Cathepsin L regulates oocyte meiosis and preimplantation embryo development

Abstract 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.


| INTRODUCTION
Embryo mortality is a major cause of subfertility in humans 1 and animals. 2 In most mammalian species, including humans, embryo losses are estimated to be 20%-40% with at least two third of these embryos are lost during the early preimplantation period of pregnancy. 3,4Successful implantation requires a viable embryo (at the blastocyst developmental stage), a receptive endometrium, as well as an embryo-endometrium effective crosstalk. 5To evolve a compartmentalized mammalian blastocyst ready for implantation, a highly organized sequence of fundamental events must occur.These events include fertilization, oocyte meiosis completion, a series of cleavage divisions, embryonic genome activation, compaction, cavitation (blastocoel formation) and cellular differentiation into the trophectoderm and inner cell mass. 6,73][14] A better understanding of the molecular mechanisms that govern these events is crucial in mitigating early pregnancy losses in humans and animals. 15thepsins are well known lysosomal proteases. 16According to the amino acid present at their active site, cathepsins are categorized into three subfamilies: aspartic (cathepsin A and G), cysteine (cathepsin B, C, F, H, K, L, O, S, V, W and X/Z) and serine (cathepsin D and E) proteases. 17,18These proteases orchestrate a variety of cell functions including immune response, autophagy, growth, as well as development. 191][32][33] However, little is known about the function of this cysteine protease in mammalian oocyte meiosis and embryo development.Using a bovine model, we showed that CTSL activity is correlated with oocyte and embryo quality and regulating such activity is an efficient approach to improve the quality and developmental competence of oocytes and preimplantation embryos.Importantly, CTSL upregulation significantly rescued, at least in part, the developmental competence of heat-shocked oocytes/embryos (notoriously known for reduced quality).

| Chemicals
All chemicals were purchased from Millipore Sigma (St. Louis, MO, USA) unless otherwise stated.

| In vitro maturation, fertilization and culture
Bovine embryos were produced in vitro as we previously described. 34iefly, bovine ovaries were obtained from a local abattoir and transported to the laboratory within 2-3 h in a 0.9% (w/v) warm sodium chloride (NaCl) solution.The ovaries were slashed, and cumulusoocyte complexes (COCs) were collected into oocyte collection medium (M-199 with Hank's salts and supplemented with 2% [v/v] calf bovine serum [ATCC, #30-2030], 1 mM L-glutamine, 0.2 USP/mL heparin, 100 IU/mL penicillin and 0.1 mg/mL streptomycin).Freshly collected immature COCs were microscopically categorized into nine morphological groups before being pooled into either good or poor quality groups as previously described. 35Briefly, the COCs were categorized into nine morphological groups: Group I, II and III (i.e., fully grown oocytes surrounded by more than three compact cumulus layers with homogenous medium brown cytoplasm) were pooled together as good quality COCs, whereas groups IV-IX (i.e., fully grown oocytes showing either abnormal or no cumulus cells and/or inhomogeneous darker or paler cytoplasm) were pooled together as poor quality COCs.Unless otherwise specified, only good quality COCs Twelve hours post-insemination (hpi), putative zygotes were stripped of their cumulus cells by vortexing for 3.5 min, washed in HEPES-TALP medium, then transferred (in groups of $30) to 500 μL/ well modified potassium simplex optimized medium (KSOM) supplemented with 3 mg/mL EFAF-BSA, essential and non-essential amino acids and 2.5 μg/mL gentamycin in four-well plates.In vitro culture (IVC) proceeded for 7-8 days at 38.5 C in a humidified atmosphere containing 5% CO 2 , 5% O 2 and 90% N 2 .HS embryos were only exposed to 41.0 C for the first 12 h of IVC period (i.e., from 12 to 24 hpi), then the temperature was kept at 38.5 C for the remainder of IVC duration. 37The cleavage, blastocyst (i.e., the sum of early blastocyst [EBL], blastocyst [BL], expanded blastocyst [XBL] and hatched blastocyst [HBL]), and HBL rates were assessed at 60, 180 and 204 hpi, respectively.
Lactoferrin from bovine milk (LF; #L9507, Sigma) was dissolved in water and added to IVM/IVC medium at 10, 100 or 1000 μg/mL to inhibit CTSL activity in good quality oocytes/embryos while recombinant human active CTSL (rCTSL; #ab198444, Abcam, MA, USA) was supplemented to IVM/IVC medium at 100, 200 or 400 ng/mL to enhance CTSL activity in good quality oocytes/embryos.The cellular uptake of the recombinant cathepsins has been previously demonstrated. 38,39

