Effects of leonurine supplementation during in vitro culture on oxidative stress, cell proliferation, apoptosis, and autophagy in bovine embryos

Abstract

A critical factor contributing to the reduced production and development of embryos during in vitro production is oxidative stress, which arises due to excessive accumulation of reactive oxygen species (ROS). In order to reduce or prevent ROS levels in the culture medium and embryos, the use of antioxidant agents is commonplace. The present study sets out to evaluate the effects of leonurine, a compound with antioxidant properties, when supplemented into the in vitro culture medium, on embryo development and quality, cell proliferation, apoptosis, autophagy, ROS and GSH levels. Blastocyst quality was evaluated by performing differential staining, ROS, GSH, TUNEL, EdU, and LC3B staining on the developing embryos. The results demonstrated that the supplementation of the culture medium with 20 µM leonurine (Leo20) resulted in a significant enhancement of blastocyst development, as evidenced by increased numbers of ICM, TE, and TCC in embryos when compared to the control group. Furthermore, the number of apoptotic cells and the apoptotic index were found to be lower in the Leo20 group than in the control group. Furthermore, the levels of ROS and LC3B were reduced, while GSH levels were elevated in the leonurine-treated group. With regard to cell proliferation, the number of EdU-positive cells and the proliferation rate were found to be significantly higher in the Leo20 group than in the control group. Gene expression analysis revealed upregulation of antioxidant-related genes (SOD, GPX4, and CAT) and pluripotency-related genes (OCT4, NANOG, and SOX2), while the expression of apoptosis-related genes (BAX and CASP3) and autophagy-related genes (ATG12 and LC3B) was downregulated. It has been demonstrated that the supplementation of leonurine to an in vitro culture medium has the potential to enhance the efficiency of blastocyst formation and to improve the viability of embryos, a phenomenon that may be attributed to the ability of leonurine to reduce oxidative stress. Consequently, the reduction of oxidative stress in embryos is hypothesized to result in increased cell proliferation and decreased rates of apoptosis and autophagy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-39889-3.

Introduction

In vitro production (IVP) of high-quality embryos is recommended as a means of enhancing the reproductive performance of dairy cows, thereby providing genetic improvements that increase yield level and shorten the generation interval^1–4. In recent decades, there has been a substantial increase in the production of in vitro bovine embryos, resulting in the development of enhanced protocols that focus on optimizing embryo yield and quality. Despite advancements, the quality of embryos and efficiency of IVP still remain lower than those of in vivo methods due to the embryos’ sensitivity to in vitro conditions, which can affect their structure, metabolism, and viability^5–9. It is evident that a multitude of morphological and molecular factors exert an influence on the success of in vitro embryo production. This phenomenon can be attributed to the occurrence of unidentified events during the process^3,10–13.

One of the significant factors that disrupts in vitro embryo production is reactive oxygen species (ROS) that arise during early embryonic development^14–17. Indeed, the synthesis of ROS is an integral component of the cellular mitochondrial respiratory chain¹⁸. However, it is important to note that oxidative stress, resulting from excessive ROS accumulation, has been identified as a significant factor associated with defects in embryo development. The generation of ROS by embryo metabolism or the culture medium has been demonstrated to induce a series of deleterious effects, including protein denaturation, apoptosis, autophagy, lipid oxidation, DNA damage, and mitochondrial impairment. These processes have been shown to culminate in the inhibition of embryo development^17,19,20. Under normal circumstances, a balance is maintained between ROS and antioxidant mechanisms^16,21–23. However, in contrast to the maternal reproductive tract, insufficient antioxidant levels in the in vitro culture environment, weakening of the cellular antioxidant mechanism, or excessive cellular oxygen production for various reasons, resulting in increased ROS accumulation, are the cause of oxidative stress^19,20,24. In order to address this issue, a number of studies have been conducted to investigate the effect of supplementing antioxidants to the in vitro culture medium on embryo development^{16,17,19,24,25}.

Leonurine (4-guanidino-n-butyl syringate), a naturally occurring alkaloid, is an active extract obtained from the plant commonly known as Chinese Motherwort (Leonurus heterophyllus)²⁶. It has been reported that leonurine exerts a protective effect in a number of pathological processes within the body, including oxidative stress, apoptosis, inflammation, and lipid and glucose metabolism. Furthermore, leonurine has been demonstrated to possess antioxidant properties, as evidenced by its substantial capacity to diminish the production of ROS within the mitochondria. The potential of leonurine to induce antioxidant stress activity has been demonstrated by an increase in endogenously produced antioxidant capacity, alongside a decrease in mitochondrial dysfunction^27,28. Indeed, it has been found that leonurine exerts an effect in suppressing the formation of ROS in vivo and increases superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels^26,28. Furthermore, a number of studies have determined that it exerts anti-apoptotic and cell proliferation-stimulating effects^27,29–31. Luo et al.²⁷ reported that supplementation of leonurine to the in vitro culture medium reduced ROS levels and increased glutathione (GSH) content in porcine embryos. These effects were shown to contribute to embryo development by alleviating oxidative stress and enhancing mitochondrial function in embryos. Similarly, Zheng et al.³⁰ demonstrated that the supplementation of leonurine to the maturation medium in bovine species decreased ROS levels and lipid content in oocytes, while increasing mitochondrial membrane potential and ATP content. Improvement in oocyte quality was associated with increased maturation rates, blastocyst formation rates, and total blastocyst cell numbers. In contrast to these studies, the present study investigates the effects of leonurine supplementation during the in vitro culture phase on bovine embryo development. This distinction is important, as in vitro maturation and in vitro culture represent fundamentally different stages characterized by distinct metabolic activities and regulatory mechanisms. For this reason, this study was carried out to evaluate the effects of leonurine, which supplemented to the culture medium during in vitro bovine embryo production, on embryo development and quality, oxidative stress, antioxidant capacity, cell proliferation, apoptosis, and autophagy, and determine the potential molecular pathways and mechanisms affected by the supplementation of leonurine during embryo development.

