Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.

A hallmark of advanced maternal age is a significant increase in meiotic chromosome segregation errors, resulting in early miscarriages and congenital disorders. These errors most frequently occur during meiosis I (MI). The spindle assembly checkpoint (SAC) prevents chromosome segregation errors by arresting the cell cycle until proper chromosome alignment is achieved. Unlike in mitosis, the SAC in oocytes is desensitized, allowing chromosome segregation in the presence of improperly aligned chromosomes. Whether SAC integrity further deteriorates with advancing maternal age, and if this decline contributes to increased segregation errors remains a fundamental question. In somatic cells, activation of the SAC depends upon Aurora kinase B (AURKB), which functions to monitor kinetochore–microtubule attachments and recruit SAC regulator proteins. In mice, oocyte‐specific deletion of AURKB (Aurkb cKO) results in an increased production of aneuploid metaphase II‐arrested eggs and premature age‐related infertility. Here, we aimed to understand the cause of the short reproductive lifespan and hypothesized that SAC integrity was compromised. In comparing oocytes from young and sexually mature Aurkb cKO females, we found that SAC integrity becomes compromised rapidly with maternal age. We show that the increased desensitization of the SAC is driven by reduced expression of MAD2, ZW10 and Securin proteins, key contributors to the SAC response pathway. The reduced expression of these proteins is the result of altered protein homeostasis, likely caused by the accumulation of reactive oxygen species. Taken together, our results demonstrate a novel function for AURKB in preserving the female reproductive lifespan possibly by protecting oocytes from oxidative stress.

The present study investigated the effect of autophagy induction and cathepsin B (CTSB) inhibition on developmental competence of poor quality oocytes. Bovine cumulus oocyte complexes (COCs) were classified as good or poor according to their morphology. Autophagy activity was detected in good and poor germinal vesicle (GV) oocytes. Then E-64, a CTSB inhibitor, rapamycin (Rapa), an autophagy inducer, and combined administration was achieved during in vitro maturation (IVM) of poor quality COCs followed by detection of autophagy activity. In the next experiment, E-64, Rapa, and E64 + Rapa, were added during IVM to good and poor quality COCs followed by in vitro fertilization and culture for 8 days to investigate whether inhibition of CTSB and/or induction of autophagy improve embryonic development and quality. Autophagy activity was significantly lower in poor quality GV oocytes than in good quality ones. E-64, Rapa and E-64 + Rapa treatment during IVM significantly increased autophagy activity in poor quality oocytes. Addition of Rapa in good quality COCs did not increase the blastocyst rate, whereas E-64 increased the blastocyst rate and total cell number (TCN) with decreasing TUNEL-positive cells. In contrast, Rapa treatment in poor quality COCs significantly increased the blastocyst rate and TCN with decreasing TUNEL-positive cells. These results indicate oocyte quality has different responses to intracellular autophagy induction and CTSB activity control by potential autophagy and catabolic status, however, synergetic effect of autophagy induction and CTSB inhibition can increase developmental competence of both good and poor quality COCs, especially rescue effect in poor quality COCs.

Lysosomal cathepsin, in particular cathepsin B (CTSB), plays an important role in implantation, pregnancy, and embryonic development. However, little is known about the mechanism related to the dynamic status of lysosomal cathepsins in bovine oocytes and preimplantation embryos. In the present study, we investigated the dynamics of gene expression, activity, and immunolocalization of CTSB, as well as the activities of lysosome, in bovine oocytes and preimplantation embryos. After gene expression analysis of several cathepsin-related genes, transcript levels of CTSB, CTSD and CTSZ were highest in Metaphase II (MII) oocytes followed by a significant decrease from the 8-cell embryo stage. Activity of CTSB showed a significant increase in 1-cell and morula stage embryos. Lysosomal activity was also significant higher in 1-cell and morula stages, which was consistent with CTSB activities. However, immunolocalization of CTSB did not show the similar pattern of CTSB and lysosomal activities. We also found significantly higher expression levels of CTSB transcript in the trophectoderm (TE) compared to inner cell mass (ICM), whereas activity and immunolocalization of CTSB showed an opposite pattern, i.e. significantly higher in ICM than TE. These patterns were confirmed by the same analysis using separated ICM and TE. Our results suggest that lysosomal CTSB has a pivotal role during embryonic development and differentiation, especially fertilization and the differentiation period.A ssisted reproductive technology, such as in vitro fertilization (IVF), offers great potential for improving the productivity of domestic animals. However, the overall efficiency of in vitro embryo production remains lower than that of in vivo production [1,2]. Not all putative zygotes obtained from in vitro maturation (IVM) and IVF have the ability to develop into blastocysts. The capacity of development is determined by the quality of the oocytes and blastocysts produced by in vitro maturation and development, with high quality oocytes and blastocysts showing the capacity for successful development [3]. In general, oocyte and embryo quality is evaluated morphologically [4]. However, this evaluation does not correlate with embryo quality [5]. Thus, it is important to understand the mechanisms of development and differentiation prior to regulating the quality of preimplantation embryos.Cathepsins (CTSs) are ubiquitous proteases, which belong to the aspartic, cysteine, or serine protease families that catalyze the hydrolysis of proteins. CTSs regulate a variety of normal biological processes such as cell death, proliferation, migration, protein turnover, and cancer [6]. Different types of CTSs have different intracellular catabolic roles during differentiation and development. Knockdown of cathepsin D (CTSD) at oocyte fertilization during zebrafish development showed diseased muscle fibers [7]. Furthermore, expression of CTSB and D was upregulated during mouse trophoblast differentiation and this was necessary for normal embryo development and ute...

In brief The Aurora protein kinases have critical functions in controlling oocyte meiotic maturation. In this study, we describe an assay for examining their activation state in oocytes and establish the best working doses of three commonly used inhibitors. Abstract Several small molecule inhibitors exist for targeting Aurora kinase proteins in somatic cells. From this point of view, we evaluate the specificity of these inhibitors in mouse oocytes, and we demonstrate that MLN 8237 and AZD 1152 are specific for Aurora kinase A and Aurora kinase C, respectively, only when used at low concentrations.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

Mammalian oocytes are transcriptionally quiescent, and meiosis and early embryonic divisions rely on translation of stored maternal mRNAs. Activation of these mRNAs is mediated by polyadenylation. Cytoplasmic polyadenylation binding element 1 (CPEB1) regulates activates mRNA polyadenylation. One message is Aurora kinase C (Aurkc), encoding a protein that regulates chromosome segregation. We previously demonstrated that AURKC levels are upregulated in oocytes lacking Aurora kinase B (AURKB), and this upregulation caused increased aneuploidy rates, a role we investigate here. Using genetic and pharmacologic approaches, we found that AURKB negatively regulates CPEB1-dependent translation of many messages. To determine why translation is increased, we evaluated Aurora kinase A (AURKA), a kinase that activates CPEB1 in other organisms. We find that AURKA activity is increased in Aurkb knockout oocytes and demonstrate that this increase drives the excess translation. Importantly, removal of one copy of Aurka from the Aurkb knockout strain background, reduces aneuploidy rates. This study demonstrates that AURKA is required for CPEB1-dependent translation, and it describes a new AURKB requirement to maintain translation levels through AURKA, a function critical to generating euploid eggs.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.