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Inhibition of cathepsin B activity prevents deterioration in the quality of in vitro aged porcine oocytes
Theriogenology 116 (2018) 103e111
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Inhibition of cathepsin B activity prevents deterioration in the quality of in vitro aged porcine oocytes Shuang Liang a, b, Hao Jiang a, b, Xing-Hui Shen c, Jia-Bao Zhang a, Nam-Hyung Kim a, b, * a
Department of Animal Science, College of Animal Sciences, Jilin University, Changchun, Jilin, 130062, China Department of Animal Science, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea c Department of Histology and Embryology, Harbin Medical University, Harbin, 150081, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2018 Received in revised form 29 April 2018 Accepted 30 April 2018 Available online 4 May 2018
The activity of cathepsin B, a member of the lysosomal protease family, directly correlates with oocyte quality and subsequent embryonic development. However, its biological function during the progression of in vitro aging of oocytes in pigs has not been demonstrated. Here, we showed that cathepsin B activity was dramatically increased during in vitro aged oocytes. The inhibition of cathepsin B activity prevented the decline of the quality of aged oocytes and improved their subsequent developmental competence. Moreover, the inhibition of cathepsin B activity reduced aging-induced mitochondrial dysfunction and attenuated oxidative stress. The inhibition of cathepsin B activity also markedly decreased early apoptosis levels and the frequency of spindle anomalies during in vitro aging of oocytes. These results demonstrate that in vitro aging of oocytes induces cathepsin B activity, which is associated with a decline in oocyte quality. The inhibition of cathepsin B activity has a beneficial effect on oocytes during the process of in vitro aging. © 2018 Published by Elsevier Inc.
Keywords: Oocyte aging Cathepsin B Pig Embryo development
1. Introduction In most mammals, oocytes liberated from follicles are arrested at metaphase of the second meiotic division (metaphase II) until fertilization or activation. If metaphase II-arrested oocytes are not fertilized within an appropriate time, they undergo a timedependent deterioration in quality, which is referred to as“postovulatory aging” [1,2]. This decline in oocyte quality decreases fertilization and increases the risk of polyspermy, ultimately resulting in poor developmental potential and early pregnancy loss [1,3,4]. Until now, the mechanism by which oocyte aging is regulated has been unclear. In pigs, as with several mammals, the aging process not only occurs in vivo, but also in vitro in culture conditions [5,6]. Many experiments involving micromanipulation and in vitro fertilization utilize cultured oocytes during assisted reproductive technologies [7e9]. In pigs, in vitro experiments are more efficient than those performed in vivo because, in vivo, pig oocytes are more difficult to obtain in the laboratory compared with other rodents
* Corresponding author. Department of Animal Science, College of Animal Sciences, Jilin University, Changchun, Jilin, 130062, China. E-mail address: [email protected] (N.-H. Kim). https://doi.org/10.1016/j.theriogenology.2018.04.035 0093-691X/© 2018 Published by Elsevier Inc.
[10,11]. Therefore, the efficiency of obtaining porcine embryos is determined by the quality of oocytes matured in vitro, and this directly influences subsequent embryo development. Previous research in pig oocytes has shown that there is a relatively large drop in embryonic developmental competence when the in vitro maturation period is prolonged [12,13]. Hence, delaying oocyte in vitro aging might improve their quality and provide more time for various clinical assisted reproductive technology outcomes in pigs. Cathepsin B is a prominent cysteine protease, and is present in lysosomes of cells under normal physiological conditions [14]. Cathepsin B plays an important role in apoptosis [15,16]. Cathepsin B released from lysosomes can induce apoptosis by activating caspases indirectly through promoting mitochondrial dysfunction [17,18]. Recently, several studies suggested the crucial roles of cathepsin B in affecting oocyte and embryo quality, and its activity could alter their developmental capacity [19e22]. Previous studies also suggested that poor quality oocytes, associated with low developmental competence, have increased cathepsin B activity and protein expression compare to good quality oocytes [21,22]. Decreasing cathepsin B activity with a specific inhibitor (E64) greatly enhanced the developmental competence of oocytes or embryos during in vitro maturation or the in vitro culture period [21,23]. Because in vitro aging causes a time-dependent
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deterioration of oocyte quality, and has a negative effect on subsequent embryonic developmental competence [24], we hypothesize that cathepsin B activity is also likely associated with in vitro aging of oocytes. In the present study, we investigated the involvement of cathepsin B in the mechanism of in vitro aging of oocytes in pigs. We examined changes in cathepsin B activity during in vitro aging of oocytes. Furthermore, we investigated whether the process of in vitro aging of oocytes was changed when the activity of cathepsin B was altered in pig oocytes by culturing with E64, an effective inhibitor of this enzyme. 2. Material and methods All chemicals and reagents used in this research were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. 2.1. In vitro maturation and aging of porcine oocytes Porcine ovaries were collected from a local abattoir and transported to the laboratory in sterile saline at 35e37 C within 2 h in a vacuum flask. Cumulus-oocyte complexes (COCs) were aspirated from follicles 3e8 mm in diameter using a 20-gauge needle attached to a 10 mL disposable syringe. COCs were subsequently collected under a stereo microscope in Tyrode's lactate-HEPES medium. Only oocytes with a homogeneous cytoplasm and compact cumulus cells were chosen and cultivated in maturation medium consisting of tissue culture medium-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) porcine follicular fluid, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 1 mg/mL insulin, 10 ng/mL epidermal growth factor, 0.5 mg/mL follicle stimulating hormone, 0.5 mg/mL luteinizing hormone, and 75 mg/mL kanamycin for 44 h at 38.5 C in an atmosphere containing 5% CO2 in air under maximum humidity. To generate in vitro aged oocytes, COCs were cultivated in maturation medium without follicle stimulating hormone and luteinizing hormone for 24 or 48 h, as described previously [25,26]. The cathepsin B inhibitor, E64, was
present in the maturation medium during the entire extended period of oocyte incubation. Oocytes were denuded by gentle pipetting in Tyrode's lactate-HEPES supplemented with 1 mg/mL hyaluronidase and 0.1% polyvinyl alcohol (PVA). 2.2. Measurement of intracellular cathepsin B activity Intracellular cathepsin B activity was detected using a commercial Magic Red cathepsin B assay kit (Catalog #938; Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's protocol. Briefly, denuded oocytes were incubated in 50 mL phosphate buffered saline (PBS)-PVA (0.1%, w/v) with 2 mL reaction mix for 20 min at 37 C while protected from light. Fluorescence signals were captured as TIFF files using a digital camera connected to a fluorescence microscope (IX70, Olympus, Tokyo, Japan). The same procedures were used for all groups of oocytes, including incubation, rinsing, and imaging. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the fluorescence intensities of the oocytes. 2.3. Activation and in vitro culturing Denuded oocytes were activated by two direct current pulses of 120 V/mm for 60 ms in 300 mM mannitol containing 0.1 mM CaCl2, 0.05 mM MgSO4, 0.01% PVA (w/v), and 0.5 mM HEPES. Following activation, the oocytes were cultured for 3 h in bicarbonatebuffered porcine zygote medium-5 (PZM-5) containing 4 mg/mL bovine serum albumin (BSA) and 7.5 mg/mL cytochalasin B to suppress extrusion of the pseudo-second polar body. Following culture, activated oocytes were thoroughly washed and cultured for 7 days at 38.5 C in bicarbonate-buffered PZM-5 supplemented with 4 mg/mL BSA in an atmosphere containing 5% CO2 in air under maximum humidity without changing the medium. The development of activated oocytes into blastocysts was examined 7 days after activation. To count total cell numbers, blastocysts were stained with 10 mg/mL Hoechst 33342 for 15 min, mounted on glass slides, and examined under an inverted fluorescence microscope.
