Effects of β-Nicotinamide Mononucleotide, Berberine, and Cordycepin on Lipid Droplet Content and Developmental Ability of Vitrified Bovine Oocytes

Oocyte vitrification is crucial for livestock reproduction, germplasm conservation, and human-assisted reproduction, but the overabundance of lipids is highly detrimental to oocyte development. It is necessary to reduce the lipid droplet content of oocytes before cryopreservation. This study analyzed the impact of β-nicotinamide mononucleotide (NMN), berberine (BER), or cordycepin (COR) on various aspects of bovine oocytes, including lipid droplet content and the expression levels of genes related to lipid synthesis in bovine oocytes, development ability, reactive oxygen species (ROS), apoptosis, and the expression levels of genes associated with endoplasmic reticulum (ER) stress, and mitochondrial function in vitrified bovine oocytes. The results of our study indicated that 1 μM NMN, 2.5 μM BER, and 1 μM COR were effective in reducing the lipid droplet content and suppressing the expression levels of genes involved in lipid synthesis in bovine oocytes. Our findings showed that the vitrified bovine oocytes treated with 1 μM of NMN had a significantly higher survival rate and better development ability compared to the other vitrified groups. Additionally, 1 μM NMN, 2.5 μM BER, and 1 μM COR decreased the levels of ROS and apoptosis, decreased the mRNA expression levels of genes involved in ER stress and mitochondrial fission but increased the mRNA expression levels of genes associated with mitochondrial fusion in the vitrified bovine oocytes. Our study results suggested that 1 μM NMN, 2.5 μM BER, and 1 μM COR effectively decreased the lipid droplet content and enhanced the development ability of vitrified bovine oocytes by lowering ROS levels, reducing ER stress, regulating mitochondrial function, and inhibiting apoptosis. Furthermore, the results showed that 1 μM NMN was more effective than 2.5 μM BER and 1 μM COR.


Introduction
Oocyte vitrification has proven to be a highly advantageous method in breeding livestock and preserving oocyte banks [1]. It boosts breeding efficiency [2] and helps to protect the genetic diversity of endangered animal species at a lower cost [3]. Furthermore, this method reduces the environmental impact and enhances animal welfare by minimizing the need for live animal transportation [4]. Meanwhile, the vitrification of oocytes helps to maintain the fertility of women suffering from illnesses [5]. Consequently, there has been rapid advancement in the development of vitrification technology for animal oocytes, including those from bovine [6], sheep [6], porcine [6], and equine [7] animals.
However, the viability of vitrified oocytes is significantly decreased due to damages caused by cryopreservation [8], which restricts the use of cryopreservation techniques. incubated with hyaluronidase (0.1%, w/v) to isolate CCs, and the oocytes exhibiting even distribution of cytoplasm and at the MII stage were selected for subsequent experiments.

Vitrification and Warming
According to the method described by Zhao [35] with slight modifications, the openpulled straw (OPS) method was used to vitrify oocytes. For vitrification and warming, the pretreatment solution consisted of Dulbecco's phosphate-buffered saline (DPBS) supplemented with ethylene glycol (EG,10%, v/v), dimethyl sulfoxide (DMSO, 10%, v/v), and FBS (10%). The vitrification solution was comprised of EG (20%, v/v) and DMSO (20%, v/v) in DPBS medium with FBS (20%, v/v), Ficoll (300 g/L), and sucrose (0.5 M). For the procedure of vitrification, oocytes were treated with the pretreatment solution for 30 s, delivered to the vitrification medium, aspirated into an OPS tube, and put in liquid nitrogen (LN 2 ) within 25 s. As for warming, the vitrified oocytes were extracted from the LN 2 , immediately incubated in 0.5 M sucrose for 5 min and then in 0.25 M sucrose for another 5 min.

