Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents) or intracellular (DNA damage and Ca²⁺ overload) stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER), cytoplasmatic, and mitochondrion‐centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double‐ and triple‐ knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase‐8 has a critical role in pro‐ and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1), RIPK3, and the mixed kinase domain‐like (MLKL) for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice.

Ovarian follicle provides a favorable environment for enclosed oocytes, which acquire their competence in supporting embryo development in tight communications with somatic follicular cells and follicular fluid (FF). Although steroidogenesis in theca (TH) and granulosa cells (GC) is largely studied, and the molecular mechanisms of fatty acid (FA) metabolism in cumulus cells (CC) and oocytes are emerging, little data is available regarding lipid metabolism regulation within ovarian follicles. In this study, we investigated lipid composition and the transcriptional regulation of FA metabolism in 3–8 mm ovarian follicles in bovine. Using liquid chromatography and mass spectrometry (MS), 438 and 439 lipids were identified in FF and follicular cells, respectively. From the MALDI-TOF MS lipid fingerprints of FF, TH, GC, CC, and oocytes, and the MS imaging of ovarian sections, we identified 197 peaks and determined more abundant lipids in each compartment. Transcriptomics revealed lipid metabolism-related genes, which were expressed constitutively or more specifically in TH, GC, CC, or oocytes. Coupled with differential lipid composition, these data suggest that the ovarian follicle contains the metabolic machinery that is potentially capable of metabolizing FA from nutrient uptake, degrading and producing lipoproteins, performing de novo lipogenesis, and accumulating lipid reserves, thus assuring oocyte energy supply, membrane synthesis, and lipid-mediated signaling to maintain follicular homeostasis.

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom‐up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

While uterine epithelium secretes eggshell matrix proteins to regulate eggshell structural organization, uterovaginal junction (UVJ) epithelium supports sperm storage in tubules (SST). Here, we examined the presence of OCX36, OC-116 and OC-17 eggshell matrix proteins in SSTs. Two experimental lines of hens displaying either a long (F+ line) or a short (F- line) potential to store sperm were used, before and 24h after insemination. Using immunohistochemistry and western blot, we analyzed the presence of OC-116, OC-17 and OCX36 proteins in the SSTs. Using lectin and calcium staining, we examined the presence in SSTs of Gal/GalNAc (Galactose/N-acetylgalactosamine) and Glc/GlcNAc (Glucose/N-acetylglucosamine) glycans, as well as calcium ions. Our results indicate that in both F+ and F- hens, the eggshell matrix proteins OC-116 and OCX36 were identified in SST cells and lumen, in contact with spermatozoa. The OC-17 protein was found associated with calcium in F+ and F- hens, only in the SST lumen 24h after insemination. Glycans Gal/GalNAc and Glc/GlcNAc were found to be more abundant in the apical cytoplasmic area of the SST cells of F+ hens than in that of F- hens after insemination. This is the first report demonstrating the presence in SSTs of the OC-116, OC-17 and OCX36 eggshell matrix proteins, and their concomitant presence with Gal/GalNAc and Glc/GlcNAc glycans, as well as with calcium. Our results suggest that the OC-116, OC-17 and OCX36 eggshell matrix proteins may be involved in sperm storage.