Fig. 1. Intracellular cathepsin B activity changes in porcine oocytes during in vitro aging. (A) Representative fluorescent images showing intracellular cathepsin B activity in oocytes after in vitro aging. Red: cathepsin B. Scale bar ¼ 100 mm. (B) Relative fluorescence intensity of cathepsin B in oocytes after in vitro aging. Relative fluorescence intensity of cathepsin B in oocytes aged for 24 h (C) and 48h (D) after E64 treatment. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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2.4. Cell proliferation analysis Cell proliferation was assessed by performing a bromodeoxyuridine (BrdU) assay as described previously [27]. Briefly, blastocysts were incubated for 6 h with 100 mM BrdU dissolved in bicarbonate-buffered PZM-5 with 4 mg/mL BSA at 38.5 C in an atmosphere containing 5% CO2 in air under maximum humidity. After incubation, blastocysts were fixed in ice-cold methanol for 20 min and permeabilized with 0.1% (v/v) Triton X-100 for 2 min. The blastocysts were then washed with PBS-PVA and treated with 2 N HCl at room temperature for 30 min. Next, the blastocysts were washed and incubated overnight with a mouse anti-BrdU monoclonal antibody (Sigma-Aldrich; B2531) diluted 1:10 at 4 C. The blastocysts were then washed with PBS containing 0.05% Tween 20 (PBS-T) and incubated for 1 h with a rabbit anti-mouse IgG Alexa Fluor 568-conjugated polyclonal antibody (1:200; Cat. A-11061; Invitrogen) at room temperature. Nuclei in the blastocysts were stained with Hoechst 33342 (10 mg/mL). Finally, the blastocysts were mounted on glass slides and examined under a confocal laser scanning microscope (Zeiss LSM 510 and 710 META, Oberkochen, Germany). Proliferating blastocyst cells were counted using Image J software.
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Prism software package (version 6.01; GraphPad, La Jolla, CA, USA). Continuous data were evaluated by a one-way analysis of variance (ANOVA) followed by a post-hoc Fisher's PLSD test. Data were normally distributed and variances were homogeneous prior to conducting ANOVA. Comparisons of data between two groups were performed with Student's t-test. Data are presented as the means ± SD and p < 0.05 was considered statistically significant. 3. Results 3.1. Changes in cathepsin B activity associated with in vitro aging of oocytes To investigate the potential involvement of cathepsin B activity in oocyte quality during in vitro aging, porcine oocytes were aged
2.5. Immunofluorescence and Annexin V analysis Denuded oocytes were fixed in 3.7% (w/v) paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for an additional 30 min at 37 C, then blocked with 1% BSA in PBS for 1 h. The oocytes were then incubated overnight at 4 C with the primary antibody against caspase 3 (Cat: ab49822; Abcam) diluted in blocking solution (1:100). After washing extensively with PBS-T, the oocytes were labeled with secondary antibodies for 1 h. Spindles were stained with an FITC-conjugated anti-a-tubulin antibody. After extensive washes with PBS-T, the nuclei were stained with Hoechst 33342 (10 mg/mL). Finally, oocytes were mounted onto glass slides and examined using a confocal laser scanning microscope. Annexin V in oocytes was detected using the Annexin VeFITC apoptosis detection kit according to the manufacturer's instructions. To differentiate between live and dead oocytes, the oocytes was stained with PI before Annexin V assay. Only PI negative oocytes were used further experiment. Fluorescence signals were detected using the laser scanning confocal microscope. 2.6. Measurement of mitochondrial membrane potential (DJm) and reactive oxygen species (ROS) generation To evaluate the DJm, denuded oocytes were incubated at 37 C with 2 mM JC-1 (Invitrogen) for 30 min while protected from light. DJm was calculated as the ratio of red florescence (corresponding to activated mitochondria; J-aggregates) to green fluorescence (corresponding to less active mitochondria; J-monomers) [28]. ROS levels were measured by a 20 ,70 -dichlorofluorescein assay as described previously [29]. Briefly, denuded oocytes were incubated for 15 min in PBS-PVA medium containing 10 mM 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA) at 37 C while protected from light. Fluorescence signals were captured as TIFF files using a digital camera connected to a fluorescence microscope. The same procedures, including incubation, rinsing, and imaging, were used for all groups of oocytes. Image J software was used to analyze the fluorescence intensities of the oocytes. 2.7. Statistical analyses All statistical analyses were performed using the GraphPad
Fig. 2. Effects of E64 exposure on porcine oocyte morphology changes during in vitro aging. (A) Representative morphological changes in aged porcine oocytes are shown. (B) Percentages of oocyte fragmentation were recorded after exposure to E64. Scale bar ¼ 100 mm. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments.