IVF Procedure of Oocytes
After 22-24 h IVM, fertilization was performed according to Brackett and Oliphant [36]. The fertilization medium was prepared by Brackett and Oliphant (BO) medium, bovine serum albumin (BSA) (20 mg/mL), heparin (20 µg/mL), penicillin (100 IU/mL), and 100 µg/mL streptomycin (100 µg/mL). Briefly, the frozen sperm were thawed at 38 • C, washed twice in BO medium, and resuspended and diluted to a concentration of 1 × 10 6 /mL with fertilization medium. For fertilization, 20-30 oocytes were transferred to 100 µL fertilization medium covered with mineral oil for [16][17][18] h, at 38.5 • C and 5%CO 2 . The hypothetical zygotes were stripped of CCs and cultured in Charles Rosenkrans medium with amino acids (CR1aa)solution for 48 h. The cleaved embryos were transferred to the CR1aa medium with 10% FBS for another 120 h to blastocysts. Half of the solution was replaced every 48 h during the culture period, and the cleavage rate and blastocyst rate were counted on day 2 and 7 after fertilization.

qRT-PCR Procedure
At least 50 oocytes were collected from each pool for RNA extraction by TriZol reagent (Invitrogen, Carlsbad, CA, USA). RNA was kept at −80 • C until use. According to the product manuals, the qRT-PCR procedure was carried out by an ABI 7500 SDS instrument (Applied Biosystems, Waltham, MA, USA) with the comparative Ct (2 −∆∆ Ct) method [37]. The primers used in the present study are shown in Table 1, and β-ACTIN was utilized as the reference gene.

ROS Staining of Oocytes
The ROS staining of bovine oocytes was carried out according to the method described by Zhao [35]. Briefly, oocytes were stained with 10 mM DCHFDA (Beyotime, Shanghai, China) for 20 min, then washed twice, and observed on the epifluorescence inverted microscope (Nikon, Tokyo, Japan) connected to a DSRi1 camera (Nikon, Tokyo, Japan). EZ-C1 Free Viewer software (Nikon, Tokyo, Japan) was utilized for fluorescence intensity.

Lipid Droplets Staining of Oocytes
With slight modifications according to the previous method [38], oocytes were washed in 0.1% PVA-PBS, incubated with 10 µg/mL Nile red solution (Solarbio, Beijing, China) for 10 min, and observed under the epifluorescence inverted microscope (Nikon, Tokyo, Japan) connected to a DSRi1 camera (Nikon, Tokyo, Japan). Fluorescent images were analyzed by the EZ-C1 Free Viewer software (Nikon, Tokyo, Japan).

PS Externalization of Oocytes
An Annexin V-FITC apoptosis detection Kit (Beyotime, Shanghai, China) was applied to measure the externalization of PS in bovine MII oocytes [35]. Oocytes were incubated in 200 µL binding buffer consisting of 10 µL of propidium iodide (PI) and 5 µL of Annexin V-FITC for 5-10 min and then examined under the epifluorescence inverted microscope (Nikon, Tokyo, Japan) connected to a DSRi1 camera (Nikon, Tokyo, Japan). According to the method described by Anguita [39], oocytes were classified into three groups based on staining signals of Annexin V, including (i) early apoptotic oocytes with an obvious Annexin V-positive signal in the membrane, (ii) survival oocytes with no Annexin V staining, and (iii) necrotic oocytes showing PI-positive red nuclei.

Effect of NMN, BER, or COR Supplementation during IVM on the mRNA Expression Levels of Genes Associated with Lipid Synthesis in Bovine Oocytes
As shown in Figure 2, the mRNA expression levels of genes promoting lipid synthesis (SREBP1, FABP3, PPARG) in the 1 µM NMN, 2.5 µM BER, and 1 µM COR groups were prominently lower than those of the fresh control group (p < 0.05), with the 1 µM NMN group being the lowest of all groups.

Effects of NMN, BER, or COR on ROS Level of Bovine Vitrified Oocytes
The representative images of ROS staining are shown in Figure 3A. Figure 3B indicates that the ROS level in vitrified bovine oocytes in the 1 μM NMN group, 2.5 μM BER group, or 1μM COR group was dramatically lower than that of the vitrification control group (p < 0.05) but higher than that of fresh control group (p < 0.05), with the 1μM NMN group being the lowest out of the 1 μM NMN group, 2.5 μM BER group, and 1μM COR group. Moreover, the results in Figure 3C show that the mRNA expression levels of antioxidant-related genes (SOD1, GPX1, and CAT) in the 1μM NMN group were significantly higher than those of the 2.5 μM BER group, 1 μM COR group, vitrification control group, and fresh control group (p < 0.05).

