Cathepsin L regulates oocyte meiosis and preimplantation embryo development.

Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.

IFNT stimulates lysosomal function via type I IFN signaling pathway in pregnant bovine leukocytes.

In brief: Interferon tau (IFNT) stimulates lysosomal activation via the Janus-activated kinase in peripheral blood leukocytes during pregnancy recognition. IFNT-mediated lysosomal activation could serve as a novel marker for early pregnancy in cattle. Abstract: IFNT is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post artificial insemination, after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in nonpregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.

Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells.

Progesterone (P4) is a well-known steroid hormone that plays a key role in oocyte growth and the maintenance of pregnancy in mammals, including cattle. Heat stress (HS) has an adverse effect on P4 synthesis through an imbalance in the cellular redox status. We have recently revealed that a standardized extract of Asparagus officinalis stem (EAS) increases P4 through non-HS induction of heat shock protein 70 (HSP70) and a synergistic increase of HSP70 by enhancing the intracellular redox balance, which was adversely affected by HS in bovine granulosa cells (GCs). Bovine GCs collected from bovine ovarian follicles were cultured at 38.5 °C and 41 °C for 12 h with or without 5 mg/mL EAS. After treatment, cells and culture suppernatant were collected for the analysis. Enzyme-linked immunosorbent assay (ELISA) was performed to detect in P4 levels. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to detect expression of steroidogenesis related genes. Fluorescence staining was used to detect mitochondrial activity and lipid droplet. P4 level was increased by EAS treatment in association with increase in steroidogenic acute regulatory protein (STAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), mitochondrial membrane activity and lipid droplet both under non-HS and HS conditions. Notably, synergistic effect of EAS with HS co-treatment was observed to show a greater increase in P4 synthesis when comparison with EAS treatment under non-HS condition. Furthermore, inhibition of HSP70 significantly reduced EAS-induced P4 synthesis, mitochondrial activity and synthesis of lipid droplets. These results suggest that P4 synthesis by EAS is mediated by the steroidogenesis pathway via HSP70-regulated activation of STAR and 3ß-HSD, together with improved mitochondrial activity and lipid metabolism in bovine GCs. Moreover, effect of EAS has a synergistic effect of with HSP70-regulated steroidogenesis pathway.

Lycopene Reduces the In Vitro Aging Phenotypes of Mouse Oocytes by Improving Their Oxidative Status.

Postovulatory aging is a major problem that limits the success of many assisted reproductive technologies (ARTs). Oxidative stress is a leading cause of oocyte aging. This study investigated the effects of lycopene supplementation of in vitro maturation (IVM) medium during the aging of mouse oocytes on the oocytes' morphology and oxidative stress status. Mouse cumulus-oocyte complexes (COCs) were collected and cultured in the IVM medium either for 17 h, (freshly matured oocytes), or for 48 h, (in vitro-aged oocytes), with or without lycopene. The rate of fragmented and degenerated oocytes and the oocyte levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), total antioxidant capacity (TAC), reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) were estimated and compared. Oocytes aged with 200 nM lycopene revealed significantly less fragmentation and degeneration, lower H2O2 and MDA levels, and higher TAC, GSH and SOD levels than those aged without lycopene. CAT levels were unchanged by lycopene treatment. Taken together, our data showed beneficial effects of lycopene during in vitro aging of mouse oocytes by reducing the oxidative stress damages that lead to their apoptosis. The present study introduces lycopene as a natural supplement to reduce the postovulatory aging-dependent abnormalities of mammalian oocytes.

Effect of autophagy induction and cathepsin B inhibition on developmental competence of poor quality bovine oocytes.

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 invitro 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 invitro 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.

Dynamic status of lysosomal cathepsin in bovine oocytes and preimplantation embryos.

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.

TLR2/4 signaling pathway mediates sperm-induced inflammation in bovine endometrial epithelial cells in vitro.

We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro, however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction.

Heat-shock-induced cathepsin B activity during IVF and culture compromises the developmental competence of bovine embryos.