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in vitro for 24 and 48 h, and intracellular cathepsin B activity was measured. The results demonstrated that intracellular cathepsin B activity in oocytes increased in a time-dependent manner (1.00 ± 0.09, 1.23 ± 0.38, 1.74 ± 0.37; Fig. 1A and B). A significant in increase in cathepsin B activity was seen in porcine oocytes aged for 24 h, with a peak occurring at around 48 h. Supplementation with E64 during the process of in vitro aging of oocytes decreased intracellular cathepsin B activity at 24 (1.00 ± 0.12 vs 0.85 ± 0.14; Fig. 1C) and 48 h (1.00 ± 0.11 vs 0.86 ± 0.13; Fig. 1D) significantly, as compared with that in control oocytes.
3.2. Inhibition of cathepsin B activity reduces the abnormal morphological changes during in vitro aging of oocytes In vitro aged oocyte quality is associated with morphological changes [25]. Therefore, we investigated oocyte morphological
changes during in vitro aging with and without E64 treatment. As shown in Fig. 2A and B, in vitro aging resulted in a time-dependent deterioration to porcine oocyte quality (3.78 ± 1.40%, 5.98 ± 0.41%, 39.6 ± 7.23%). When in vitro aged oocytes were exposed to E64, the percentage of morphological defects was not dramatically changed when compared with control oocytes at 24 h (5.98 ± 0.41% vs 4.90 ± 0.38%). However, the percentage of morphological defects was dramatically lower compared with control oocytes at 48 h (39.60 ± 7.23% vs 30.20 ± 3.83%).
3.3. Inhibition of cathepsin B activity improves the embryonic developmental potential of in vitro aged oocytes To extend our finding that inhibiting cathepsin B activity ameliorated the time-dependent deterioration of porcine oocyte quality, we monitored their developmental potential after
Fig. 3. Developmental competence of in vitro aged porcine oocytes after E64 exposure. (A) Blastocyst formation is shown at day 7. Scale bar ¼ 100 mm. (B) Blastocyst formation rate. (C) Total cell number in each blastocyst. (D) Hatching rate. (E) Cell proliferation rate in each blastocyst. The numbers of embryos examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments.
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Fig. 4. Effects of E64 exposure on the mitochondrial membrane potential (DJm) in porcine oocytes during in vitro aging. (A) Representative fluorescent images of JC-1 stained oocytes after in vitro aging. Scale bar ¼ 50 mm. (B) Quantification of JC-1 fluorescence intensity. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments.
parthenogenesis (Fig. 3A). The results showed that inhibiting cathepsin B activity during in vitro aging of oocytes ameliorated declines in their subsequent developmental competence. When in vitro aged oocytes were treated with E64, the proportion reaching the blastocyst stage was increased (58.50 ± 6.24%, 26.25 ± 3.59%, 36.00 ± 2.94%; Fig. 3B). In addition, the number of cells per blastocyst tended to be higher in the E64 treated aged oocytes than in the non-treated aged oocytes (46.25 ± 5.02%, 33.55 ± 5.62%, 37.24 ± 5.88%; Fig. 3C). Moreover, the hatching rate
of blastocysts derived from E64-treated aged oocytes was also increased as compared with non-treated aged oocytes (28.50 ± 3.41%, 13.75 ± 2.22%, 19.5 ± 1.30%; Fig. 3D). However, no significant changes in blastocyst formation between E64-treated and -untreated aged oocytes were observed at 48 h (5.33 ± 2.08% vs 7.33 ± 1.53%; Fig. S1). Further analyses revealed that inhibiting cathepsin B activity during in vitro aging of oocytes also increased cell proliferation in these blastocysts (42.26 ± 6.62%, 30.56 ± 6.10%, 37.79 ± 5.21%; Fig. 3E).
Fig. 5. Effects of E64 exposure on the production of reactive oxygen species (ROS) in porcine oocytes during in vitro aging. (A) Representative fluorescence images of ROS in oocytes after in vitro aging. Green: ROS. Scale bar ¼ 50 mm. (B) Quantification of ROS levels. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 6. Effects of E64 exposure on apoptosis in porcine oocytes during in vitro aging. (A) Representative images of Annexin V staining in oocytes after in vitro aging. Green: Annexin V. Scale bar ¼ 20 mm. (B) The percentages of Annexin V positive oocytes were recorded after exposure to E64. (C) Representative fluorescence images of CASPASE 3 levels in oocytes after in vitro aging. Scale bar ¼ 20 mm. (D) Quantitative analysis of CASPASE 3 fluorescence intensity. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.4. Inhibition of cathepsin B activity maintains mitochondrial function and reduces the generation of ROS during in vitro aging of oocytes Because mitochondria play crucial roles in maintaining normal cell metabolic functions [30], we evaluated mitochondrial function (as indicated by DJm) during in vitro aging of oocytes with and without E64 treatment. Representative images of JC-1 staining are shown in Fig. 4A. The DJm in aged oocytes was rapidly reduced, whereas it increased markedly in the presence of E64 (1.06 ± 0.18, 0.70 ± 0.15, 0.88 ± 0.15; Fig. 4B). In addition, compared to non-treated oocytes, those treated with E64 displayed significantly lower ROS levels during in vitro aging of oocytes (1.00 ± 0.32, 1.78 ± 0.57, 1.30 ± 0.35; Fig. 5). These results suggest that inhibiting cathepsin B activity during in vitro aging of oocytes could maintain mitochondrial function and reduce ROS generation.
3.5. Inhibition of cathepsin B activity inhibits apoptosis during in vitro aging of oocytes Because in vitro aged oocyte is closely related to apoptosis [25,31], we next analyzed apoptotic status in in vitro aged oocytes. The result showed that the percentages of early apoptotic (Annexin V positive) oocytes were significantly increased during in vitro aging. However, the percentages of early apoptotic oocytes were decreased in oocytes treated with E64 (28.33 ± 3.22%, 46.00 ± 3.61%, 37.33 ± 2.52%; Fig. 6A and B). Furthermore, immunostaining analysis revealed that CASPASE 3 expression was increased and E64 treatment dramatically reduced this upregulation of caspase 3 expression during in vitro aging of oocytes (1.00 ± 0.26, 1.98 ± 0.19, 1.43 ± 0.30; Fig. 6C and D).