| Detection of intracellular CTS activity
The intracellular activity of CTSL was assayed in COCs, denuded oocytes or embryos using Magic Red ® CTSL detection kit (#941, Immunochemistry Technologies, LLC, Bloomington, MN, USA) according to the manufacturer's protocols.Briefly, the targeted cells (COCs, denuded oocytes or embryos) were incubated in the reaction mix (    The number of replicates was included in our statistical model; there was no significant variance between the different replicates.COCs, cumulus-oocyte complexes; CTSL, cathepsin L; HS, heat shock; SEM, standard error of the mean. The oocytes were denuded from their cumulus cells using 1% hyaluronidase solution with vortexing for 3.5 min. 25In a similar way to CTSL detection assay, the intracellular activity of CTSB and CTSK was detected in bovine COCs using Magic Red ® CTSB detection kit (#938, Immunochemistry Technologies) and CTSK detection kits (#940, Immunochemistry Technologies).

| Cell livability assessment
The percentage of dead cells in COCs/embryos was determined on the basis of plasma membrane integrity using a supravital staining (propidium iodide [PI]; #P4170, Sigma) as previously described 40

| Blastocyst total cell number assessment
Blastocysts were harvested at 180 hpi and washed twice in PBS-PVP.
The nuclei of blastocysts were then stained via Hoechst 33342, mounted onto a glass slide, and observed under epifluorescence microscope with an excitation filter of 365 nm.Z-stacks were reconstructed into a 3D structure for every blastocyst and the total cell number (i.e., the number of nuclei) was determined using a Microscopy Image Analysis Software (Imaris ® 9.9 Release, Bitplane AG, Zurich, Switzerland).Results showed that metaphase II (MII) oocytes (i.e., the oocytes showing two sets of DNA with chromosomal alignment at MII plate 41 ) had higher CTSL activities than prophase I-arrested germinal vesicle (GV) oocytes or those failed to reach MII and were arrested at MI stage ( p < 0.0001; Figure 1C).It is well known that the exposure of bovine oocytes to elevated temperatures, either in vivo or in vitro, disrupts their quality and developmental competence. 36,42,43Indeed, the exposure of COCs to HS during IVM (22-24 h) resulted in a lower percentage of oocytes reaching MII stage when compared to COCs in vitro matured at the homeothermic temperature of cows ( p < 0.01; Figure 2A).We

| CTSL inhibition during IVM disturbs oocyte development and compromises the quality of produced embryos
Because our results suggest that CTSL associates with proper oocyte maturation, we then explored the possible involvement of active CTSL in the capacity of oocytes to be fertilized and undergo normal early embryo development.In order to test this, we assessed oocyte developmental competence after CTSL inhibition using LF.5][46] Accordingly, LF was added to IVM medium at different concentrations; we identified that a concentration of 1000 μg/mL causes a significant reduction of CTSL activity in COCs ( p < 0.0001; Figure S1A).On the other hand, the intracellular activity of other cysteine cathepsins (CTSB and CTSK) in COCs showed no significant difference in the absence or presence of LF in IVM medium (Figure S1B) indicating that both LF and CTSL activity detection assay are specific and efficient to inhibit and detect CTSL, respectively.To exclude the possibility of drug cytotoxicity, LF-treated COCs were assessed for their viability using PI staining.There was no significant difference in the percentage of dead cells in LF-treated COCs when compared to non-treated COCs (Figure S1C).
Next, COCs were in vitro matured in the absence (control) or presence of LF (1000 μg/mL) for 22-24 h followed by IVF and IVC.
Our results showed that LF supplementation during IVM decreased the percentage of oocytes that reach the MII stage ( p < 0.05; Figure 3A) and embryo cleavage rate after IVF ( p < 0.05; Figure 3B).
In fact, cleaved embryos derived from LF-treated COCs showed a significant reduction in blastocyst ( p < 0.001) and HBL (p < 0.05) rates when compared to those derived from the non-treated control COCs (Figure 3B).Of note, the blastocyst and HBL rates were calculated in respectively (Figure 3D).Moreover, a further analysis of the blastocyst components (i.e., EBL, BL, XBL and HBL) at 180 and 204 hpi showed a dissimilar composition between the groups; the proportion of EBL (p < 0.001) and BL ( p < 0.01) was significantly higher in LF group than the control group, and neither XBL nor HBL were observed in LF group at 180 hpi.Further, at 204 hpi, the proportion of EBL was higher (p < 0.001) in LF group when compared to controls, indicating a delay in the speed of embryo development (Figure 3E).In line with the poor development observed in the LF group, our analysis revealed that these blastocysts had significantly lower total cell numbers than the control embryos ( p < 0.0001; Figure 3F).

