Velvet antler water extract protects porcine oocytes from lipopolysaccharide‐induced meiotic defects

Abstract Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram‐negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS‐induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS‐induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS‐induced oxidative stress.

However, the approach concerning how to prevent oocytes from LPS-induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS-induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS-induced oxidative stress.

| INTRODUCTION
Gram-negative bacteria possess an outer protective cell wall that harbours several pathogen-associated molecular patterns (PAMPs) to trigger the inflammation and cause other toxic effects when they infect a host or are lysed by antibiotics. 1 Lipopolysaccharide (LPS), as a core component of the outer membrane of gram-negative bacteria, exists extensively in numerous industrial and external environments, including livestock farms, lumbermills and cotton mills. 2,3 LPS is a prototypical PAMP which is identified by toll-like receptor 4 (TLR-4) protein in complex with CD14 and soluble myeloid differentiation factor 2 (MD2) on the cell surface. 4,5 After LPS binds to its receptor, inflammatory molecules such as chemokines IL-8, TNF-α, IL-6 and cytokines IL-1β are released. 4 The LPS-induced molecules cause severe pathology by exerting profound regulatory effects on the cellular functions. 6 In addition, many studies have validated the adverse influences of LPS on the folliculogenesis, oocyte development, ovulation, luteal function, ovarian steroidogenesis, estrus behaviour, and puberty onset in female animals. 7 Notably, a recent study has found that exposure to LPS destructed the oocyte meiotic maturation due to the reduced m6A levels in pigs. 8 In general, LPS exposure seriously damages the physiological health and female reproductive performance of animals. [8][9][10] Velvet antler (VA), a tissue that is separated from elk or deer, is one of the most famous animal-derived medicine materials. [11][12][13] As a invigorator of yang and qi acting through the kidney meridian, VA has been widely used in the traditional Chinese medicine to reinforce virility, replenish the crucial essence, nourish the blood, strengthen bones, and accelerate male and female sexual functions. [14][15][16] Previous reports have indicated that VA had antiinflammatory effects on human rheumatoid arthritis fibroblast-like synoviocytes and alleviated the Parkinson's disease by inhibiting oxidative stress in mice, 17,18 further demonstrating that VA had many beneficial components and pharmacological actions. [19][20][21][22] Also, other studies have shown that VA could rescue the sarcoplasmic reticulum Ca 2+ -ATPase activity and thus enhanced the cardiac function in rats with heart failure. 23,24 However, whether VA could improve the quality of oocytes under oxidative stress from diverse environmental factors is largely elusive.
In the present study, we examined the potential impacts of velvet antler water extract (VAWE) on the quality of porcine oocytes exposed to LPS. We evaluated the maturation competency and fertilization ability of oocytes. We also assessed the cytoskeleton organization, mitochondrial integrity, and cortical granule dynamics during nuclear and cytoplasmic maturation of oocytes.

| Porcine oocyte collection
Porcine ovaries were obtained from a local abattoir. Within 2 h after slaughtering, abattoir-derived porcine ovaries were collected and then transported to the laboratory in a physiological saline (0.9% NaCl) containing 500 IU/ml of streptomycin sulphate and penicillin G. The cumulus-oocyte complexes (COCs) were aspirated with 10 ml dispos-

| Statistical analysis
All percentages or values from at least three repeated experiments were expressed as mean ± SEM or SD, and the number of oocytes was labelled in parentheses as (n). Data were analysed by unpairedsamples t-test, provided by GraphPad Prism 7 statistical software. The level of significance was accepted as P < 0.05.