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3.6. Inhibition of cathepsin B activity maintains the spindle morphology of in vitro aged oocytes Because decreased oocyte quality is always accompanied by spindle defects [3,25], we examined whether the meiotic spindle morphology was affected by E64 treatment during in vitro aging of oocytes. As shown in Fig. 7A, control oocytes showed normal spindle morphology, whereas aged oocytes showed disrupted spindle morphology. However, the frequency of abnormal spindle morphology in aged oocytes was dramatically reduced when E64 was added during the process of in vitro aging of oocytes (27.20 ± 3.56%, 43.20 ± 4.44%, 36.60 ± 3.65%; Fig. 7B). 4. Discussion Postovulatory mammalian oocytes undergo a marked quality decline during the process of aging, both in vivo and under in vitro culture conditions [3,32,33]. These aged oocytes show lower fertilization rates and pre-implantation developmental competence [3,12]. In the present study, we showed that cathepsin B activity was significantly more abundant after aging, and was associated with the aging-dependent decline in porcine oocyte quality. The inhibition of cathepsin B activity significantly improved the quality of aged pig oocytes and subsequent early embryo development. These improvements were achieved by maintaining normal morphology and mitochondrial function, reducing ROS generation, and inhibiting the apoptosis that otherwise occurs during in vitro aging of oocytes. Cathepsin B is a lysosomal cysteine protease expressed in various types of cells. It plays an important role in the degradation of intracellular proteins in lysosomes [23,34,35]. Previous studies
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clearly showed that oocytes and embryos with low developmental competence exhibited increased cathepsin B activity compare to those of high developmental competence [20,21]. These results suggest that cathepsin B activity is inversely correlated with the quality of oocytes and embryos. With in vitro aging, we found that cathepsin B activity was increased, suggesting that this contributed to the dysfunction observed in aged pig oocytes. Importantly, the subsequent embryonic development potential was markedly increased when cathepsin B activity was blocked with an inhibitor. These results suggest that increased cathepsin B activity is a phenomenon during the process of in vitro aging of oocytes in pigs. The oocyte aging process is accompanied by age-related mitochondrial dysfunction, which is directly responsible for the high levels of developmental retardation and delay, or arrest of pre-implantation embryos, produced in vitro [3,36]. The results of previous studies suggested that cathepsin B activity was implicated in destabilization of the mitochondrial outer membrane and loss of DJm [37,38]. Cathepsin B-induced mitochondrial membrane degradation could ultimately lead to the release of proapoptotic factors into the cytoplasm [39,40]. DJm is generally used as an indicator of mitochondrial function and the quality of oocytes [3,41]. DJm reflects the pumping of hydrogen ions across the mitochondrial inner membrane during electron transport, which is the driving force for ATP production [42]. In concordance with previous results implicating cathepsin B activity in mitochondrial function, our results show that inhibiting cathepsin B activity prevents the change of DJm during in vitro aging of oocytes in pigs. However, the detailed mechanisms underlying the interplay between cathepsin B and mitochondrial function in postovulatory oocytes deserves further investigation. A previous study has shown that oxidative stress is a factor that is likely to be
Fig. 7. Effects of E64 exposure on spindle morphology in porcine oocytes during in vitro aging. (A) Representative images of spindle morphology in oocytes after in vitro aging. Green: a-tubulin; Blue: DNA. Scale bar ¼ 20 mm. (B) The percentages of abnormal spindles in oocytes were recorded after exposure to E64. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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involved during in vitro aging of oocytes [24]. High levels of intracellular ROS are commonly considered to be a major factor responsible for negative signs of aging in oocytes [43]. Therefore, increased intracellular ROS production during in vitro aging of oocytes is a crucial factor that affects oocyte quality [43]. Given that cathepsin B activity is associated with ROS production by facilitating mitochondrial membrane disruption [44], we hypothesized that the inhibition of cathepsin B activity might preserve oocyte quality by preventing the increase in ROS production. A previous study suggested that inhibiting cathepsin B activity protected mitochondria, decreased oxidative stress, and attenuated liver cell injury in vivo [45]. Porcine oocytes have relatively more intracellular lipids compared with oocytes of other species, making them highly sensitive to ROS-induced damage [46]. In the present study, we found that the inhibition of cathepsin B activity efficiently prevented the production of ROS in in vitro aged oocytes. Cathepsin B is released from lysosomes and contributes to apoptosis by indirectly inducing mitochondrial dysfunction [47]. In bovine oocytes, inhibiting cathepsin B activity ameliorated heat shock-induced apoptosis and improved their subsequent developmental competence [19]. The end point of oocyte aging is cell death through an apoptotic pathway [31,48]. Therefore, it is reasonable to speculate that cathepsin B induces apoptosis during the in vitro aging process. Furthermore, previously studies have suggested that in vitro aged oocytes suffer from oxidative stress, which always induces apoptosis [49,50]. In the present study, we observed that inhibiting cathepsin B activity inhibited the apoptosis in aged oocytes. These results suggest that inhibiting cathepsin B activity can modulate intracellular ROS levels and inhibit signs of apoptosis, thus suppress the aging process in porcine oocytes. Previous studies demonstrated that the in vitro aging process in oocytes was commonly associated with defective spindle assembly and chromosome alignment [3,25,51], which is consistent with our results. We also observed that inhibiting cathepsin B activity reduced the frequency of spindle defects, further supporting the association of cathepsin B with the in vitro aging process in oocytes. In fact, decreased mitochondrial function and increased oxidative stress is often associated with an age-related higher incidence of meiotic spindle abnormalities during oocyte maturation [52]. Therefore, inadequate mitochondria function and increases oxidative stress in oocytes may be key factors contributing to spindle disorganization. It is therefore possible that inhibiting cathepsin B activity ameliorates mitochondrial function and reduces oxidative stress probably protects spindle from the effect of aging. These results further demonstrate that cathepsin B activity plays a crucial role during the process of in vitro aging in pig oocytes. In conclusion, our findings indicate that an increase in cathepsin B activity is one phenomenon of the process of in vitro aging in pig oocytes. The inhibition of cathepsin B activity prevents multiple changes associated with oocyte aging. This provides a potential new pathway of delaying the aging of pig oocytes.
Conflicts of interest The authors declare that they have no competing interests.
Acknowledgements This study was supported by a grant from the Next-Generation BioGreen 21 Program (PJ011126), Rural Development Administration (RDA), Republic of Korea.
