BCL2L15 Depletion Inhibits Endometrial Receptivity via the STAT1 Signaling Pathway

Abstract

In domestic ruminants, endometrial receptivity is critical for a successful pregnancy and economic efficiency. Although the endometrium undergoes major cellular changes during peri-implantation, the precise mechanisms regulating goat endometrial receptivity remain unknown. In this study, we investigated the functional roles and signal transduction of the B-cell lymphoma 2 (Bcl-2)-like protein 15 (BCL2L15) in the regulation of endometrial receptivity in vitro. Our results showed that BCL2L15 was up-regulated in goat endometrial epithelial cells (EECs) under progesterone (P4), estradiol (E2), and interferon-tau (IFN-τ) treatments. Our knockdown of BCL2L15 by specific shRNA that significantly hampered endometrial receptivity. In the absence of BCL2L15, the signal transducer and activator of transcription (STAT)1 and STAT3 pathway were activated. Additionally, pretreatment with the STAT1 inhibitor, fludarabine, restored the effect of silencing BCL2L15 on the endometrial receptivity, but not the STAT3 inhibitor Stattic. Overall, these results suggested that BCL2L15 is the key regulator of endometrial receptivity in goats, regulating the endometrial receptivity through the STAT1 pathway. Understanding the function of BCL2L15-STAT1 in endometrial receptivity is important to the exploration of new targets for the diagnosis and treatment of early pregnancy failure, and improving the success rates for artificial reproduction.

Essential Role of CRIM1 on Endometrial Receptivity in Goat

“…According to our previous description, the EECs were cultured in fresh DMEM/F-12 plus 0.1% bovine serum albumin (BSA) when cell density reached 70–80% confluence [ 70 ]. Then, P 4 (10 −7 M, Sigma, St. Louis, MO, USA) and E 2 (10 −9 M, Sigma, St. Louis, MO, USA) were added to the medium.…”

“…Recombinant lentiviral vectors encoding the CRIM1 shRNA (shCRIM1), the ATG7 shRNA (shATG7) and negative control short hairpin RNA (shN) were constructed as previously described [ 70 ]. The sequences of shCRIM1, shATG7 and shN are shown in Supplementary Table S1 .…”

“…Single GTC suspensions were labeled with CellTracker CM-DiI (1 μM, Yeasen Biotech Co., Ltd., Shanghai, China) and seeded at 2500 cells per well in non-adherent round-bottomed 96-well plates to encourage spheroid development. The GTC spheroids (approximately 50 spheroids per well) were delivered onto EECs, which were treated with hormones and IFN-τ as previously described [ 70 ]. The co-culture system was shaken for 10 min at 110 rpm after 1 h of incubation.…”

“…The method employed for the subsequent steps followed the method described in our previous publication [ 70 ]. Briefly, after blocking in 5% BSA in PBS for 1 h, the samples were incubated with primary antibodies at 37 °C for 2 h, including anti-SPP1 (Wanleibio Co., Ltd. Shenyang, China, WL02378, diluted 1:150) and anti-TFEB (ABclonal A7311, diluted 1:200).…”

“…SEM analysis was performed following previous studies [ 70 ]. Briefly, the cell samples were fixed with glutaraldehyde for 48 h, then dehydrated in a graded ethanol series and critical-point dried.…”