| RESULTS
3.1 | VAWE promotes the in vitro maturation of porcine oocytes exposed to LPS During in vitro maturation (IVM), we added different doses of LPS (10, 25, 50, or 100 μg/ml) in the culture medium to study its effects on the porcine oocyte meiotic progression. It was shown that a large number of cumulus cells were fully expanded around the oocytes in the control group, but became less in the LPS-exposed group ( Figure 1A). We further calculated the frequency of first polar body extrusion (PBE), and observed that exposure to different concentrations of LPS remarkably reduced the PBE rate in varying degrees (control: 81.3 ± 2.9%, n = 104; 10 μg/ml LPS: 67.2 ± 4.4%, n = 171, P < 0.05; 25 μg/ml LPS: 64.7 ± 1.0%, n = 151, p < 0.001; 50 μg/ml LPS: 58.1 ± 1.9%, n = 171, P < 0.001; 100 μg/ml LPS: 56.1 ± 3.5%, n = 156, P < 0.01; Figure 1B). For subsequent studies, 25 μg/ml LPS was chosen because it permitted a certain proportion of oocytes to reach the M II stage.
3.3 | VAWE maintains the actin polymerization in LPS-exposed oocytes On account of the fact that microfilament takes a vital part in the meiotic cell polarization and spindle positioning, we used phalloidin to display the polymerization of actin filaments. According to the results from fluorescent images, a uniform distribution of actin filaments was observed on the plasma membrane with strong signals in the control group ( Figure 3A). By comparison, porcine oocytes exposed to LPS displayed less actin signals, as indicated along the line drawn through the oocyte by fluorescence profiling and quantification (19.2 ± 0.7%, n = 41 VS 12.2 ± 0.5%, n = 32, P < 0.0001; Figure 3A-C). Strikingly, VAWE supplementation increased the actin signals on the plasma membrane in LPSexposed oocytes as assessed by the measurement of fluorescence intensity (12.2 ± 0.5%, n = 32 VS 19.9 ± 0.8%, n = 28, P < 0.0001; Figure 3C). To Sum up, these data imply that the recovery of VAWE on the oocyte meiotic defects induced by LPS involves in the actin dynamics.
3.4 | VAWE restores the distribution and function of mitochondria in porcine oocytes exposed to LPS Given that mitochondria provide the energy for many biological processes including cytoskeleton assembly during oocyte maturation, its distribution has been considered as one of the major indexes for the oocyte cytoplasmic maturation. In the control group, we found that most of mitochondria in the oocytes aggregated in the subcortical region around lipid droplets, whereas this particular localization pattern disappeared in oocytes exposed to LPS ( Figure 4A). Based on the quantitative fluorescence intensity analysis, we validated that signals of mitochondria in LPS-exposed oocytes prominently declined compared to the control oocytes (44.4 ± 1.7%, n = 22 VS 19.4 ± 0.9%, n = 34, P < 0.0001), which was prevented by the treatment with VAWE (19.4 ± 0.9%, n = 34 VS 38.6 ± 1.7%, n = 23, P < 0.0001; Figure 4A,B). Moreover, mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining to assess the mitochondrial function. When mitochondria had a high membrane potential, they fluoresced in red to represent the aggregates of JC-1, and mitochondria with a low membrane potential fluoresced in green to show the monomers of JC-1 ( Figure 4C). As measured by fluorescence intensity, the ratio of red to green fluorescence in LPS-exposed oocytes was dramatically lower than that in control oocytes, but elevated after VAWE supplementation (5.6 ± 0.2%, n = 21, P < 0.0001 VS 2.6 ± 0.1%, n = 31 VS 4.2 ± 0.2%, n = 30, P < 0.0001; Figure 4C,D). In brief, these results indicate that VAWE recovers the impairment of mitochondria in oocytes exposed to LPS.