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Inhibition of cathepsin B activity prevents deterioration in the quality of in vitro aged porcine oocytes Shuang Liang a, b, Hao Jiang a, b, Xing-Hui Shen c, Jia-Bao Zhang a, Nam-Hyung Kim a, b, * a
Department of Animal Science, College of Animal Sciences, Jilin University, Changchun, Jilin, 130062, China Department of Animal Science, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea c Department of Histology and Embryology, Harbin Medical University, Harbin, 150081, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2018 Received in revised form 29 April 2018 Accepted 30 April 2018 Available online 4 May 2018
The activity of cathepsin B, a member of the lysosomal protease family, directly correlates with oocyte quality and subsequent embryonic development. However, its biological function during the progression of in vitro aging of oocytes in pigs has not been demonstrated. Here, we showed that cathepsin B activity was dramatically increased during in vitro aged oocytes. The inhibition of cathepsin B activity prevented the decline of the quality of aged oocytes and improved their subsequent developmental competence. Moreover, the inhibition of cathepsin B activity reduced aging-induced mitochondrial dysfunction and attenuated oxidative stress. The inhibition of cathepsin B activity also markedly decreased early apoptosis levels and the frequency of spindle anomalies during in vitro aging of oocytes. These results demonstrate that in vitro aging of oocytes induces cathepsin B activity, which is associated with a decline in oocyte quality. The inhibition of cathepsin B activity has a beneficial effect on oocytes during the process of in vitro aging. © 2018 Published by Elsevier Inc.
Keywords: Oocyte aging Cathepsin B Pig Embryo development
1. Introduction In most mammals, oocytes liberated from follicles are arrested at metaphase of the second meiotic division (metaphase II) until fertilization or activation. If metaphase II-arrested oocytes are not fertilized within an appropriate time, they undergo a timedependent deterioration in quality, which is referred to as“postovulatory aging” [1,2]. This decline in oocyte quality decreases fertilization and increases the risk of polyspermy, ultimately resulting in poor developmental potential and early pregnancy loss [1,3,4]. Until now, the mechanism by which oocyte aging is regulated has been unclear. In pigs, as with several mammals, the aging process not only occurs in vivo, but also in vitro in culture conditions [5,6]. Many experiments involving micromanipulation and in vitro fertilization utilize cultured oocytes during assisted reproductive technologies [7e9]. In pigs, in vitro experiments are more efficient than those performed in vivo because, in vivo, pig oocytes are more difficult to obtain in the laboratory compared with other rodents
* Corresponding author. Department of Animal Science, College of Animal Sciences, Jilin University, Changchun, Jilin, 130062, China. E-mail address: [email protected] (N.-H. Kim). https://doi.org/10.1016/j.theriogenology.2018.04.035 0093-691X/© 2018 Published by Elsevier Inc.
[10,11]. Therefore, the efficiency of obtaining porcine embryos is determined by the quality of oocytes matured in vitro, and this directly influences subsequent embryo development. Previous research in pig oocytes has shown that there is a relatively large drop in embryonic developmental competence when the in vitro maturation period is prolonged [12,13]. Hence, delaying oocyte in vitro aging might improve their quality and provide more time for various clinical assisted reproductive technology outcomes in pigs. Cathepsin B is a prominent cysteine protease, and is present in lysosomes of cells under normal physiological conditions [14]. Cathepsin B plays an important role in apoptosis [15,16]. Cathepsin B released from lysosomes can induce apoptosis by activating caspases indirectly through promoting mitochondrial dysfunction [17,18]. Recently, several studies suggested the crucial roles of cathepsin B in affecting oocyte and embryo quality, and its activity could alter their developmental capacity [19e22]. Previous studies also suggested that poor quality oocytes, associated with low developmental competence, have increased cathepsin B activity and protein expression compare to good quality oocytes [21,22]. Decreasing cathepsin B activity with a specific inhibitor (E64) greatly enhanced the developmental competence of oocytes or embryos during in vitro maturation or the in vitro culture period [21,23]. Because in vitro aging causes a time-dependent
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deterioration of oocyte quality, and has a negative effect on subsequent embryonic developmental competence [24], we hypothesize that cathepsin B activity is also likely associated with in vitro aging of oocytes. In the present study, we investigated the involvement of cathepsin B in the mechanism of in vitro aging of oocytes in pigs. We examined changes in cathepsin B activity during in vitro aging of oocytes. Furthermore, we investigated whether the process of in vitro aging of oocytes was changed when the activity of cathepsin B was altered in pig oocytes by culturing with E64, an effective inhibitor of this enzyme. 2. Material and methods All chemicals and reagents used in this research were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. 2.1. In vitro maturation and aging of porcine oocytes Porcine ovaries were collected from a local abattoir and transported to the laboratory in sterile saline at 35e37 C within 2 h in a vacuum flask. Cumulus-oocyte complexes (COCs) were aspirated from follicles 3e8 mm in diameter using a 20-gauge needle attached to a 10 mL disposable syringe. COCs were subsequently collected under a stereo microscope in Tyrode's lactate-HEPES medium. Only oocytes with a homogeneous cytoplasm and compact cumulus cells were chosen and cultivated in maturation medium consisting of tissue culture medium-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) porcine follicular fluid, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 1 mg/mL insulin, 10 ng/mL epidermal growth factor, 0.5 mg/mL follicle stimulating hormone, 0.5 mg/mL luteinizing hormone, and 75 mg/mL kanamycin for 44 h at 38.5 C in an atmosphere containing 5% CO2 in air under maximum humidity. To generate in vitro aged oocytes, COCs were cultivated in maturation medium without follicle stimulating hormone and luteinizing hormone for 24 or 48 h, as described previously [25,26]. The cathepsin B inhibitor, E64, was
present in the maturation medium during the entire extended period of oocyte incubation. Oocytes were denuded by gentle pipetting in Tyrode's lactate-HEPES supplemented with 1 mg/mL hyaluronidase and 0.1% polyvinyl alcohol (PVA). 2.2. Measurement of intracellular cathepsin B activity Intracellular cathepsin B activity was detected using a commercial Magic Red cathepsin B assay kit (Catalog #938; Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's protocol. Briefly, denuded oocytes were incubated in 50 mL phosphate buffered saline (PBS)-PVA (0.1%, w/v) with 2 mL reaction mix for 20 min at 37 C while protected from light. Fluorescence signals were captured as TIFF files using a digital camera connected to a fluorescence microscope (IX70, Olympus, Tokyo, Japan). The same procedures were used for all groups of oocytes, including incubation, rinsing, and imaging. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the fluorescence intensities of the oocytes. 2.3. Activation and in vitro culturing Denuded oocytes were activated by two direct current pulses of 120 V/mm for 60 ms in 300 mM mannitol containing 0.1 mM CaCl2, 0.05 mM MgSO4, 0.01% PVA (w/v), and 0.5 mM HEPES. Following activation, the oocytes were cultured for 3 h in bicarbonatebuffered porcine zygote medium-5 (PZM-5) containing 4 mg/mL bovine serum albumin (BSA) and 7.5 mg/mL cytochalasin B to suppress extrusion of the pseudo-second polar body. Following culture, activated oocytes were thoroughly washed and cultured for 7 days at 38.5 C in bicarbonate-buffered PZM-5 supplemented with 4 mg/mL BSA in an atmosphere containing 5% CO2 in air under maximum humidity without changing the medium. The development of activated oocytes into blastocysts was examined 7 days after activation. To count total cell numbers, blastocysts were stained with 10 mg/mL Hoechst 33342 for 15 min, mounted on glass slides, and examined under an inverted fluorescence microscope.