Results

Effect of different leonurine doses on the early development of bovine embryos

In the present study, the optimal dose of leonurine to supplement to the culture medium during in vitro embryo production was determined to be effective on blastocyst development. The numbers of presumptive zygotes transferred into in vitro culture media and blastocyst formation rates included in the groups are presented in Table 1. The highest blastocyst formation rate was observed in the Leo20 group. The blastocyst formation rates in the Leo5 and Leo10 groups were comparable to those of the control group, whereas the Leo40 and Leo60 groups exhibited the lowest blastocyst formation rates. Moreover, it was determined that when the leonurine dose exceeded 20 µM, the blastocyst formation rate decreased. Thus, it was determined that 20 µM leonurine, when supplemented to the in vitro culture medium, was the most effective dose for embryo development, as indicated by the blastocyst formation rates.

Table 1.

Obtained blastocyst formation rates with different doses of leonurine supplemented to the in vitro culture medium.

Group	Leo5	Leo10	Leo20	Leo40	Leo60	0 (Con)
Blastocyst formation rates (%)	26.73a (27/101)	28.28a (28/99)	36.89b (38/103)	17.34c (17/98)	18.75c (18/96)	26.60a (29/109)

^a−c : Superscripts represent the statistical difference at P < 0.05.

Effect of leonurine on early embryo development and cell count

Following the determination of the optimal dose in the study, the applications were continued in two groups: leonurine (Leo20, n = 1017) and control (Con, n = 1000). There was no statistical difference in maturation and cleavage rates between the Leo20 and control groups. The blastocyst formation rates were found to be higher in the Leo20 group (35.39%, 360/1017) in comparison to the control group (27.60%, 276/1000) (P < 0.0001, Fig. 1).

Fig. 1.

Effect of leonurine supplementation during in vitro culture on the development of bovine embryos and on ICM, TE, and TCC of bovine embryos. (A) Representative images of Day-7 IVEP embryos (20X), (B) rate of blastocyst formation, (C) images of differential staining (Hoechst 33342 + PI) of Day-7 IVEP blastocysts supplemented with 0 (Control) and 20 µM of leonurine. ICM (D), TE (E) numbers, and TCC (F) of blastocysts on day 7 in the Leo20 (n = 30) and Con (n = 30) groups. R = 2. Data are presented as the mean ± SD. The green and orange dots represent the distribution of measurements in the Leo20 and Control groups, respectively. Blastocyst formation rate = numbers of blastocyst / numbers of presumptive zygotes transferred into culture media. Abbreviations: ICM, inner cell mass; TE, trophectoderm; TCC, total cell count; IVEP, in vitro embryo production; PI, propidium iodide. Scale bar = 100 μm.

The number of cells determined by differential staining in blastocysts developing in groups to evaluate the effect of leonurine on blastocyst development is shown in Fig. 1. The number of inner cell mass (ICM), trophectoderm (TE), and total cell count (TCC) in the Leo20 group was found to be statistically higher than in the control group. In the leonurine and control groups, the ICM counts were 48.20 ± 11.46 and 39.93 ± 8.08, respectively; the TE counts were 92.40 ± 20.58 and 78.66 ± 17.74, respectively; and the TCC values were 140.60 ± 30.35 and 118.60 ± 22.78, respectively.

Effect of leonurine on apoptosis in blastocysts

In order to evaluate the effect of leonurine on apoptosis in embryos at the blastocyst stage, the presence and number of apoptotic cells in embryos developed after the supplementation of leonurine to the in vitro culture medium were determined by TUNEL analysis (Fig. 2). The number of cells demonstrating apoptosis, as well as the apoptotic index ratio, in blastocysts developed following the supplementation of leonurine into the in vitro culture medium, were found to be significantly lower in the Leo20 group in comparison to the control group. In the leonurine and control groups, the number of apoptotic cells was determined as 8.83 ± 3.07 and 12.10 ± 4.22, respectively, and the apoptotic index was 6.47% and 10.88%, respectively. Furthermore, quantitative real-time PCR findings revealed statistical differences in the mRNA expression levels of apoptosis-related genes. In the Leo20 group, the levels of the apoptosis-related mRNAs BAX (3-fold decrease) and CASP3 (2-fold decrease) were found to be lower than in the control group (P < 0.0001). No statistical difference was detected between BCL2 levels.

Fig. 2.

Effect of leonurine supplementation during in vitro culture on apoptotic cell number and apoptotic index of bovine embryos. (A) Representative images of TUNEL staining of Day-7 IVEP blastocysts supplemented with 0 (Control) and 20 µM of leonurine. Arrows indicate cells undergoing apoptosis. Apoptotic cell (B), TCC (C), and apoptotic index ratio (D) of blastocysts in the Leo20 (n = 30) and Con (n = 30). R = 2. Data are presented as the mean ± SD. The green and orange dots represent the distribution of measurements in the Leo20 and Control groups, respectively. Effect of leonurine on the mRNA expression levels of apoptosis-related genes, BAX (E), CASP3 (F), and BCL2 (G), quantified with RT-qPCR. Data are derived from at least three independent replicates. Abbreviations: BAX, BCL2-related x protein; BCL2, B-cell lymphoma gene-2; CASP3, cysteine aspartic acid protease-3; DAPI, 4’,6-diamidino-2-phenylindole; IVEP, in vitro embryo production; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling; TCC, total cell count. Scale bar = 100 μm.

Effect of leonurine on antioxidant capacity in blastocysts

In order to ascertain the effect of leonurine on the antioxidant capacity of in vitro produced bovine embryos, the levels of ROS (CM-H2DCFDA) and GSH (CMF2HC) were analyzed. The mean intensity of fluorescence, evaluated to ascertain the level of ROS in blastocyst-stage embryos, was determined to be lower in the Leo20 group (20.38 ± 6.50) in comparison to the control group (38.06 ± 16.50) (P < 0.0001). However, GSH levels were found to be higher in the Leo20 group (29.50 ± 6.05) than in the control group (18.51 ± 8.50) (P < 0.0001). The findings demonstrated that the supplementation of leonurine to the culture medium led to a reduction in ROS levels by approximately 1.9 times and an increase in GSH levels by 1.6 times, in comparison to the control group (Fig. 3). As expected, RT-qPCR results demonstrated substantial increases in the mRNA expression levels of antioxidant-related genes, including SOD, GPX, and CAT, in the Leo20 group in comparison with the control group. It was determined that the mRNA expression levels of SOD, GPX, and CAT in embryos from the leonurine group increased by 3-, 1.34-, and 1.66-fold, respectively, compared with the control group.