| Supplementation of rCTSL during IVM improves oocyte development under normal and HS conditions
Because CTSL activity is lower in poor quality versus good quality oocytes and its inhibition perturbs oocyte developmental competence, we asked whether the enhancement of intracellular active CTSL in COCs/oocytes could improve oocyte developmental  S2).Accordingly, we supplemented the IVM medium with 100 or 200 ng/mL rCTSL followed by IVF and IVC; a non-supplemented group served as control.We observed a significant increase in the percentage of MII oocytes upon supplementation of IVM medium with 200 ng/mL rCTSL ( p < 0.05; Figure 4A).Further, increasing CSTL levels during oocyte maturation improved embryo development post-fertilization ( p < 0.05; Figure 4B).Moreover, embryos originating from rCTSL-treated COCs showed faster cell progression than those from the non-treated COCs (Figure 4C), which resulted in a higher percentage of HBL (recorded at 180 and 204 hpi; p < 0.01; Figure 4D).In addition, the blastocysts produced from rCTSL group had more cells than those produced from The total number of analysed embryos is specified in each cell stage bar.CTSL activity represents the raw integrated density values of its fluorescence intensity in the defined cells.These values were normalized against those of the two cell-stage embryos.Different letters denote a significant variance (p < 0.05) between the successive embryonic stages when compared using one-way ANOVA followed by Tukey's multiple comparisons test.The number of replicates was included in our statistical model; there was a significant variance ( p = 0.0001) between the different replicates.CTSL, cathepsin L; IVC, in vitro culture; SEM, standard error of the mean.
the control group (p < 0.0001; Figure 4E).Given the improving effect of rCTSL on oocyte developmental competence, we asked whether rCTSL supplementation during IVM can improve the developmental competence of HS-exposed COCs which are associated with poor quality and developmental competence (Figure 4B).Accordingly, a group of COCs was exposed to HS during IVM (22-24 h) in the absence or presence of rCTSL followed by IVF and IVC.Our results showed that increasing the levels of CSTL ameliorates the adverse effects of HS on oocyte developmental competence.Indeed, increasing CSTL levels during oocyte maturation of HS-exposed COCs significantly improved the developmental competence (i.e., increased cleavage and blastocyst rates) of the produced embryos postfertilization (Figure 4B) and tended to increase (p = 0.058) the average total cell number of the produced blastocysts (Figure 4E).The quality of early embryos is largely determined on the basis of cleavage speed and their morphology. 47,48Because of the variance in CTSL activity between successive cleavage-stage embryos identified here, we examined embryo morphology to ascribe embryo quality.The embryo morphology assessment routinely evaluates the cytoplasmic appearance, size and symmetry of blastomeres, degree of fragmentation, presence of vacuoles and the thickness of zona pellucida. 49To investigate if there is an association between embryo morphology and CTSL activity, we assayed CTSL activity in embryos of similar stages at specific time-points after insemination.We observed that the embryos showing morphological abnormalities (e.g., low cytoplasmic homogeneity, asymmetrical blastomeres, high degree of fragmentation or vacuoles) had lower CTSL activity ( p < 0.001; Figure 6A).1][52] To ensure that the lower CTSL activity in morphologically-abnormal embryos reflects their lower quality, we exposed putative zygotes $12 h after insemination to a physiologically relevant heat shock 37 during the first 12 h of IVC followed by assessing CTSL activity.Expectedly, CTSL activity was significantly lower in HS-exposed embryos (i.e., poor quality embryos) when compared to controls ( p < 0.0001; Figure 6B).