Heat stress can cause significant reproductive dysfunction in mammals and previous studies report that expression and activity of cathepsin B (CTSB), a lysosomal cysteine protease, is negatively correlated with the developmental competence of bovine oocytes and embryos. However, the relationship between heat shock (HS) and CTSB remains largely unknown. Here, we investigated the effects of HS during IVF and early embryonic stages of IVC on CTSB activity and developmental competence in bovine embryos. HS (40 °C for 6 h during IVF and 20 h during IVC) caused a significant increase in CTSB activity irrespective of the developmental stage or duration of HS. The developmental rate to the blastocyst stage was also significantly decreased by HS. Additionally, HS during IVC significantly increased the number of apoptotic cells in blastocysts. Notably, these HS-induced changes in blastocyst development and quality were significantly improved by inhibition of CTSB activity, indicating a key role for CTSB. These results showed that CTSB activity plays an essential role in HS-induced dysfunction in bovine embryo development, and that inhibition of this activity could enhance the developmental competence of heat-shocked embryos.

Activation of lysosomal cathepsins in pregnant bovine leukocytes.

In ruminants, interferon-tau (IFNT)-mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy.

Different thermotolerances in in vitro-produced embryos derived from different maternal and paternal genetic backgrounds.

The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro-produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre-incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre-incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.

Pyridoxine supplementation during oocyte maturation improves the development and quality of bovine preimplantation embryos.

Recently, inhibition of cathepsin B (CTSB) activity during in vitro maturation (IVM) and culture (IVC) improved the developmental competence and quality of bovine oocytes and embryos. E-64 is a widely used inhibitor to inhibit CTSB activity, however, E-64 inhibits not only CTSB activity but also the activities of other proteases including cathepsin L (CTSL), papain, calpain, and trypsin. Pyridoxine, the catalytically active form of vitamin B6, plays a crucial role in several cellular processes and has the ability to inhibit CTSB activity. However, whether pyridoxine has an improving effect during IVM of bovine oocytes is still unknown. In this study, we investigated the effect of pyridoxine supplementation during IVM on the developmental competence of bovine oocytes and the quality of the produced blastocysts. Supplementation of pyridoxine to the maturation medium significantly decreased the activity of CTSB in both bovine cumulus cells and oocytes. Moreover, pyridoxine improved both the blastocyst and hatched blastocyst rates. In addition, the presence of pyridoxine during IVM also significantly improved the quality of the produced embryos by increasing the total cell number as well as decreasing the CTSB mRNA expression and apoptotic rate. These results indicate that pyridoxine is a promising tool to improve the developmental competence of bovine oocytes and subsequent embryo quality.

Status of autophagy, lysosome activity and apoptosis during corpus luteum regression in cattle.

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, LC3ß, 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.

Effects of downregulating DNA methyltransferase 1 transcript by RNA interference on DNA methylation status of the satellite I region and in vitro development of bovine somatic cell nuclear transfer embryos.

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.

Gene silencing of DNA methyltransferases by RNA interference in bovine fibroblast cells.

A highly methylated genome, like a somatic donor cell, is observed in somatic cell nuclear transfer (SCNT) embryos. The aberrant DNA methylation status causes global gene expression failure, resulting in low developmental competence of SCNT embryos. In addition, recent studies have uncovered the relationship between DNA methylation status and reprogramming efficiency. Because DNA methylation is performed by DNA methyltransferases (DNMTs), developing a technique which specifically inhibits DNMTs is necessary for further SCNT studies. In the present study, we examined the potential use of RNA interference for knockdown of DNMT mRNA in bovine fibroblast cells that were commonly used as karyoplast donors in SCNT studies. We designed three siRNAs corresponding to DNMT1, DNMT2 and DNMT3a mRNA. In Experiment 1, to optimize transfection conditions, fluorescence and cell viability after transfection were evaluated at different concentrations of transfection reagent using a FITC-labeled nonsilencing control siRNA. Although fluorescence was observed in all groups transfected except for the negative control group, transfection with a higher concentration of transfection reagent significantly decreased in cell viability (P<0.05). In Experiment 2, the amount of DNMT mRNA was measured by real-time PCR at 0, 48 and 96 h after siRNA transfection into the cells. The levels of each DNMT mRNA were significantly decreased at 48 and 96 h after transfection (P<0.01). Furthermore, decreased expression of DNMT1 protein was confirmed by western blotting. In Experiment 3, the DNA methylation statuses were analyzed in each of the siRNA-transfected groups. The DNMT1 siRNA-transfected group had a significantly decreased level of DNA methylation (P<0.05), but the other groups did not. Our data demonstrate that RNA interference with siRNA can be analyzed the function and expression of DNMT genes in bovine fibroblast cells. The present study provides useful information for further SCNT studies.