Fig. 1. Intracellular cathepsin B activity changes in porcine oocytes during in vitro aging. (A) Representative fluorescent images showing intracellular cathepsin B activity in oocytes after in vitro aging. Red: cathepsin B. Scale bar ¼ 100 mm. (B) Relative fluorescence intensity of cathepsin B in oocytes after in vitro aging. Relative fluorescence intensity of cathepsin B in oocytes aged for 24 h (C) and 48h (D) after E64 treatment. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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2.4. Cell proliferation analysis Cell proliferation was assessed by performing a bromodeoxyuridine (BrdU) assay as described previously [27]. Briefly, blastocysts were incubated for 6 h with 100 mM BrdU dissolved in bicarbonate-buffered PZM-5 with 4 mg/mL BSA at 38.5 C in an atmosphere containing 5% CO2 in air under maximum humidity. After incubation, blastocysts were fixed in ice-cold methanol for 20 min and permeabilized with 0.1% (v/v) Triton X-100 for 2 min. The blastocysts were then washed with PBS-PVA and treated with 2 N HCl at room temperature for 30 min. Next, the blastocysts were washed and incubated overnight with a mouse anti-BrdU monoclonal antibody (Sigma-Aldrich; B2531) diluted 1:10 at 4 C. The blastocysts were then washed with PBS containing 0.05% Tween 20 (PBS-T) and incubated for 1 h with a rabbit anti-mouse IgG Alexa Fluor 568-conjugated polyclonal antibody (1:200; Cat. A-11061; Invitrogen) at room temperature. Nuclei in the blastocysts were stained with Hoechst 33342 (10 mg/mL). Finally, the blastocysts were mounted on glass slides and examined under a confocal laser scanning microscope (Zeiss LSM 510 and 710 META, Oberkochen, Germany). Proliferating blastocyst cells were counted using Image J software.
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Prism software package (version 6.01; GraphPad, La Jolla, CA, USA). Continuous data were evaluated by a one-way analysis of variance (ANOVA) followed by a post-hoc Fisher's PLSD test. Data were normally distributed and variances were homogeneous prior to conducting ANOVA. Comparisons of data between two groups were performed with Student's t-test. Data are presented as the means ± SD and p < 0.05 was considered statistically significant. 3. Results 3.1. Changes in cathepsin B activity associated with in vitro aging of oocytes To investigate the potential involvement of cathepsin B activity in oocyte quality during in vitro aging, porcine oocytes were aged
2.5. Immunofluorescence and Annexin V analysis Denuded oocytes were fixed in 3.7% (w/v) paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for an additional 30 min at 37 C, then blocked with 1% BSA in PBS for 1 h. The oocytes were then incubated overnight at 4 C with the primary antibody against caspase 3 (Cat: ab49822; Abcam) diluted in blocking solution (1:100). After washing extensively with PBS-T, the oocytes were labeled with secondary antibodies for 1 h. Spindles were stained with an FITC-conjugated anti-a-tubulin antibody. After extensive washes with PBS-T, the nuclei were stained with Hoechst 33342 (10 mg/mL). Finally, oocytes were mounted onto glass slides and examined using a confocal laser scanning microscope. Annexin V in oocytes was detected using the Annexin VeFITC apoptosis detection kit according to the manufacturer's instructions. To differentiate between live and dead oocytes, the oocytes was stained with PI before Annexin V assay. Only PI negative oocytes were used further experiment. Fluorescence signals were detected using the laser scanning confocal microscope. 2.6. Measurement of mitochondrial membrane potential (DJm) and reactive oxygen species (ROS) generation To evaluate the DJm, denuded oocytes were incubated at 37 C with 2 mM JC-1 (Invitrogen) for 30 min while protected from light. DJm was calculated as the ratio of red florescence (corresponding to activated mitochondria; J-aggregates) to green fluorescence (corresponding to less active mitochondria; J-monomers) [28]. ROS levels were measured by a 20 ,70 -dichlorofluorescein assay as described previously [29]. Briefly, denuded oocytes were incubated for 15 min in PBS-PVA medium containing 10 mM 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA) at 37 C while protected from light. Fluorescence signals were captured as TIFF files using a digital camera connected to a fluorescence microscope. The same procedures, including incubation, rinsing, and imaging, were used for all groups of oocytes. Image J software was used to analyze the fluorescence intensities of the oocytes. 2.7. Statistical analyses All statistical analyses were performed using the GraphPad
Fig. 2. Effects of E64 exposure on porcine oocyte morphology changes during in vitro aging. (A) Representative morphological changes in aged porcine oocytes are shown. (B) Percentages of oocyte fragmentation were recorded after exposure to E64. Scale bar ¼ 100 mm. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments.