| CTSL inhibition during IVC perturbs the development and quality of early embryos
Linking the embryo quality to intracellular CTSL activity led us to hypothesize that CTSL is involved in regulating early embryo development.To test our hypothesis, we first investigated the effect of CTSL inhibition on embryo development.Putative zygotes were in vitro cultured in the absence (control) or presence of LF (1000 μg/mL) for 7-8 days.Our data revealed that LF-treated zygotes have a lower developmental competence when compared to non-treated zygotes (p < 0.05; Figure 7A).It should be emphasized that 1000 μg/mL LF supplementation to IVC medium has the capability of inhibiting CTSL (p < 0.0001; Figure S3A).Moreover, there was no significant difference in the percentage of dead cells in LF-treated embryos when compared to non-treated embryos (Figure S3B).
To uncover how the reduction of intracellular CTSL activity negatively affects early embryo development, we monitored cleavage progression at specific time points post-insemination.There was a delay in embryonic cell progression upon CTSL reduction (Figure 7B,C); such delay was highly apparent by the complete lack of HBL at 180 hpi in LF group (Figure 7D).Moreover, we found reduced total cell numbers in blastocysts from the LF group when compared to the control group ( p < 0.05; Figure 7E).Different letters in line graphs (C) denote a significant variance between the successive time-points within each cell stage in the same group (i.e., capital letters refer to control and small letters refer to LF) when compared using one-way ANOVA followed by Tukey's multiple comparisons test.(E) Evaluation of blastocyst total cell number; asterisks denote a significant variance (* means p < 0.05) between both groups when compared using Student's t-test.CTSL, cathepsin L; HBL, hatched blastocyst; IVF, in vitro fertilization; IVM, in vitro maturation; LF, lactoferrin; SEM, standard error of the mean.
IVC period.Results showed that the rCTSL supplementation to the IVC medium (at 100 ng/mL) significantly improved the developmental competence of HS-exposed embryos (Figure 8A) and tended to increase ( p = 0.12) the average total cell number of the produced blastocysts (Figure 8D) when compared to non-treated HS-exposed blastocysts.