Fig. 3.

Effect of leonurine on the intracellular ROS and GSH levels in bovine embryos. (A) Representative images of embryos stained with CMF2HC to assess intracellular GSH levels and with CM-H2DCFDA to detect ROS levels in the Day-7 IVEP blastocysts supplemented with 0 (Control) and 20 µM of leonurine. (B) Relative intensity of fluorescence changed of ROS levels Day-7 blastocysts in the Leo20 (n = 30) and Con (n = 30) groups. ROS fluorescence intensity level in Leo20-treated blastocysts significantly decreased to 1.9-fold compared to Con group. (C) Relative intensity of fluorescence changed of GSH levels Day-7 blastocysts in the Leo20 (n = 30) and Con (n = 30) groups. GSH fluorescence intensity level in Leo20-treated blastocysts significantly increased to 1.6-fold compared to Con group. Effect of leonurine on the mRNA expression levels of antioxidant enzyme-related genes, SOD (D), GPX4 (E), and CAT (F), quantified with RT-qPCR. Data are derived from at least three independent replicates. Abbreviations: ROS, reactive oxygen species; CM-H2DCFDA, 5-(and-6)-chloromethyl-2´,7´-dichlorodihydro-fluorescein diacetate; GSH, glutathione; CMF2HC, 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin; IVEP, in vitro embryo production; SOD, superoxide dismutase; GPX4, glutathione peroxidase − 4; CAT, catalase. Scale bar = 100 μm.

Effect of leonurine on cell proliferation in blastocysts

The effect of leonurine supplementation on cell proliferation competence in blastocysts was also investigated during the in vitro culture phase, and for this purpose, the number of EdU-positive cells in blastocysts was determined. In the Leo20 group, the ratio of EdU-positive cells to the total number of cells was found to be statistically higher than in the control group, and the cell proliferation rate was found to be approximately 1.5 times higher than in the control group (Fig. 4). The number of EdU-positive cells was determined as 47.32 ± 7.94 and 28.64 ± 9.82 in the leonurine and control groups, respectively. However, RT-qPCR results demonstrated significant increases in mRNA expression levels of genes associated with pluripotency and cell proliferation, such as OCT4, NANOG, and SOX2, in the Leo20 group. However, no differences were detected in GATA3 and CDX2 mRNA expression levels. It was determined that the mRNA expression levels of OCT4, NANOG, and SOX2 in embryos from the leonurine group increased by 1.9-, 2.8-, and 2.5-fold, respectively.

Fig. 4.

Effect of leonurine on the cell proliferation competence in bovine embryos. (A) Representative images of EdU staining of Day-7 IVEP blastocysts supplemented with 0 (Control, n = 30) and 20 µM of leonurine (n = 30). EdU-positive cell (B), TCC (C), and cell proliferation rate (D) of blastocysts in the Leo20 (n = 30) and Con (n = 30). R = 2. Data are presented as the mean ± SD. The green and orange dots represent the distribution of measurements in the Leo20 and Control groups, respectively. Effect of leonurine on the mRNA expression levels of pluripotency-related genes, OCT4 (E), NANOG (F), and SOX2 (G), quantified with RT-qPCR. Data are derived from at least three independent replicates. Abbreviations: EdU, 5-ethynyl-20-deoxyuridine; DAPI, 4’,6-diamidino-2-phenylindole; TCC, total cell count; OCT4, POU class 5 transcription factor 1; NANOG, homeobox transcription factor; SOX2, SRY (sex determining region Y)-Box2. Scale bar = 100 μm.

Effect of leonurine on autophagy in blastocysts

The present study sought to evaluate the effect of leonurine supplementation during the in vitro culture phase on the autophagy level in developing blastocysts. To this end, the LC3B fluorescence intensity level associated with autophagy was determined in blastocysts. The mean LC3B fluorescence intensity was found to be lower in the Leo20 group (4.98 ± 2.61) in comparison to the control group (7.74 ± 3.51) (P < 0.01). The findings of the study demonstrated that the supplementation of leonurine to the culture medium led to a reduction in the LC3B fluorescence intensity level by approximately 1.55 times in comparison with the control group (Fig. 5). As expected, RT-qPCR results demonstrated a significant decrease in mRNA expression levels of autophagy-related genes, including ATG12 (approximately 3 times) and LC3B (approximately 5 times), in the Leo20 group compared to the control group.

Fig. 5.

Effect of leonurine on the autophagy in bovine embryos. (A) Representative images of LC3B staining of Day-7 IVEP blastocysts supplemented with 0 (Control) and 20 µM of leonurine. Scale bar = 200 μm. (B) Relative intensity of fluorescence changed of LC3B levels Day-7 blastocysts in the Leo20 (n = 30) and Con (n = 30) groups. LC3B fluorescence intensity level in Leo20-treated blastocysts significantly decreased to 1.55-fold compared to Con group. Effect of leonurine on the mRNA expression levels of autophagy-related genes, ATG12 (C), and LC3B (E), quantified with RT-qPCR. Data are derived from at least three independent replicates. Abbreviations: ATG12: autophagy related gene-12, LC3B: light chain 3 beta IVEP, in vitro embryo production. Scale bar = 100 μm.