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in vitro for 24 and 48 h, and intracellular cathepsin B activity was measured. The results demonstrated that intracellular cathepsin B activity in oocytes increased in a time-dependent manner (1.00 ± 0.09, 1.23 ± 0.38, 1.74 ± 0.37; Fig. 1A and B). A significant in increase in cathepsin B activity was seen in porcine oocytes aged for 24 h, with a peak occurring at around 48 h. Supplementation with E64 during the process of in vitro aging of oocytes decreased intracellular cathepsin B activity at 24 (1.00 ± 0.12 vs 0.85 ± 0.14; Fig. 1C) and 48 h (1.00 ± 0.11 vs 0.86 ± 0.13; Fig. 1D) significantly, as compared with that in control oocytes.
3.2. Inhibition of cathepsin B activity reduces the abnormal morphological changes during in vitro aging of oocytes In vitro aged oocyte quality is associated with morphological changes [25]. Therefore, we investigated oocyte morphological
changes during in vitro aging with and without E64 treatment. As shown in Fig. 2A and B, in vitro aging resulted in a time-dependent deterioration to porcine oocyte quality (3.78 ± 1.40%, 5.98 ± 0.41%, 39.6 ± 7.23%). When in vitro aged oocytes were exposed to E64, the percentage of morphological defects was not dramatically changed when compared with control oocytes at 24 h (5.98 ± 0.41% vs 4.90 ± 0.38%). However, the percentage of morphological defects was dramatically lower compared with control oocytes at 48 h (39.60 ± 7.23% vs 30.20 ± 3.83%).
3.3. Inhibition of cathepsin B activity improves the embryonic developmental potential of in vitro aged oocytes To extend our finding that inhibiting cathepsin B activity ameliorated the time-dependent deterioration of porcine oocyte quality, we monitored their developmental potential after
Fig. 3. Developmental competence of in vitro aged porcine oocytes after E64 exposure. (A) Blastocyst formation is shown at day 7. Scale bar ¼ 100 mm. (B) Blastocyst formation rate. (C) Total cell number in each blastocyst. (D) Hatching rate. (E) Cell proliferation rate in each blastocyst. The numbers of embryos examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments.
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Fig. 4. Effects of E64 exposure on the mitochondrial membrane potential (DJm) in porcine oocytes during in vitro aging. (A) Representative fluorescent images of JC-1 stained oocytes after in vitro aging. Scale bar ¼ 50 mm. (B) Quantification of JC-1 fluorescence intensity. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments.
parthenogenesis (Fig. 3A). The results showed that inhibiting cathepsin B activity during in vitro aging of oocytes ameliorated declines in their subsequent developmental competence. When in vitro aged oocytes were treated with E64, the proportion reaching the blastocyst stage was increased (58.50 ± 6.24%, 26.25 ± 3.59%, 36.00 ± 2.94%; Fig. 3B). In addition, the number of cells per blastocyst tended to be higher in the E64 treated aged oocytes than in the non-treated aged oocytes (46.25 ± 5.02%, 33.55 ± 5.62%, 37.24 ± 5.88%; Fig. 3C). Moreover, the hatching rate
of blastocysts derived from E64-treated aged oocytes was also increased as compared with non-treated aged oocytes (28.50 ± 3.41%, 13.75 ± 2.22%, 19.5 ± 1.30%; Fig. 3D). However, no significant changes in blastocyst formation between E64-treated and -untreated aged oocytes were observed at 48 h (5.33 ± 2.08% vs 7.33 ± 1.53%; Fig. S1). Further analyses revealed that inhibiting cathepsin B activity during in vitro aging of oocytes also increased cell proliferation in these blastocysts (42.26 ± 6.62%, 30.56 ± 6.10%, 37.79 ± 5.21%; Fig. 3E).
Fig. 5. Effects of E64 exposure on the production of reactive oxygen species (ROS) in porcine oocytes during in vitro aging. (A) Representative fluorescence images of ROS in oocytes after in vitro aging. Green: ROS. Scale bar ¼ 50 mm. (B) Quantification of ROS levels. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 6. Effects of E64 exposure on apoptosis in porcine oocytes during in vitro aging. (A) Representative images of Annexin V staining in oocytes after in vitro aging. Green: Annexin V. Scale bar ¼ 20 mm. (B) The percentages of Annexin V positive oocytes were recorded after exposure to E64. (C) Representative fluorescence images of CASPASE 3 levels in oocytes after in vitro aging. Scale bar ¼ 20 mm. (D) Quantitative analysis of CASPASE 3 fluorescence intensity. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.4. Inhibition of cathepsin B activity maintains mitochondrial function and reduces the generation of ROS during in vitro aging of oocytes Because mitochondria play crucial roles in maintaining normal cell metabolic functions [30], we evaluated mitochondrial function (as indicated by DJm) during in vitro aging of oocytes with and without E64 treatment. Representative images of JC-1 staining are shown in Fig. 4A. The DJm in aged oocytes was rapidly reduced, whereas it increased markedly in the presence of E64 (1.06 ± 0.18, 0.70 ± 0.15, 0.88 ± 0.15; Fig. 4B). In addition, compared to non-treated oocytes, those treated with E64 displayed significantly lower ROS levels during in vitro aging of oocytes (1.00 ± 0.32, 1.78 ± 0.57, 1.30 ± 0.35; Fig. 5). These results suggest that inhibiting cathepsin B activity during in vitro aging of oocytes could maintain mitochondrial function and reduce ROS generation.
3.5. Inhibition of cathepsin B activity inhibits apoptosis during in vitro aging of oocytes Because in vitro aged oocyte is closely related to apoptosis [25,31], we next analyzed apoptotic status in in vitro aged oocytes. The result showed that the percentages of early apoptotic (Annexin V positive) oocytes were significantly increased during in vitro aging. However, the percentages of early apoptotic oocytes were decreased in oocytes treated with E64 (28.33 ± 3.22%, 46.00 ± 3.61%, 37.33 ± 2.52%; Fig. 6A and B). Furthermore, immunostaining analysis revealed that CASPASE 3 expression was increased and E64 treatment dramatically reduced this upregulation of caspase 3 expression during in vitro aging of oocytes (1.00 ± 0.26, 1.98 ± 0.19, 1.43 ± 0.30; Fig. 6C and D).