| DISCUSSION
The present study, using a bovine oocyte/embryo model, provides novel evidence that CTSL plays a pivotal role in regulating oocyte mei-   impact on their quality and, in turn, their developmental competence.
In addition, our results demonstrate that upregulating CTSL activity in the oocytes or zygotes shows great potential in improving the quality of preimplantation mammalian embryos with clear applied implications for enhancing assisted reproductive technologies.
Our findings demonstrate a positive correlation between CTSL activity and mammalian preimplantation embryo development.Consistent with our results, monitoring the activity of CTSL protease in zebrafish embryos from the start of fertilization until hatching revealed a time-dependent increase in CTSL activity during the entire process of embryogenesis. 53Moreover, CTSL is required for Caenorhabditis elegans embryo development and its disruption results in the accumulation of yolk platelets in the cytoplasm of early embryos, leading to a high percentage of arrested non-viable embryos and decelerated progression of viable embryos. 54,55Several potential mechanisms can explain the pivotal role of CTSL in early embryo development.
Given that protein and amino acid turnover is essential during oocyte maturation 56 and early embryo development, 57,58 it is possible that intracellular active CTSL, as a cysteine protease, is involved in the regulation of protein degradation and synthesis, which are necessary for oocyte and embryo development and quality.For example, CTSL is essential for the degradation of the inherited sperm histones in sea urchin embryos, a process required for the initiation of cell cleavage. 59,60Consequently, interfering with CTSL function prevents the post-fertilization degradation of these histones and perturbed early embryo development. 61,624][65][66] Since autophagy is required for oocyte meiosis and embryo development under normal and HS conditions, 20,[67][68][69][70][71][72][73][74][75] it is plausible that CTSL regulates oocyte and embryo development via autophagy activation.These mechanisms (i.e., regulating the protein turnover machinery and autophagy) may also explain why CTSL upregulation in oocytes and early embryos protects, at least partially, against the adverse effects of HS.However, further research is necessary to fully comprehend the underlying molecular mechanisms underpinning the involvement of CTSL in regulating embryo quality and enhancing its tolerance to adverse conditions.
We previously demonstrated that the activities of cysteine proteases, CTSB and CTSK, are inversely correlated with oocyte and embryo developmental competence. 20,23,24In a stark contrast to CTSB and CTSK, we found that CTSL activity correlates with oocyte and embryo developmental competence.Although lysosomal cysteine proteases are involved in the breakdown of a wide range of intracellular and extracellular proteins, 16 cathepsins may differ in their substrate specificities and their roles in physiological and pathological processes. 17,18For example, CTSB is involved in the degradation of extracellular matrix proteins (e.g., collagen, elastin and fibronectin) 76 and its proteolytic activity (at its normal physiological level) is essential for several cellular functions. 77However, excessive CTSB activity is detrimental for both oocyte and early embryo development in cattle, [23][24][25] sheep 78,79 and pigs. 80On the contrary, CTSL has a wider range of substrates than CTSB 81 and has been implicated in several physiological and pathological processes including bone remodelling, angiogenesis and tumour invasion. 82Moreover, CTSL has been identified as an important embryotrophin in bovine preimplantation embryos. 83termining the target substrates of the aforementioned cathepsins is essential to understand how these cathepsins differ in their roles in oocyte and early embryo development.
While our data provide several lines of evidence that the intracellular CTSL activity is a prerequisite for the acquisition of oocyte developmental competence, we did not reveal whether such beneficial effect of CTSL is attributed to its activity in oocytes themselves and/or their surrounding cumulus cells.A bi-directional communication exists between the oocyte and cumulus cells throughout folliculogenesis and oogenesis 84 ; such bi-directional communication allows the continuous flow of essential nutrients and signals between the oocyte and its surrounding cumulus cells and thereby enhancing their functions. 85,86Therefore, further investigation is required to address this question.
Reducing CTSL activity by LF treatment in oocytes and zygotes disturbs early embryo development.These findings are in accordance with previous observations that using LF, to inhibit bovine herpesvirus 1, during embryo culture is accompanied by embryo developmental suppression. 87Although LF is a selective inhibitor of CTSL, 44,45 it can also promote CTSG activity. 880][91][92] Moreover, our observations revealed that LF does not affect CTSB or Lactoferrin is expressed in the mucosal epithelium of the reproductive organs and is also detected in seminal plasma, oviductal, uterine, cervical and vaginal fluids. 93A recent study identified a negative correlation between LF levels in the cervical fluid and IVF success rates in women; such negative correlation was highly apparent when the age of patient was ≥35 years. 94It is well established that oocytes from females of advanced reproductive age are of lower quality than those of younger females. 95Therefore, it is tempting to speculate that LF accumulation in genital tract secretions of women with advanced reproductive age, above its physiological levels, contributes to ageassociated reduced oocyte/embryo quality, at least in part, by inhibiting intracellular CTSL activity in oocytes and zygotes.
2 μL of DMSO diluted stock solution in 500 μL culture medium [either OMM or KSOM]) for 20-25 min at 38.5 C in a humidified atmosphere containing 5% CO 2 .For DNA labelling, 1 μg/mL Hoechst 33342 (#639, Immunochemistry Technologies) was added to the cell suspension and coincubated for further 5-10 min.The stained cells were rinsed twice in U R E 1 Intracellular CTSL activity relates to the quality of COCs/oocytes.Detection of intracellular CTSL activity in good and poor quality bovine COCs (A) or denuded oocytes (B) either pre-or post-IVM conduction.(C) Quantification of CTSL activity during the different meiotic stages of bovine oocytes.CTSL activity represents the mean values of fluorescence intensity in the indicated cells.Scale bar represents 50 μm.The data of four independent experiments are presented as mean ± SEM.The total number of analysed COCs/oocytes is specified in each graph.Asterisks denote a significant variance (* means p < 0.05, and **** means p < 0.0001) between the different groups when compared using either Student's t-test (A & B) or one-way ANOVA followed by Tukey's multiple comparisons test (C).The number of replicates was included in our statistical model; there was a significant variance (p = 0.0089) between the different replicates (C).COCs, cumulus-oocyte complexes; CTSL, cathepsin L; GV, germinal vesicle; IVM, in vitro maturation; MII, metaphase II; SEM, standard error of the mean.phosphate buffered saline (PBS, 10 mM potassium phosphate [KPO 4 ], 0.9% [w/v] NaCl, pH 7.4) to which 1 mg/mL polyvinylpyrrolidone (PVP) was added (PBS-PVP).The COCs were then mounted onto a glass slide using a Vectashield ® mounting medium (Vector Laboratories Inc., CA, USA), while oocytes/embryos were transferred to a pre-heated (at 38.5 C) 50 μL drop of PBS-PVP under oil in a 35 mm glass-bottom dish (MatTek Corporation, Ashland, MO, USA).Subsequently, the cells were imaged at 200X using a DMi8 S Platform inverted epifluorescence microscope supported with LAS X software (Leica Microsystems Inc, Buffalo Grove, IL); Magic Red (CTSL activity) was observed as red colour using an excitation filter of 550-590 nm, while Hoechst 33342 (DNA) was observed as blue colour using an excitation filter of 365 nm.The images were captured, then fluorescence emission (red = 592-668 nm; blue = 435-485 nm) intensity (pixels) was analysed using Image J software (FIJI ® ; National Institutes of Health, Bethesda, MD, USA); the mean values of CTSL fluorescent intensity (i.e., sum of pixel values/area of selection) were applied for measuring CTSL activity in the defined cells.CTSL fluorescent intensity was measured in the whole area of oocytes/embryos including their zona pellucida.Due to the unequal cumulus layers surrounding COCs, the measurement was applied to the area of oocyte (including zona pellucida) as well as the first two cumulus cell layers.When comparing CTSL activity levels between embryonic stages, we used raw integrated density values (i.e., sum of pixel values within the selection) to account for blastocoel presence in blastocyst stage embryos.Of note, the images of CTSL activity within the same experiments were captured under the same image setting.