Discussion

In vitro culture (IVC) of embryos renders them highly vulnerable to oxidative stress, a phenomenon attributable to the disparity between the in vivo environment and the in vitro production processes^15,16,20. Although the development of the IVC environment is relatively complete, embryos produced in vitro have less developmental potential than embryos developing in vivo because they lack the protective maternal endocrine environment provided to embryos developing in vivo^19,32. The increase and accumulation of ROS triggered by oxidative stress can cause changes in various developmental parameters (e.g. blastocyst formation rate, cell count, cellular survival, and cell proliferation) in the embryo³³. Furthermore, the imbalance and/or cellular stress of biological chemicals designed to prevent the damage warned by ROS has been shown to cause damage to DNA, proteins, and lipid layers, reducing embryo development and quality^25,34. Therefore, the supplementation of antioxidants at various stages of in vitro embryo production has been demonstrated to be an effective measure in the prevention of excessive ROS accumulation and the enhancement of embryo development^17,19,20,24. The present study investigates the effect of leonurine, when supplemented to the culture medium, on the early embryo development of cattle.

The initial step in the study was to ascertain the effective dose of leonurine for the purpose of promoting embryo development. During the process of determining the optimal dosage, it was observed that the highest rate of blastocyst formation was recorded in the group that supplemented 20 µM leonurine in the in vitro culture medium. Consequently, this dosage was selected for the subsequent phases of the study. Zheng et al.³⁰ also stated that the supplementation of 20 µM leonurine to the in vitro maturation medium in cattle was a more efficacious dose for the purpose of blastocyst development. However, Zheng et al.³⁰ focused on different points from the presented study because they supplemented leonurine to the maturation medium. Moreover, it has been documented that the supplementation of 20 µM leonurine into the in vitro maturation medium has a detrimental effect on the blastocyst formation rate, and that excessive leonurine accumulation has a deleterious effect on oocyte development³⁰. Luo et al.²⁷ reported that the supplementation of high doses (60 µM) of leonurine to in vitro culture in pigs resulted in toxic effects and adversely affected embryo development. In the present study, it was hypothesized that the low blastocyst formation rate observed in the groups administered high doses could be attributed to leonurine exerting a deleterious effect on embryo development. Furthermore, following the determination of the dosage, the blastocyst formation rate of the Leo20 group (35.39%) was found to be higher than that of the control group (27.60%). The number of cells in blastocysts developing in cattle is of significant importance to the quality of the embryo, subsequent developmental stages, and the formation of implantation^35–38. This is because as the number of cells increases, the implantation capacity also increases in parallel³⁹. In the present study, cell counts were determined in blastocysts developed by means of differential staining. These results revealed that the numbers of ICM, TE and TCC were higher in the Leo20 group than in the control group. Luo et al.²⁷ have demonstrated that the supplementation of leonurine to the in vitro culture media has been shown to significantly increase the blastocyst formation rate in pigs. Zheng et al.³⁰ reported that the supplementation of 20 µM leonurine to the in vitro maturation media resulted in enhanced blastocyst formation rate and TCC in cattle when compared to the control group. The findings of these studies suggest that leonurine enhances blastocyst formation rates by inhibiting the accumulation of intracellular ROS and enhancing mitochondrial function during oocyte or embryo development, attributable to its antioxidant properties. Furthermore, leonurine protects embryos from oxidative stress during early embryo development^27,30. Similarly, in the present study, it is thought that leonurine enhances blastocyst development rates by protecting embryos from oxidative stress, a process that is facilitated by its antioxidant properties.

In this study, the mRNA expression levels of ROS, GSH, and antioxidant-related genes were investigated in embryos developed after in vitro embryo production to reveal the antioxidant activity of leonurine. The results of this study demonstrated a decline in intracellular ROS levels and an increase in intracellular GSH levels in blastocysts developed following the supplementation of leonurine into the culture medium. Furthermore, it was determined that the mRNA expression levels of antioxidant genes such as SOD, GPX4, and CAT in the Leo20 group were significantly increased in comparison with the control group. Oxidative stress, characterized by an accumulation of ROS, is a pivotal factor influencing embryo development. Embryos exhibit heightened sensitivity to oxidative stress during the initial phases of embryonic development, a period characterized by embryonic genome activation. Oxidative stress has been documented to frequently result in the arrest of embryos during this stage^20,40,41. Mitochondria, which play a pivotal role in regulating cellular energy, are imperative for cell division, pluripotency, and differentiation in embryos during early development⁴². Indeed, during endogenous metabolic reactions, oxygen radicals such as superoxide anion, hydrogen peroxide, hydroxyl radicals, and oxygen peroxides are produced as normal molecular oxygen products in the mitochondria⁴³. However, under normal conditions, a balance exists between ROS levels and antioxidant mechanisms. Disruption to this balance can result in adverse effects on oocyte or embryo development^18,44. GSH is the most abundant endogenous antioxidant in cells; however, as the early development period progresses, its concentration remains low due to the low GSH synthesis capacity of preimplantation embryos^45–47. Furthermore, an increase in ROS leads to a decline in GSH levels and a disruption in the antioxidant defense mechanism^38,48. Consequently, the utilization of antioxidant supplements has been identified as a necessity during various phases of in vitro embryo production^16,18. The present study demonstrated that the supplementation of leonurine into the culture media during in vitro embryo production in cattle resulted in an augmentation of the antioxidant capacity of the developing embryos. Luo et al.²⁷ reported that the supplementation of leonurine to the culture medium during early embryo development reduced intracellular ROS levels and increased GSH levels in blastomeres. Furthermore, it has been indicated that the antioxidant properties of leonurine may be attributable to its capacity to enhance SOD and CAT activity, whilst concomitantly reducing NAPDH oxidase 4 activity and malondialdehyde content^27–29. In the present study, the expression levels of the antioxidant genes SOD, GPX4, and CAT mRNA were found to be higher in the Leo20 group than in the control group. SOD and CAT are antioxidant enzymes necessary for the processing of ROS such as superoxide and hydrogen peroxide, and an increase in the transcription level of these enzymes indicates oxidative stress⁴⁹. The results of this study suggest that leonurine may enhance antioxidant activity, thereby reducing oxidative stress and potentially enhancing the developmental competence of bovine blastocysts. Furthermore, it has been demonstrated that the augmentation of antioxidant capacity subsequent to the supplementation of leonurine exerts a favorable influence on biochemical and metabolic reactions, including DNA synthesis and repair, protein synthesis, and consequently affects cell proliferation and apoptosis by diminishing cellular ROS accumulation during the initial stages of embryo development^27,50.