| VAWE rescues the abnormal distribution of cortical granules and ovastacin in LPS-exposed oocytes
As another index of oocyte cytoplasmic maturation, the dynamics of cortical granules (CGs), a particular group of membrane-bound secretory F I G U R E 2 Effects of VAWE supplementation on the spindle/ chromosome structure in LPS-exposed porcine oocytes. (A) Metaphase I oocytes were immunostained with anti-α-tubulin-FITC antibody to represent the spindle morphology and counterstained with propidium iodide (PI) to exhibit the chromosome alignment. Scale bar, 5 μm. (B) The percentage of aberrant spindles was recorded in control, LPS-exposed and VAWE-supplemented oocytes. (C) The percentage of misaligned chromosomes was recorded in control, LPS-exposed and VAWE-supplemented oocytes. Data in (B) and (C) were expressed as mean percentage (mean ± SEM) of at least three independent experiments. ***P < 0.001; ****P < 0.0001 vesicles that mainly exist in the cortex of mammalian oocytes to function for prevention of polyspermy, 25 was assessed by LCA-FITC staining.
Besides, we monitored the behaviour of ovastacin, one key component of CGs in LPS-exposed oocytes. Fluorescent imaging and intensity measurement analysis revealed that the dynamics of ovastacin was similar to that of CGs, showing the lower intensity of ovastacin signals in LPS-exposed oocytes than those in the controls (18.6 ± 0.8%, n = 19 VS 9.0 ± 0.4%, n = 21, P < 0.0001; Figure 5C,D).
Altogether, our data illustrate that VAWE promotes the cytoplasmic maturation of oocytes exposed to LPS.
3.6 | VAWE improves the sperm binding ability and fertilization capacity in porcine oocytes exposed to LPS When ovastacin is exocytosed from CGs to extracellular space before fertilization, it would harden zona pellucida surrounding the oocyte, F I G U R E 3 Effects of VAWE supplementation on the actin polymerization in LPS-exposed porcine oocytes. (A) Representative images of actin filaments in control, LPS-exposed and VAWE-supplemented oocytes. Scale bar, 30 μm. (B) The graphs showed the fluorescence intensity profiling of actin filaments in control, LPS-exposed and VAWE-supplemented oocytes. Lines were drawn through the oocytes, and pixel intensities were quantified along the lines. (C) The fluorescence intensity of actin signals was quantified in control, LPS-exposed and VAWE-supplemented oocytes. Data in (C) were shown as mean value (mean ± SD) of at least three independent experiments. ****P < 0.0001 bringing about unsuccessful sperm binding and fertilization. As the dynamics of ovastacin is impaired in LPS-exposed oocytes, we performed sperm-oocyte binging assay to evaluate their sperm binding ability. Sperm heads were stained with Hoechst to count the number of sperm binding to the zona pellucida surrounding oocytes ( Figure 6A). The quantitative results displayed that in the control group abundant sperm closely bound to the zona pellucida of unfertilized oocytes, while the number of sperm significantly declined in LPS-exposed group (125.4 ± 8.2%, n = 15 VS 64.3 ± 5.7%, n = 10, P < 0.0001; Figure 6A,B). By contrast, supplementation of VAWE increased the number of sperm binding to the LPS-exposed oocytes (64.3 ± 5.7%, n = 10 VS 115.3 ± 9.6%, n = 15, P < 0.0001; Figure 6A,B).
Since weakened sperm binding ability predicts the reduced fertilization potential, we next conducted IVF experiments to confirm it.
3.7 | VAWE decreases ROS levels to suppress DNA damage and early apoptosis in porcine oocytes exposed to LPS It has been reported that LPS-induced mitochondrial dysfunction produces high levels of reactive oxygen species (ROS) in a variety of cells F I G U R E 4 Effects of VAWE supplementation on the distribution and function of mitochondria in LPS-exposed porcine oocytes. (A) Representative images of mitochondrial distribution in control, LPS-exposed and VAWE-supplemented oocytes. Scale bar, 40 μm. (B) The fluorescence intensity of mitochondrial signals was measured in control, LPS-exposed and VAWE-supplemented oocytes. (C) Mitochondrial membrane potential (ΔΨm) was tested by JC-1 staining in control, LPS-exposed and VAWE-supplemented oocytes (Red, high ΔΨm; Green, low ΔΨm). Scale bar, 40 μm. (D) The ratio of red to green fluorescence intensity was calculated in control, LPS-exposed and VAWE-supplemented oocytes. Data in (B) and (D) were presented as mean values (mean ± SD) of at least three independent experiments. ****P < 0.0001 to impair their functions. [26][27][28][29] In view of this, we asked whether the improvement of LPS-exposed oocyte quality by VAWE is related to the removal of ROS. By DCFH staining, ROS levels were compared between control and LPS-exposed oocytes. The results showed that ROS signals were weak in control oocytes, but became stronger following LPS exposure (4.7 ± 0.5%, n = 18 VS 8.3 ± 0.7%, n = 20, P < 0.001; Figure 7A,B). Nevertheless, supplementation of VAWE sharply reduced the excessive ROS in LPS-exposed oocytes (8.3 ± 0.7%, n = 20 VS 5.7 ± 0.3%, n = 23, P < 0.001; Figure 7A,B), indicating that VAWE effectively inhibits LPS-induced oxidative stress in porcine oocytes.