2
Heat shock perturbs CTSL activity in COCs/ oocytes.Bovine COCs were in vitro matured at either 38.5 C (control group) or 41 C (HS group) for 22-24 h followed by quantification of oocytes meiotic progression (A) and detection of intracellular CTSL activity in COCs/denuded oocytes (B).CTSL activity represents the mean values of fluorescent intensity in the indicated cells.Scale bar represents 50 μm.The data of three independent experiments are presented as mean ± SEM.The total number of analysed COCs/oocytes is specified in each graph.Asterisks denote a significant variance (** means p < 0.01, and **** means p < 0.0001) between the different groups when compared using either Student's t-test (A) or Chi-square contingency test (B).

3
with minor modifications.Briefly, COCs/embryos were incubated in a Legend on next page.culture medium (OMM/KSOM), supplemented with 10 μg/mL PI as well as 1 μg/mL Hoechst 33342, for 10-15 min at 38.5 C in a humidified atmosphere containing 5% CO 2 .The samples were then washed twice in PBS-PVP, mounted onto a glass slide, and observed using epifluorescence microscopy.Using an excitation filter of 550-590 nm, the dead cells (i.e., with degenerated plasma membrane) showed a red fluorescence (PI-positive), while the viable cells (i.e., with intact plasma membrane) did not show a red fluorescence (PI-negative); Hoechst 33342 was incorporated in such reaction for DNA labelling (blue).To confirm that the red colour appeared in PIpositive cells was not a result of autofluorescence phenomenon, positive and negative controls were implicated in each experimental design.Positive control COCs/embryos were incubated in RQ1 RNase-free DNase (50 U/mL) at 38.5 C in a humidified atmosphere containing 5% CO 2 for 1 h prior to running PI/Hoechst 33342 staining reaction.On the other hand, negative control COCs/embryos were incubated with Hoechst 33342 only (no PI staining was added to such reaction).