The supplementation of leonurine into the culture medium during in vitro embryo production resulted in an enhancement of the blastocyst formation rate and total cell count, as well as cell proliferation in developing embryos. This is due to the finding that the cell proliferation rate in blastocysts developed after the supplementation of leonurine to the culture medium was higher than that in the control group, with the proliferation rate being approximately 1.5 times higher. However, levels of pluripotency and proliferation-related gene expression (OCT4, NANOG, SOX2) were found to be at least 2–3 times higher than in the control group. As Luo et al. (2020) reported, the supplementation of leonurine to the culture medium in pigs resulted in an increase in the cell proliferation rate in blastocysts. As posited by Zheng et al.³⁰, the supplementation of leonurine into the environment during the maturation of oocytes in cattle resulted in a substantial augmentation in the number of EdU-positive cells observed within developing blastocysts, in comparison to the control group. Luo et al.²⁷ reported that the effect of leonurine on cell proliferation is likely to be mediated by the regulation of the mTOR/MAPK/PI3K/AKT/GSK3β pathways, with these mechanisms improving embryo development and differentiation. It has been demonstrated that pluripotency-related genes in bovine embryos are important factors in sustaining the proliferation and differentiation of embryonic stem cells and pluripotent stem cells^51–53. OCT4 is a transcription factor belonging to the POU transcription family domain (POU5F1) and is predominantly expressed in pluripotent cells. OCT4 has been demonstrated to play a pivotal role in the maintenance of cells in an undifferentiated state, exerting its influence through the modulation of the expression of various loci implicated in pluripotency and cellular differentiation⁵⁴. Furthermore, OCT4 has been demonstrated to function as a regulator of cell lineage specification following the morula stage and is required for the maintenance of pluripotency and NANOG expression⁵⁵. While NANOG is not considered a prerequisite for the effective separation of TE and ICM, its role is vital for the establishment and preservation of pluripotent epiblast formation⁵⁶ and the maintenance of pluripotency in bovine embryos⁵⁷. It has been asserted that SOX2 begins to accumulate gradually in ICM cells alongside blastocyst expansion in bovine embryos. Concurrently, it has been documented that a deficiency in SOX2 expression results in a decline in cell number in blastocysts and a disruption of the pluripotency gene network in bovine blastocysts⁵⁸. Consequently, it was determined that the supplementation of leonurine into the culture medium may enhance cell proliferation and stimulate the expression of mRNA genes associated with blastocyst development.

The delicate equilibrium between cell proliferation and death (apoptosis, programmed cell death) is pivotal for successful embryo development⁵⁹. The degree of apoptosis has been identified as a significant criterion affecting embryo development and quality, as well as post-implantation development potential⁶⁰. During in vitro embryo production, elevated levels of ROS (reactive oxygen species) in cells have been observed to induce mitochondrial-mediated apoptosis (programmed cell death), while glutathione deficiency/depletion has been shown to trigger this process as well^61,62. Mitochondria are vital organelles in embryos, and excessive ROS has been demonstrated to cause mitochondrial dysfunction. Mitochondrial dysfunction has been demonstrated to exert a deleterious effect on the development of embryos, resulting in aberrant apoptosis and autophagy. The apoptotic pathway is initiated in embryos through the expression of various genes encoding pro-apoptotic and anti-apoptotic proteins by mitochondria^47,63. Li et al.²⁸ reported that leonurine exerts a dual effect on apoptosis. The first is anti-apoptotic activity, which is mediated by the activation of the PI3K/AKT signaling pathway and an increase in the BCL-2/BAX ratio. The second is pro-apoptotic activity, which is mediated by an increase in the BAX/BCL-2 ratio, a decrease in mitochondrial transmembrane potential, and the initiation of the apoptotic caspase cascade signal. The study revealed that the apoptotic index was elevated in the control group following TUNEL staining in developing blastocysts. However, it was found that the supplementation of leonurine to the culture medium resulted in a reduction of the apoptotic index. Furthermore, the mRNA expression levels of apoptosis-related genes such as BAX and CASP3 (pro-apoptotic) were found to be lower in the Leo20 group than in the control group. Zheng et al.³⁰ reported that the supplementation of leonurine to the medium during in vitro maturation in cattle resulted in elevated BCL-2 expression levels in oocytes, accompanied by substantial reductions in the BAX/BCL-2 ratio and CASP3 expression. It has been reported that the supplementation of leonurine during in vitro maturation has been demonstrated to successfully protect oocytes from apoptosis³⁰. Liu et al.²⁹ reported that leonurine reduced the expression level of pro-apoptotic protein BAX mRNA (myocardial injury) and increased the expression level of anti-apoptotic protein BCL-2 mRNA. Furthermore, leonurine has been posited as a potential therapeutic agent for the mitigation of apoptosis induced by ROS accumulation²⁹. Xu et al.³¹ reported that leonurine exerts anti-apoptotic effects by activating the PI3K/AKT signaling pathway, increasing BCL-2 levels and decreasing CASP3 levels. Luo et al.²⁷ posited that the supplementation of leonurine to the culture medium during in vitro embryo production in pigs may have averted apoptosis by enhancing mitochondrial function. In consideration of the aforementioned data, it has been determined that the supplementation of leonurine into the culture medium during the in vitro production of bovine embryos diminishes the probability of embryo apoptosis, consistent with the previously documented effects pertaining to the apoptosis mechanism.