Effects of NMN, BER, or COR on ROS Level of Bovine Vitrified Oocytes
The representative images of ROS staining are shown in Figure 3A. Figure 3B indicates that the ROS level in vitrified bovine oocytes in the 1 µM NMN group, 2.5 µM BER group, or 1 µM COR group was dramatically lower than that of the vitrification control group (p < 0.05) but higher than that of fresh control group (p < 0.05), with the 1 µM NMN group being the lowest out of the 1 µM NMN group, 2.5 µM BER group, and 1 µM COR group. Moreover, the results in Figure 3C show that the mRNA expression levels of antioxidantrelated genes (SOD1, GPX1, and CAT) in the 1 µM NMN group were significantly higher than those of the 2.5 µM BER group, 1 µM COR group, vitrification control group, and fresh control group (p < 0.05).   Figure 4 shows that the mRNA expression levels of ER stress marker genes XBP1 and ATF4) in the 1 μM NMN group, 2.5 μM BER group, 1 μM COR group, a control group were distinctly lower than those of the vitrification control group ( with 1 μM NMN being the lowest of all groups (p < 0.05).  Figure 4 shows that the mRNA expression levels of ER stress marker genes (GRP78, XBP1 and ATF4) in the 1 µM NMN group, 2.5 µM BER group, 1 µM COR group, and fresh control group were distinctly lower than those of the vitrification control group (p < 0.05), with 1 µM NMN being the lowest of all groups (p < 0.05).  Figure 5 shows that the mRNA expression levels of genes (MFN1 and MFN2) promoting mitochondrial fusion in the 1 μM NMN group, 2.5 μM BER group, and 1 μM COR group were distinctly higher than those of the vitrification control group (p < 0.05), with the 1 μM NMN group being the highest of all groups. Meanwhile, the mRNA expression levels of genes (DRP1 and FIS1) promoting mitochondrial fission in the 1 μM NMN group, 2.5 μM BER group, and 1 μM COR group were distinctly lower than those of the vitrification control group (p < 0.05), with the 1 μM NMN group being the lowest in all groups.   Figure 5 shows that the mRNA expression levels of genes (MFN1 and MFN2) promoting mitochondrial fusion in the 1 µM NMN group, 2.5 µM BER group, and 1 µM COR group were distinctly higher than those of the vitrification control group (p < 0.05), with the 1 µM NMN group being the highest of all groups. Meanwhile, the mRNA expression levels of genes (DRP1 and FIS1) promoting mitochondrial fission in the 1 µM NMN group, 2.5 µM BER group, and 1 µM COR group were distinctly lower than those of the vitrification control group (p < 0.05), with the 1 µM NMN group being the lowest in all groups.  Figure 5 shows that the mRNA expression levels of genes (MFN1 and MFN2) promoting mitochondrial fusion in the 1 μM NMN group, 2.5 μM BER group, and 1 μM COR group were distinctly higher than those of the vitrification control group (p < 0.05), with the 1 μM NMN group being the highest of all groups. Meanwhile, the mRNA expression levels of genes (DRP1 and FIS1) promoting mitochondrial fission in the 1 μM NMN group, 2.5 μM BER group, and 1 μM COR group were distinctly lower than those of the vitrification control group (p < 0.05), with the 1 μM NMN group being the lowest in all groups.