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3.6. Inhibition of cathepsin B activity maintains the spindle morphology of in vitro aged oocytes Because decreased oocyte quality is always accompanied by spindle defects [3,25], we examined whether the meiotic spindle morphology was affected by E64 treatment during in vitro aging of oocytes. As shown in Fig. 7A, control oocytes showed normal spindle morphology, whereas aged oocytes showed disrupted spindle morphology. However, the frequency of abnormal spindle morphology in aged oocytes was dramatically reduced when E64 was added during the process of in vitro aging of oocytes (27.20 ± 3.56%, 43.20 ± 4.44%, 36.60 ± 3.65%; Fig. 7B). 4. Discussion Postovulatory mammalian oocytes undergo a marked quality decline during the process of aging, both in vivo and under in vitro culture conditions [3,32,33]. These aged oocytes show lower fertilization rates and pre-implantation developmental competence [3,12]. In the present study, we showed that cathepsin B activity was significantly more abundant after aging, and was associated with the aging-dependent decline in porcine oocyte quality. The inhibition of cathepsin B activity significantly improved the quality of aged pig oocytes and subsequent early embryo development. These improvements were achieved by maintaining normal morphology and mitochondrial function, reducing ROS generation, and inhibiting the apoptosis that otherwise occurs during in vitro aging of oocytes. Cathepsin B is a lysosomal cysteine protease expressed in various types of cells. It plays an important role in the degradation of intracellular proteins in lysosomes [23,34,35]. Previous studies
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clearly showed that oocytes and embryos with low developmental competence exhibited increased cathepsin B activity compare to those of high developmental competence [20,21]. These results suggest that cathepsin B activity is inversely correlated with the quality of oocytes and embryos. With in vitro aging, we found that cathepsin B activity was increased, suggesting that this contributed to the dysfunction observed in aged pig oocytes. Importantly, the subsequent embryonic development potential was markedly increased when cathepsin B activity was blocked with an inhibitor. These results suggest that increased cathepsin B activity is a phenomenon during the process of in vitro aging of oocytes in pigs. The oocyte aging process is accompanied by age-related mitochondrial dysfunction, which is directly responsible for the high levels of developmental retardation and delay, or arrest of pre-implantation embryos, produced in vitro [3,36]. The results of previous studies suggested that cathepsin B activity was implicated in destabilization of the mitochondrial outer membrane and loss of DJm [37,38]. Cathepsin B-induced mitochondrial membrane degradation could ultimately lead to the release of proapoptotic factors into the cytoplasm [39,40]. DJm is generally used as an indicator of mitochondrial function and the quality of oocytes [3,41]. DJm reflects the pumping of hydrogen ions across the mitochondrial inner membrane during electron transport, which is the driving force for ATP production [42]. In concordance with previous results implicating cathepsin B activity in mitochondrial function, our results show that inhibiting cathepsin B activity prevents the change of DJm during in vitro aging of oocytes in pigs. However, the detailed mechanisms underlying the interplay between cathepsin B and mitochondrial function in postovulatory oocytes deserves further investigation. A previous study has shown that oxidative stress is a factor that is likely to be
Fig. 7. Effects of E64 exposure on spindle morphology in porcine oocytes during in vitro aging. (A) Representative images of spindle morphology in oocytes after in vitro aging. Green: a-tubulin; Blue: DNA. Scale bar ¼ 20 mm. (B) The percentages of abnormal spindles in oocytes were recorded after exposure to E64. The numbers of oocytes examined in each experimental group are shown in the bars. Statistically significant differences are represented with different letters (p < 0.05). Data are expressed as means ± SD from at least three separate experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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involved during in vitro aging of oocytes [24]. High levels of intracellular ROS are commonly considered to be a major factor responsible for negative signs of aging in oocytes [43]. Therefore, increased intracellular ROS production during in vitro aging of oocytes is a crucial factor that affects oocyte quality [43]. Given that cathepsin B activity is associated with ROS production by facilitating mitochondrial membrane disruption [44], we hypothesized that the inhibition of cathepsin B activity might preserve oocyte quality by preventing the increase in ROS production. A previous study suggested that inhibiting cathepsin B activity protected mitochondria, decreased oxidative stress, and attenuated liver cell injury in vivo [45]. Porcine oocytes have relatively more intracellular lipids compared with oocytes of other species, making them highly sensitive to ROS-induced damage [46]. In the present study, we found that the inhibition of cathepsin B activity efficiently prevented the production of ROS in in vitro aged oocytes. Cathepsin B is released from lysosomes and contributes to apoptosis by indirectly inducing mitochondrial dysfunction [47]. In bovine oocytes, inhibiting cathepsin B activity ameliorated heat shock-induced apoptosis and improved their subsequent developmental competence [19]. The end point of oocyte aging is cell death through an apoptotic pathway [31,48]. Therefore, it is reasonable to speculate that cathepsin B induces apoptosis during the in vitro aging process. Furthermore, previously studies have suggested that in vitro aged oocytes suffer from oxidative stress, which always induces apoptosis [49,50]. In the present study, we observed that inhibiting cathepsin B activity inhibited the apoptosis in aged oocytes. These results suggest that inhibiting cathepsin B activity can modulate intracellular ROS levels and inhibit signs of apoptosis, thus suppress the aging process in porcine oocytes. Previous studies demonstrated that the in vitro aging process in oocytes was commonly associated with defective spindle assembly and chromosome alignment [3,25,51], which is consistent with our results. We also observed that inhibiting cathepsin B activity reduced the frequency of spindle defects, further supporting the association of cathepsin B with the in vitro aging process in oocytes. In fact, decreased mitochondrial function and increased oxidative stress is often associated with an age-related higher incidence of meiotic spindle abnormalities during oocyte maturation [52]. Therefore, inadequate mitochondria function and increases oxidative stress in oocytes may be key factors contributing to spindle disorganization. It is therefore possible that inhibiting cathepsin B activity ameliorates mitochondrial function and reduces oxidative stress probably protects spindle from the effect of aging. These results further demonstrate that cathepsin B activity plays a crucial role during the process of in vitro aging in pig oocytes. In conclusion, our findings indicate that an increase in cathepsin B activity is one phenomenon of the process of in vitro aging in pig oocytes. The inhibition of cathepsin B activity prevents multiple changes associated with oocyte aging. This provides a potential new pathway of delaying the aging of pig oocytes.
Conflicts of interest The authors declare that they have no competing interests.
Acknowledgements This study was supported by a grant from the Next-Generation BioGreen 21 Program (PJ011126), Rural Development Administration (RDA), Republic of Korea.
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