The classification of animal cell death is typically into three categories: type I (apoptotic), type II (autophagic, lysosomal cell death), and necrotic⁶⁴. Autophagy, a process in which eukaryotic cells selectively eliminate misfolded, damaged, or excess cytoplasmic products and degrade or recycle macromolecules and organelles, plays an important role in the cellular response to oxidative stress^65,66. In recent years, a significant body of research has indicated that excessive ROS accumulation in embryos can induce autophagy, thereby impeding embryo development^{27,30,32,58,66,67}. Autophagy, which can be conceptualized as an environmental response to cells’ demand for clearance within the cell, serves as a reliable metric for evaluating the quality of embryo development by examining its formation within the cell⁶⁸. Methods such as immunofluorescence staining and autophagy-related gene expression can be used to assess autophagy in preimplantation embryos⁶⁷. Light chain 3 beta (LC3B, essential for autophagy formation) is an RNA-binding protein that interacts with target mRNAs during autophagy, triggering their rapid degradation⁶⁸. In the present study, the level of autophagy-related protein LC3B in blastocysts developed after the supplementation of leonurine to in vitro culture was analyzed by immunofluorescence staining. The results showed that the intensity of fluorescence decreased in the Leo20 group compared to the control group. Furthermore, mRNA expression levels of autophagy-related genes such as ATG12 and LC3B were also found to be lower than in the control group. Consequently, it was determined that the supplementation of leonurine into the culture medium during in vitro embryo production in cattle exerts a suppressive effect on autophagy in blastocysts. Luo et al.²⁷ in pigs and Zheng et al.³⁰ in cattle have determined that the level of autophagy-related marker LC3B decreases after the supplementation of leonurine. These researchers emphasized that autophagy may be reduced by improving ROS levels through the use of leonurine, which has an antioxidant effect, due to the significant impact of ROS in inducing autophagy.

In conclusion, it was determined that the supplementation of leonurine into the culture medium during in vitro embryo production in cattle was efficacious in enhancing the blastocyst formation rate. Furthermore, this supplementation led to an augmentation in embryo quality and an increase in the total cell count (including ICM and TE) in developing blastocysts. Moreover, the supplementation of leonurine as an antioxidant into the culture medium resulted in a decline in ROS levels and an augmentation in GSH levels within the blastocysts. Consequently, it was hypothesized that the mitigation of oxidative stress in embryos would result in a reduction in the occurrence of apoptosis and autophagy. As a result, it was determined that the supplementation of leonurine into the culture medium during in vitro bovine embryo production may prove advantageous in enhancing the developmental competence of embryos.

Materials and methods

Chemicals

In this study, media with undefined contents (BO-Wash, BO-IVM, BO-SemenPrep, BO-IVF, BO-IVC, BO-Oil) prepared by a commercial company (IVF Bioscience, UK and Denmark) were used during in vitro embryo production^3,69. All applications were carried out in accordance with the protocols of the commercial company. The leonurine (SML0670, CAS Number:24697-74-3) utilized in the study was obtained from Sigma-Aldrich (USA). Unless otherwise specified, all other chemical substances utilized in the study were obtained from Sigma-Aldrich (USA).

Collection of oocytes, in vitro maturation, and fertilization

In the study, ovaries from Holstein cattle were collected from local slaughterhouses and transported to the laboratory within 2–3 h in lactated Ringer’s solution supplemented with 50 µg/mL gentamicin (Gentacure, Teknovet, Turkiye) at 30–33 °C. The ovaries were washed three to four times with 0.9% NaCl in order to remove any residual blood, tissue material and the transport medium. Cumulus oocyte complexes (COCs) were aspirated from antral follicles with a diameter of 3–8 mm using an 18-gauge needle and a disposable 10-mL syringe from the ovarian cortex and the obtained cumulus oocyte complexes within oocyte washing medium (BO-Wash, IVF Bioscience) evaluated under a stereomicroscope according to the quality criteria established⁷⁰. In the study, only category I (A-quality) COCs were utilized, and the selected oocytes were taken into 4-well dishes containing in vitro maturation medium (BO-IVM, IVF Bioscience) at 38.5 °C and 6% CO₂ for a duration of 22–24 h. A maximum of 45–55 oocytes were transferred in one well of each 4-well dish. The degree of cumulus expansion of all oocytes’ following maturation was assessed, and Grade 1 is characterized by very little expansion and little morphological change compared to the pre-maturation period; Grade 2 is characterized by partial cumulus expansion and unexpanded areas; and Grade 3 is characterized by complete or near complete cumulus expansion and homogeneous distribution of cumulus cells. Only Grade II and III oocytes were transferred to the in vitro fertilization medium^3,37. Sperm selection prior to in vitro fertilization was performed using the sperm washing method⁶⁹. First, two frozen semen straws were thawed in a 37 °C water bath for 30 s and thawed semen were added to the centrifuge tube containing 2 mL of semen preparation medium (BO-SemenPrep, IVF Bioscience) and then centrifuged at 328 g for 5 min. The supernatant after centrifugation was removed. Then, 2 mL of sperm preparation medium was added to the tube again and centrifuged at 328 g for 5 min, and then the supernatant was again removed. Finally, after both methods, the final concentration of spermatozoa for fertilization was calculated as 1 × 10⁶/mL. Thereafter, the oocytes and spermatozoa were then incubated for 18 h at 38.5 °C and 6% CO₂ within in vitro fertilization medium (BO-IVF, IVF Bioscience).

In vitro culture

Subsequent to the process of fertilization, the cumulus cells were removed by using a vortex. Fertilized oocytes (presumptive zygotes) were transferred into a 2 mL tube with 1 mL oocyte washing medium (BO-Wash, IVF Bioscience) and vortexed at a fast speed for 2 min. After this procedure, presumptive zygotes were controlled and transferred to in vitro culture medium (BO-IVC, IVF Bioscience) covered with mineral oil (BO-Oil, IVF Bioscience) and incubated for a period of 7–8 days at 38.5 °C, 6% CO₂, 6% O₂, 88% N₂, and in a humid environment. The following procedures were performed during the in vitro culture step.