Effects of NMN, BER, or COR on Apoptosis Level of Bovine Vitrified Oocytes
The representative images of Annexin V-FITC staining are shown in Figure 6A. The apoptosis percentage of vitrified bovine oocytes in the 1 µM NMN group (12.77 ± 3.73%) was significantly lower than that of the 2.5 µM BER group (22.22 ± 1.99%, p < 0.05), 1 µM COR group (21.82 ± 1.38%, p < 0.05), and vitrification control group (37.50 ± 4.63%, p < 0.05) and comparable to the fresh control group (10.20 ± 3.36%, p > 0.05) ( Figure 6B). Moreover, the anti-apoptotic gene mRNA expression level of BCL2 in the 1 µM NMN group was distinctly higher than those of other groups (p > 0.05), in contrast to the pro-apoptosis gene expression of BAX ( Figure 6C).

Effects of NMN, BER, or COR on Apoptosis Level of Bovine Vitrified Oocytes
The representative images of Annexin V-FITC staining are shown in Figure 6A apoptosis percentage of vitrified bovine oocytes in the 1 μM NMN group (12.77 ± 3. was significantly lower than that of the 2.5 μM BER group (22.22 ± 1.99%, p < 0.05), COR group (21.82 ± 1.38%, p < 0.05), and vitrification control group (37.50 ± 4.63%, p < and comparable to the fresh control group (10.20 ± 3.36%, p > 0.05) ( Figure 6B). More the anti-apoptotic gene mRNA expression level of BCL2 in the 1 μM NMN group distinctly higher than those of other groups (p > 0.05), in contrast to the pro-apoptosis expression of BAX ( Figure 6C).

Discussion
Wang and colleagues [40] found that after treatment with NMN, the lipid dr content in mouse oocytes significantly decreased. Similarly, our results in Figure 1B cated that NMN treatment also led to a significant reduction in lipid droplet conte bovine oocytes, which could be attributed to the reduced expression of genes su

