingly, transcripts encoding these proteins were minimally changed or unchanged. miRNA are small, noncoding RNA that regulate protein expression primarily by repressing translation of their targets, without affecting transcript abundance. Our recent miRNA profiling study showed that the most abundant miRNA in day 4 CL, miR-125b-5p, decreased four-fold by day 6. We hypothesized that predicted targets of this miRNA would include the apoptotic proteins that changed without corresponding change in transcript abundance. Indeed, predicted targets (miRDB, TargetScan, and miRTargetLink 2.0) of miR-125b-5p included BAK1, NUP37, and NUP93. To determine whether miR-125b-5p acts as a prosurvival factor in luteal cells, we transfected luteal cells with either mimics or inhibitors of miR-125b-5p and then treated them with interferon gamma (IFNG), which induces apoptosis in luteal cells.  While controls and cells transfected with the miR-125b-5p inhibitor decreased in number in response to IFNG treatment (fold change = 0.673; P = 0.0016), in cells transfected with the miR-125b-5p mimic, cell number did not change in response to IFNG (fold change = 0.853; P = 0.4673). This suggests that miR-125b-5p confers resistance to cell death induced by IFNG. In summary, upregulation of proteins associated with nuclear transport and apoptosis on day 6 may indicate increased potential for susceptibility to cellular death after acquisition of luteolytic capacity. Furthermore, abundant expression of miR-125b-5p likely acts as a prosurvival factor in early CL, inhibiting apoptotic mechanisms and preventing cell death in response to PGF2A. Overall, this study contributes to our knowledge of PGF2A responsiveness and could be used to develop technology to address pregnancy loss. This project was supported by USDA Agriculture and Food Research Initiative Predoctoral Fellowship no. 2017-67011-26062 and Penn State start-up funding to CHKH.