Determination of the effect of different leonurine doses on early embryonic development

Initially, different concentrations of leonurine were supplemented to the culture medium during the in vitro culture phase, and the optimal leonurine concentration in terms of embryo development and blastocyst formation rates (numbers of blastocysts/numbers of presumptive zygotes taken into culture medium x 100) were identified (Fig. 6). In order to achieve this objective, presumptive zygotes were randomly divided into six groups prior to being transferred to the culture medium for in vitro culture^27,30. The leonurine was dissolved in dimethyl sulfoxide (DMSO, D5879, Sigma-Aldrich) and subsequently prepared in specific concentrations. The presumptive zygotes were then cultured and divided into groups, to which 5 (Leo5, n = 101), 10 (Leo10, n = 99), 20 (Leo20, n = 103), 40 (Leo40, n = 98), and 60 (Leo60, n = 96) µM, and 0 (Con, n = 109) µM leonurine were supplemented to the groups in sequence. The control group was also supplemented with DMSO at the same final concentration as the treatment groups. The dose determination trial was conducted in two replications.

Fig. 6.

Schematic representation of experimental design. Collection of cumulus-oocyte complexes (COCs) from ovaries obtained from the slaughterhouse, fertilization of oocytes, and in vitro culture of the resulting presumptive zygotes. (A) After fertilization, presumptive zygotes are allocated into different groups (Leo5, 10, 20, 40, 60, and Con) to determine the most effective leonurine dose for embryo development. (B) Following in vitro culture, the potential molecular pathways influenced by leonurine—related to antioxidant activity, apoptosis, cell proliferation, and autophagy—are analyzed using RT-qPCR. (C) To assess embryo quality, various staining procedures are applied, including differential staining, TUNEL, and EdU assays, as well as measurements of ROS, GSH, and LC3B levels.

Following the completion of the in vitro embryo production steps, the developmental stages and qualities of the embryos in each group were evaluated⁷¹. The blastocyst formation rates were determined in the groups, and the leonurine dose that resulted in the highest blastocyst formation rate (Leo20, 20 µM) was selected for the other steps of the study. Subsequent to the completion of these procedures, in vitro embryo production processes were initiated utilizing new replicas from the selected dose (Leo20, n = 1017) and control group (Con, n = 1000). The ensuing procedures were then applied to the obtained blastocysts. These applications were performed in 12 replications.

Determination of cell numbers by differential staining

Following the determination of the developmental stages of the embryos following in vitro culture, 30 blastocysts from each group were subjected to the differential staining method^3,37,69. The blastocysts were firstly washed 3 times in a 0.1% PBS-PVA solution and then transferred to 0.2% Triton X-100 (for 30 s, T8787, Sigma-Aldrich, 2 µl/ml). Following permeabilization, the blastocysts were placed in a solution containing propidium iodide (for 30 s, PI, P4170, Sigma-Aldrich, 100 µg/ml) and then washed in PBS-PVA before being transferred to a solution containing 4% paraformaldehyde (158127, Sigma-Aldrich, w/v) + Hoechst 33,342 (for 15 min, B2261, Sigma-Aldrich, 25 µg/ml). After this procedure, the blastocysts were washed 3 times again in PBS-PVA and placed in a glycerol droplet on a polylysine-coated slide. The prepared specimens were then imaged under a fluorescence microscope (Fluorescence microscope Visoria B and camera Leica K3M mono, Leica Microsystems, Germany). The inner cell mass (ICM), trophectoderm (TE), and total cell count (TCC) of the blastocysts were subsequently determined.

Cell death and determination of apoptotic index

The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) method (In Situ Apoptosis Kit, Green-FITC, E-CK-A320, Elabscience) was utilized to detect cell death and apoptotic index in blastocysts identified after in vitro culture^3,69. The staining of 30 blastocysts from each group was conducted using this method. The blastocysts were firstly washed 3 times in a 0.1% PBS-PVA solution and then transferred to a 3.7% paraformaldehyde solution (at the room temperature for 20 min). Subsequent to this procedure, the blastocysts were washed again in a solution of PBS-PVA and then transferred to a permeabilization solution composed of 0.2% Triton X-100 (v/v, at 37 °C for 10 min). The blastocysts were then washed again in PBS-PVA solution and transferred to TdT Equilibration Buffer solution (at 37 °C for 20 min). The blastocysts were subsequently transferred directly into the TUNEL reaction mixture (at 37 °C for 60 min) without undergoing a washing process. Following this, the blastocysts were washed in PBS-PVA and incubated in DAPI (at the room temperature for 5 min, 4′,6-diamidino-2-phenylindole, D9542, Sigma-Aldrich, 25 µg/ml) in a dark environment to ascertain the total number of cells. After this process, the blastocysts were washed in PBS-PVA solution and transferred to a glycerol droplet on a polylysine-coated slide. Following staining, the blastocysts were examined under a fluorescence microscope (Fluorescence microscope Zeiss Axioscope 5 and camera Axiocam 208 color, Zeiss, Germany). Under a fluorescence microscope, the observation was made that apoptotic cells emitted green light. The images obtained were recorded and the apoptotic index ratio was calculated.

Intracellular ROS and GSH levels assays

The levels of ROS and GSH were subsequently determined in blastocysts following in vitro culture^3,38,72. The staining of 30 blastocysts from each group was conducted using these methods. In order to ascertain the levels of intracellular ROS and GSH, the blastocysts were subjected to an incubation period of one hour in IVC media, to which 10 µM CM-H2DCFDA (at 37 °C for 1 h, 5-(and-6)-chloromethyl-2´,7´-dichlorodihydro-fluorescein diacetate, 21884, Sigma-Aldrich, w/v) and 10 µM CMF2HC (at 37 °C for 1 h, 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin, C12881, ThermoFisher Scientific, w/v) had been administered. Then, the blastocysts were washed 3 times in a 0.1% PBS-PVA solution. Following staining, the embryos were examined under a fluorescence microscope (Fluorescence microscope Zeiss Axioscope 5 and camera Axiocam 208 color, Zeiss, Germany). The ROS and GSH fluorescence intensity of the embryos was analyzed using the fluorescence microscope’s own Zen software program (ZEN lite, 3.11, Zeiss).