| DISCUSSION
Velvet antler has a wide range of pharmacological effects which are implicated in promoting the reproductive system development, enhancing immune function, boosting the cell proliferation, and intensifying the anti-inflammatory and analgesic effects. 12,20,30 In a previous study, VAWE has been found to improve wound healing in a streptozotocin-induced diabetic rat model. 12 In addition, VAWE recovers the mucosal barrier function of colitis, 21 and protects against cell infection by down-regulation of proinflammatory cytokines (TNF-α and IL-6) and reduction of phagocytosis. 31,32 Of note, LPS is known as to be responsible for most biological attributes of bacterial endotoxins, which are diffusely present in the environment. LPS induces the release of inflammatory molecules and severely impairs the reproductive health of female animals. 33,34 Therefore, we hypothesized that VAWE has a protective role against the oxidative damage induced by LPS exposure. 26,27 F I G U R E 5 Effects of VAWE supplementation on the localization of CGs and ovastacin in LPS-exposed porcine oocytes. (A) Representative images of CG distribution in control, LPS-exposed and VAWE-supplemented oocytes. Scale bar, 40 μm. (B) The fluorescence intensity of CGs was quantified in control, LPS-exposed and VAWE-supplemented oocytes. (C) Representative images of ovastacin distribution in control, LPS-exposed and VAWE-supplemented oocytes. Scale bar, 40 μm. (D) The fluorescence intensity of ovastacin signals was measured in control, LPS-exposed and VAWE-supplemented oocytes. Data in (B) and (D) were expressed as mean values (mean ± SD) of at least three independent experiments. ****P < 0.0001 Our findings revealed that LPS exposure impeded the porcine In addition to the nuclear maturation for ensuring the genomic stability, oocytes need to complete the cytoplasmic maturation to acquire the fertilization ability and subsequent embryonic development potential. The distribution patterns mitochondria and CGs are two well-accepted indexes for assessing the oocyte cytoplasmic maturation. 35 Our data demonstrated that VAME supplementation recovered the cytoplasmic maturation of LPS-exposed oocytes by maintaining the normal distribution and function of mitochondria in oocytes, which might be critical for the generation of energy for oocyte development. 36,37 Furthermore, we evidenced that VAME supplementation also advanced the cytoplasmic maturation of LPS-exposed oocytes by restoring the dynamics of CGs and their component ovastacin. As a component of CGs, ovastacin could destroy the sperm binding site in the zona pellucida of oocytes by cleaving the N-terminal domain of ZP2 to prevent post-fertilization polyspermy. 38,39 However, if it is released before fertilization, it would prematurely cleave the ZP2 to impair F I G U R E 6 Effects of VAWE supplementation on the sperm binding ability and fertilization potential of LPS-exposed porcine oocytes. (A) Representative images of sperm binding to the matured oocytes or 2-cell embryos in control, LPS-exposed and VAWE-supplemented groups. Scale bar, 40 μm. (B) The number of sperm binding to the surface of zona pellucida surrounding control, LPS-exposed and VAWE-supplemented oocytes was counted. (C) Representative images of 2-cell embryos developed from in vitro fertilized control, LPS-exposed and VAWEsupplemented oocytes. Scale bars, 120 μm (A-C); 30 μm (D-F). (D) The in vitro fertilization rate was quantified in control, LPS-exposed and VAWE-supplemented oocytes. Data in (B) and (D) were shown as mean percentage (mean ± SEM) of at least three independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001 the sperm binding and fertilization. Consistently, we verified that VAME supplementation enhanced the sperm binding ability and fertilization capability of LPS-exposed oocytes.
As mitochondrial dysfunction is usually associated with perturbed redox homeostasis, our findings finally illustrated that VAME supplementation reduced the LPS-induced high levels of F I G U R E 7 Effects of VAWE supplementation on the ROS level, DNA damage accumulation and apoptosis in LPS-exposed porcine oocytes. (A) Representative images of ROS levels were shown in control, LPS-exposed and VAWE-supplemented oocytes. Scale bar, 40 μm. (B) The fluorescence intensity of ROS signals was measured in control, LPS-exposed and VAWE-supplemented oocytes. (C) Representative images of DNA damage were shown in control, LPS-exposed and VAWE-supplemented oocytes. Scale bar, 5 μm. (D) The fluorescence intensity of γH2AX signals was measured in control, LPS-exposed and VAWE-supplemented oocytes. (E) Representative images of apoptotic oocytes were shown in control, LPS-exposed and VAWE-supplemented groups. Scale bar, 40 μm. (F) The fluorescence intensity of Annexin-V signals was measured in control, LPS-exposed and VAWE-supplemented oocytes. Data in (B), (D) and (F) were presented as mean values (mean ± SD) of at least three independent experiments. ***P < 0.001; ****P < 0.0001