Discussion
Wang and colleagues [40] found that after treatment with NMN, the lipid droplet content in mouse oocytes significantly decreased. Similarly, our results in Figure 1B indicated that NMN treatment also led to a significant reduction in lipid droplet content in bovine oocytes, which could be attributed to the reduced expression of genes such as FABP3, SREBP1, and PPARG (as shown in Figure 2). Previous studies have established a positive correlation between the expression levels of FABP3 [11], SREBP1 [41], and PPARG [42] and lipid accumulation. Wang [43] also reported that NMN could inhibit lipid accumulation by reducing the expression levels of SREBP1 and FASN. According to Uddin [26], NMN also was found to regulate the expression levels of ACC1, MPC1, and CD36, leading to increased lipid absorption and transport. All these findings suggested that NMN could decrease the lipid droplet content of bovine oocytes by reducing the expression levels of FABP3, SREBP1, and PPARG.
As shown in Figure 1C, the lipid droplet content in bovine oocytes was notably reduced in the BER group due to BER's role in regulating lipid metabolism. BER has previously been demonstrated to decrease lipid accumulation in porcine oocytes [31], colon cancer cells [27], and mouse liver tissue [44] by suppressing the expression of genes that drive lipid buildup, such as SCAP [27], SREBP-1 [27,31], PPARG [31], SCD1, and FASN [45]. Our results, consistent with previous findings, demonstrated that the expression of FABP3, SREBP-1, and PPARG were reduced in bovine oocytes exposed to BER (Figure 2). This reduction in gene expression contributed to the decrease in lipid droplet content observed in oocytes treated with BER.
Our results showed that the lipid droplet content of bovine oocytes was observably reduced in the COR group ( Figure 1D). Previous studies have shown that COR has a significant impact on reducing lipid accumulation, which is in line with our findings [24,46,47]. This decrease in lipid accumulation may be attributed to the inhibitory effect of COR on gene expression levels that drive lipid accumulation (Figure 2). In agreement with these findings, Gong [48] found that the expression levels of genes involved in lipid synthesis (SREBP1-c, ACC, SCD-1, and CD36), absorption, and transport were significantly reduced by COR. Furthermore, according to Xu [29], the expression of genes responsible for lipid droplet formation such as FSP27, RAB5, and RAB11 were also suppressed by COR. These studies collectively help to shed light on the observed decrease in lipid droplet content in oocytes treated with COR.
As shown in Table 2, our findings revealed that 1 µM NMN increased the maturation rate of bovine oocytes. Analogously, NMN could improve the percentage of the polar body extrusion of heat-stressed porcine oocytes via keeping the spindle/chromosome structure, restoring the distribution of cortical granules, and protecting the dynamic polymerization of the actin cytoskeleton [30]. The survival rate of vitrified bovine oocytes improved significantly when treated with 1 µM NMN, which may be attributed to the reduction of lipid droplet content. This observation is supported by previous studies in both bovines [49] and pigs [14], which showed that reducing lipid droplet content can enhance the survival rate of vitrified oocytes. Additionally, our results indicated that the developmental ability of vitrified bovine oocytes treated with 1 µM NMN improved significantly, likely because of the inhibition of ER stress and improvement of mitochondrial function. As shown in Figure 4, our results indicate that the process of vitrification led to a significant increase in the mRNA expression levels of GRP78, XBP1, and ATF4, which are markers of ER stress activation in bovines [50], pigs [51], and mice [52]. Our results also showed that treatment with NMN was able to decrease the expression levels of GRP78, XBP1, and ATF4 (Figure 4). This is in line with previous research, which demonstrated that NMN could alleviate heat stress in bovine mammary epithelial cells by reducing ER stress and minimizing mitochondrial damage [19]. Moreover, the balance of mitochondrial fusion and fission is crucial for maintaining mitochondrial quality [19] and an imbalance in this balance can result in a decline in oocyte quality [53,54]. The positive regulation of mitochondrial fusion in mammals is achieved through the genes MFN1 and MFN2 [55], while mitochondrial fission is linked to the genes DRP1 and FIS1 [56], and inhibition of these genes can inhibit mitochondrial fission [57]. As shown in Figure 5, vitrification caused a disruption in the balance between mitochondrial fusion and fission by inhibiting the expression of MFN1 and MFN2 while increasing the expression of DRP1 and FIS1. Similarly, the expression levels of MFN1, MFN2, DRP1, and FIS1 were also affected by heat stress [58], ischemia [59], vitrification temperatures, and cryoprotective agent concentrations [60]. The results showed that NMN could restore this balance of mitochondrial fusion and fission by upregulating MFN1 and MFN2 and downregulating DRP1 and FIS1 (Figure 5), similar to previous researches [19,59]. Furthermore, NMN has been proven to reduce the damages of stress such as high-fat diet [40], senescence [33], toxicants [61], and heat stress [30], and improve the quality of oocytes in mice [61] and pigs [30] by reducing meiotic defects, rescuing mitochondrial function, and eliminating the accumulated ROS to suppress apoptosis.