Each experiment was repeated 3 - 1 ( 1 |
Figure 1A).To clarify whether the reduction in CTSL activity could be corrected through the incorporation of these COCs into the physiological maturation process, both immature good and poor quality COCs were subjected to the routine IVM procedures followed by CTSL activity detection.In a similar pattern to the immature COCs, the poor quality mature COCs had lower intracellular CTSL activities when compared to their good quality counterparts ( p < 0.0001; Figure1A).To elucidate whether the disturbances in CTSL activities in relation to COCs quality is attributed to the oocytes and/or the cumulus cells, oocytes were denuded of their granulosa cells, either pre-or post-IVM, and CTSL activity was assayed.In line with what we observed in COCs, CTSL activity was lower in both immature and mature denuded poor quality oocytes when compared to the good quality oocytes ( p < 0.0001; Figure1B).We then investigated whether CTSL activity is altered in relation to oocyte meiotic progression by measuring CTSL activity at various stages of oocyte meiosis.
relation to the number of embryos that reached at least the 2-cell stage at 60 hpi (i.e., cleavage rate) to eliminate the possibility that the poor developmental competence of LF-treated COCs was a reflection of oocyte maturation failure.The above results indicate that low levels of CTSL in COCs/oocytes is associated with poor potency of zygotes to develop into blastocysts.To better understand this phenomenon, we did a detailed characterization of early embryonic development by assessing the number of embryonic cells (i.e., the 2 cell, 3-4 cell, 5-8 cell, 9-16 cell, morula and blastocyst) at specific time points postinsemination (i.e.,48, 84, 132, 180 and 204 hpi).We observed a delay in the cleavage speed of embryos derived from LF-treated COCs (i.e., LF group) when compared to those derived from the non-treated COCs (i.e., control group; Figure3C).In the control group, the majority of 3-4 cell, 5-8 cell, 9-16 cell and morula stage embryos was recorded at 48, 84, 84 and 132 hpi, respectively, while their counterparts in LF group peaked at 84, 132, 204 and 204 hpi, U R E 5 CTSL activity in relation to early embryo development.Detection of intracellular CTSL activity in the developing embryos at the indicated time-points during IVC period.Scale bar represents 50 μm.The data of three independent experiments are presented as mean ± SEM.
U R E 6 CTSL activity is related to early embryo quality.(A) Detection of intracellular CTSL activity in good and poor quality cleaved embryos.(B) Upon conducting the normal IVM and IVF procedures, the putative zygotes were in vitro cultured at either 38.5 C (control group) or 41 C (HS group) for 12 h followed by the detection of intracellular CTSL activity in the developing embryos.CTSL activity represents the mean values of its fluorescence intensity in the defined cells.Scale bar represents 50 μm.The data of three independent experiments are presented as mean ± SEM.The total number of analysed embryos is specified in each graph.Asterisks denote a significant variance (*** means p < 0.001 and **** means p < 0.0001) between the different groups when compared using Student's ttest.The number of replicates was included in our statistical model; there was a significant variance ( p = 0.0001) between the different replicates.The number of replicates was included in our statistical model; there was no significant variance between the different replicates.CTSL, cathepsin L; HS, heat shock; IVF, in vitro fertilization; IVM, in vitro maturation; SEM, standard error of the mean.

3. 4 | 7
The activity of CTSL correlates with the development and quality of preimplantation embryos Our data strongly support the notion that intracellular CTSL activity in COCs plays an important role in acquiring the oocyte developmental competence.Therefore, we wondered if CTSL would also play a role during early embryonic development.We first measured CTSL activity in preimplantation embryos and found a positive correlation (r = 0.69, p < 0.001) between CTSL activity and embryo developmental stage up to the EBL stage, with a decline thereafter (Figure5).Of note, Legend on next page.CTSL activity was detected at the time-points at which each embryo developmental stage reaches its peak (Figure3C), and the values of CTSL fluorescent intensities for the successive embryonic stages were normalized against those of the 2 cell-stage embryos (i.e., our starting point).

3. 6 |
Figure 8C).Furthermore, the blastocyst derived from rCTSL-treated zygotes showed higher total cell number than those derived from the non-treated zygotes ( p < 0.001; Figure8D).Concurrently, another group of putative zygotes was exposed to 41 C for the first 12 h of IVC (in the absence or presence of rCTSL), then returned to control temperature (38.5 C) for the remainder of the osis and preimplantation embryo development.We show that the depletion of CTSL activity in the oocytes or zygotes has a negative

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Legend on next page.