The results in Table 3 indicated that the maturation rate of bovine oocytes was improved by 2.5 µM BER. Analogously, Dai and colleagues [31] also reported this phenomenon in pigs and they also suggested that this was related to BER promoting oocyte lipid metabolism. The use of 2.5 µM BER led to an improvement in both cleavage rate and blastocyst rate of vitrified bovine oocytes, likely due to an enhancement in lipid metabolism. This is in line with previous research which showed that BER improves the maturation rate of porcine oocytes in vitro by activating the expression of miR-192 and promoting lipid metabolism, resulting in an improved cleavage rate and blastocyst rate [31]. Additionally, another study found that the supplementation of 2.5 µM BER significantly promoted the development of two-cell embryos to eight-cell embryos in mice by reducing ROS and apoptosis [34]. Meanwhile, our results showed that the treatment with BER resulted in a reduction in the expression levels of GRP78, XBP1, ATF4, DRP1, and FIS1 and an increase in the expression levels of MFN1 and MFN2 (Figures 4 and 5), which suggested that BER reduced the injuries and improved the quality of vitrified oocytes. Similarly, previous research reported that BER could decrease ER stress by decreasing the expression of ATF4 and XBP1 [62] and maintain proper mitochondrial morphology by inhibiting the activation of DRP1 [63] in mammal cells.
As shown in Table 4, our results demonstrated that the maturation rate of bovine oocytes was effectively improved by 1 µM COR. Liu [32] also reported that COR was effective in improving the maturation rate of small porcine oocytes (<3 mm) in vitro [32]. The cleavage rate and blastocyst rate of bovine vitrified oocytes treated with 1 µM COR were effectively improved, which may be due to the reduction of ER stress ( Figure 4) and the rescuing of mitochondrial function ( Figure 5). Our results align with previous findings that COR has the ability to inhibit the expression of ER stress activation marker genes such as ATF4 and CHOP [64,65], reverse DRP1-mediated aberrant mitochondrial fragmentation, and maintain normal mitochondrial morphology [66]. However, other studies have found that the development ability of oocytes in bovines [67,68] and pigs [69] was inhibited by COR in a dose-dependent manner, likely due to the inhibition of polyadenylation at high concentrations of COR. The different results could be explained by the differences in COR concentrations and culture systems used [69].
As shown in Table 5, our results suggested that the blastocyst rate of the 1 µM NMN group was distinctly higher than that of the 2.5 µM BER group and 1 µM COR group, which may be due to the stronger ability of NMN to regulate the gene expression levels of lipid synthesis (Figure 2), antioxidants ( Figure 3C), ER stress (Figure 4), mitochondrial function ( Figure 5), and anti-apoptosis ( Figure 6C).
Previous research has established that vitrification causes a significant increase in the level of ROS in oocytes [70,71], leading to lipid peroxidation and damaging DNA, proteins, and enzyme activity [72]. Similarly, vitrification obviously facilitated the ROS generation of bovine oocytes in our results ( Figure 3B). Our results, as depicted in Figure 3B, also demonstrated that NMN, BER, and COR were effective in reducing the level of ROS in vitrified bovine oocytes, as a result of their ability to increase the mRNA expression of antioxidant-related genes (SOD1, GPX1, and CAT) ( Figure 3C). Moreover, NMN has been shown to reduce the level of ROS by increasing the activity of SOD, which detoxifies ROS, and decreasing the expression levels of NOX1 and NOX4 that generate ROS [59,73]. Furthermore, BER reduces the excessive production of mitochondrial ROS by activating the PGC-1α signaling pathway, which supports mitochondrial energy balance [74]. Additionally, COR has also been demonstrated to reduce ROS levels [66,75], due to the increased activity of SOD and CAT [76]. Furthermore, the ROS level of the NMN group was dramatically lower compared to both the BER group and COR group, which was due to the better capability of NMN to regulate the mRNA expression levels of antioxidant genes ( Figure 3C).
Vitrification exposes oocytes to various forms of stress, such as oxidative stress, ER stress, and osmotic stress, ultimately leading to apoptosis [77]. PS externalization, a commonly used marker for apoptosis [78], was also detected in this study. Figure 6 illustrates that vitrification markedly increased the level of apoptosis in vitrified bovine oocytes; however, the addition of NMN, BER, or COR significantly reduced this level, similar to previous research [19,[79][80][81][82]. NMN can inhibit apoptosis by reducing the BAX/BCL2 ratio and blocking the cleavage of CASPASE-3 [19]. Similarly, BER reduces apoptosis by suppressing the expression of BAX, which is a pro-apoptotic marker gene, and promoting the expression of BCL2, which is a marker gene of anti-apoptosis [83]. Additionally, COR reduces apoptosis through inhibiting the pro-apoptotic IRE1-JNK pathway [65]. Furthermore, the level of apoptosis in the 1 µM NMN group was comparable to that of the fresh control group and obviously lower than both the BER group and the COR group, which may be due to NMN's stronger regulatory effect on genes related to apoptosis ( Figure 6C).

Conclusions
In conclusion, the supplementation of 1 µM NMN, 2.5 µM BER, or 1 µM COR during IVM was found to be effective in reducing the lipid droplet content of bovine oocytes by regulating the expression of lipid metabolism genes and the supplementation also improved the development ability of vitrified bovine oocytes by reducing the generation of ROS, reducing ER stress, regulating mitochondrial function, and inhibiting apoptosis. Furthermore, 1 µM NMN was more effective than 2.5 µM BER and 1 µM COR.