F I G U R E 8
Enhancing CTSL activities in early embryos improves their development under normal and heat shock conditions.The putative zygotes were in vitro cultured with rCTSL (0, 100 or 200 ng/mL) at either 38.5 C (control groups) or 41 C (HS groups) for 7-8 days.The data of three independent experiments are presented as mean ± SEM.The total number of analysed embryos is specified in each graph.(A) Assessment of cleavage, blastocyst and HBL rates.The data were analysed using one-way ANOVA with Tukey's multiple comparisons test.Different letters denote a significant variance ([a] means p < 0.05, [b] means p < 0.01, [c] means p < 0.001 and [d] means p < 0.0001) between each group when compared to the control group (zygotes in vitro cultured at 38.5 C with 0 ng/mL rCTSL), while asterisks denote a significant variance (** means p < 0.01 and *** means p < 0.001) between the indicated groups.(B, C) Quantification of embryonic cell progression in relation to the total number of cleaved embryos (n = 65 for control and n = 69 for rCTSL).Asterisks denote a significant variance (* means p < 0.05, ** means p < 0.01 and *** means p < 0.001) between the corresponding stages of control and rCTSL groups at each time-point when compared using Fisher's exact test.(D) Evaluation of blastocyst total cell number; asterisks denote a significant variance (* means p < 0.05 and **** means p < 0.0001) between each group when compared to the control group (zygotes in vitro cultured at 38.5 C with 0 ng/mL rCTSL) using one-way ANOVA followed by Tukey's multiple comparisons test.CTSL, cathepsin L; HBL, hatched blastocyst; HS, heat shock; rCTSL, recombinant CTSL; SEM, standard error of the mean.CTSK, the only cathepsins identified to play roles in bovine oocytes and embryos.Furthermore, enhancing CTSL activity using rCTSL improved oocyte and early embryo development.Taken together, our results suggest that LF impairs oocyte and embryo development by inhibiting CTSL activity.
Inhibition of CTSL activity during oocyte maturation results in impaired early embryo development.Bovine COCs were in vitro matured without-(control group) or with 1000 μg/mL LF (LF group) followed by the normal IVF and IVC procedures.The data of three independent experiments are presented as mean ± SEM.The total number of analysed oocytes/embryos is specified in each graph.
(A) Quantification of oocyte meiotic progression; asterisks denote a significant variance (* means p < 0.05) between both groups when compared using Chi-square contingency test.(B) Assessment of cleavage, blastocyst and HBL rates; asterisks denote a significant variance (* means p < 0.05, ** means p < 0.01 and *** means p < 0.001) between both groups when compared using Student's t-test.(C-E) Quantification of embryonic cell progression in relation to the total number of cleaved embryos (n = 144 for control and n = 76 for LF).Asterisks in stacked bar graphs (C & E) denote a significant variance (* means p < 0.05, ** means p < 0.01 and *** means p < 0.001) between the corresponding stages of control and LF groups at each time point when compared using Fisher's exact test.Different letters in line graphs (D) denote a significant variance between the successive time points within each cell stage in the same group (i.e., capital letters refer to control and small letters refer to LF) when compared using one-way ANOVA followed by Tukey's multiple comparisons test.(F) Evaluation of blastocyst total cell number; asterisks denote a significant variance (**** means p < 0.0001) between both groups when compared using Student's t-test.COCs, cumulus-oocyte complexes; CTSL, cathepsin L; HBL, hatched blastocyst; IVC, in vitro culture; IVF, in vitro fertilization; LF, lactoferrin; SEM, standard error of the mean.
Upregulating CTSL activities in COCs/oocytes enhances their developmental competence under normal and heat shock conditions.Bovine COCs were in vitro matured with rCTSL (0, 100 or 200 ng/mL) at either 38.5 C (control groups) or 41 C (HS groups) for 22-24 h followed by the normal IVF and IVC procedures.The data of three independent experiments are presented as mean ± SEM.
control group (COCs in vitro matured at 38.5 C with 0 ng/mL rCTSL), while asterisks denote a significant variance (* means p < 0.05, ** means p < 0.01, *** means p < 0.001 and **** means p < 0.0001) between the indicated groups.(C, D) Quantification of embryo cell progression in relation to the total number of cleaved embryos (n = 71 for control and n = 75 for rCTSL).Asterisks denote a significant variance (* means p < 0.05, ** means p < 0.01 and *** means p < 0.001) between the corresponding stages of control and rCTSL groups at each time-point when compared using Fisher's exact test.(E) Evaluation of blastocyst total cell number; asterisks denote a significant variance (*** means p < 0.001 and **** means p < 0.0001) between each group when compared to the control group (COCs in vitro matured at 38.5 C with 0 ng/mL rCTSL) using one-way ANOVA followed by Tukey's multiple comparisons test.COCs, cumulus-oocyte complexes; CTSL, cathepsin L; HBL, hatched blastocyst; HS, heat shock; IVC, in vitro culture; IVF, in vitro fertilization; rCTSL, recombinant CTSL; SEM, standard error of the mean.competence.To this end, rCTSL was added to the IVM medium at different concentrations followed by assessing CTSL activity.Our results indicate that concentrations of 100 and 200 ng/mL, but not 400 ng/ mL (likely compromises cell viability), significantly increased CTSL activity in COCs ( p < 0.0001; Figure