Determination of cell proliferation by measurement of 5-ethynyl-20-deoxyuridine (EdU)

The 5-ethynyl-20-deoxyuridine (EdU-647) cell proliferation assay kit (Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 dye, C10340, ThermoFisher Scientific) was utilized to ascertain cell proliferation in blastocysts^27,32. Thirty blastocysts obtained from each group were stained using this method. First, the blastocysts were incubated in IVC media containing 10 µM EdU (at 37 °C for 4 h). The blastocysts were then transferred to 3.7% paraformaldehyde (at room temperature for 30 min) and washed 3 times in 3% PBS-BSA solution, followed by permeabilization (at room temperature for 30 min, 0.5% Triton X-100, v/v). The blastocysts were then washed again in a solution of PBS-BSA and transferred to a Click-iT reaction cocktail solution (at room temperature for 30 min). Subsequent to this process, the blastocysts were washed again in a solution of PBS-BSA and then transferred to a solution of Hoechst 33,342 (at room temperature for 30 min, 10 µg/mL) to determine the TCC. Finally, the blastocysts were thoroughly rinsed in a solution of PBS and then transferred to a polylysine-coated slide. Following staining, the blastocysts were examined under a fluorescence microscope (Fluorescence microscope Zeiss Axioscope 5 and camera Axiocam 208 color, Zeiss, Germany). The presence of EdU-positive cells was detected under a fluorescence microscope, with these cells exhibiting red-light emissions. The images obtained were recorded and the cell proliferation rate was calculated.

Immunofluorescence staining

To ascertain the level of autophagy in blastocysts following in vitro culture, 30 embryos obtained from each group were subjected to staining^27,32. The blastocysts were initially washed 3 times in a 0.1% PBS-PVA solution, followed by fixation in a 3.7% paraformaldehyde solution (at room temperature for 30 min). The blastocysts were then washed again in PBS-PVA solution and left to undergo permeabilization (at room temperature for 30 min, 0.5% Triton X-100, v/v). After this procedure, the blastocysts were incubated in 1% PBS-BSA solution at the room temperature for one hour. Subsequent to this process, the blastocysts were subjected to an overnight incubation at 4 °C in the presence of the LC3B primary antibody (Autophagosome Marker, ab48394, Abcam). The blastocysts were then washed in PBS-PVA solution and transferred to a solution containing secondary antibody [Goat anti-rabbit IgG (H + L) Flour 488 conjugated, ab150077, Abcam] and incubated at room temperature for one hour. Following this procedure, the blastocysts were washed one final time in PBS-PVA solution and then examined under a fluorescence microscope (Fluorescence microscope Zeiss Axioscope 5 and camera Axiocam 208 color, Zeiss, Germany). The LC3B fluorescence intensity of the blastocysts was analyzed using the Zen software program (ZEN lite, 3.11, Zeiss) that is integrated into the fluorescence microscope.

Quantitative real-time polymerase chain reaction (RT-qPCR) analysis

Gene expression analysis was conducted using eight pools of 10 blastocysts per group³. The blastocysts underwent a freezing process in 1 ml of TRI Reagent (93289, Sigma-Aldrich), subsequently being stored at -80 °C in order to facilitate the isolation of RNA. Total RNA extraction was conducted using TRI Reagent, following the manufacturer’s protocol. RNA samples were treated with DNase-I (ThermoFisher Scientific, Germany) according to the manufacturer’s instructions to remove any DNA contamination. cDNA synthesis was performed using the iScript™ cDNA Synthesis Kit (170–8891, Bio-Rad, USA), and 1 µg of RNA obtained after DNase treatment was used for each sample. Primers (Supplementary Table 1) were obtained from a commercial supplier (B-Oligo, Turkey). Primer efficiency was calculated by combining cDNA samples. The Light Cycler Nano System (Roche Diagnostics, Germany) and the iTaq Universal SYBR Green qPCR Kit (Bio-Rad, USA) were utilized for the RT-qPCR process. The following components were incorporated into the reaction mixture: The master mix contained 10 µL, the forward and reverse primers 50 pMol each, and the cDNA 4 µL, creating a total volume of 20 µL. The steps applied for RT-qPCR are as follows: The temperature was set to 98 °C for 3 min, followed by 45 cycles of 15 s at 95 °C, 30 s at the annealing temperature, 30 s at 72 °C, and a melting program at a rate of 0.1 °C/10 seconds between 60 and 95 °C. The specificity of the RT-qPCR product target sequence was confirmed by analyzing band sizes on a 1% agarose gel. All reactions were performed in triplicate for each sample.

Statistical analyses

The analysis was conducted utilizing SPSS 25.0 (IBM Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.) statistical software. The Kolmogorov-Smirnov test was utilized to verify the prerequisites of normality of variables and homogeneity of variances. The relationships between categorical variables were analyzed using the Chi-square test. The results of the variables demonstrating a normal distribution were presented as the mean ± standard deviation (SD), and the One-way ANOVA post-hoc Tukey HSD test was employed to evaluate the variables.

Ct values obtained from RT-qPCR results performed with primers with primer activities between 1.9 and 2.2 were normalized using the 2-ΔΔCt method according to Livak and Schmittengen⁷³ criteria with the Ct value obtained for the specified reference gene (housekeeping). After normalization, the Anderson-Darling normality test was performed to determine whether the values showed a normal distribution. Intergroup differences were evaluated using ANOVA and Duncan’s test. Results with P < 0.05 were considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

HA: Conceptualization, Methodology, Formal analysis, Investigation, Project administration, Writing - Original Draft. FS: Methodology, Investigation. YED: Methodology, Investigation. GS: Methodology, Investigation. MHC: Methodology, Investigation. MFC: Methodology, Formal analysis, Investigation. HY: Methodology, Formal analysis. ETBK: Methodology, Investigation. BEG: Methodology, Investigation. OFY: Methodology, Investigation. SS: Methodology, Investigation. MAD: Methodology, Investigation. KKA: Conceptualization, Methodology, Investigation, Project administration, Writing - Original Draft, Writing - Review & Editing.

Funding

This work was supported by the Scientific Research Project Coordinator of Selcuk University [grant number 23401109] and The Scientific and Technological Research Council of Türkiye [grant number 124O204].

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All methods in this study were performed in accordance with the relevant guidelines and approved by the Local Ethics Committee (Selcuk University Local Ethics Committee for Animal Experiments, Approval Number: 2023/08/093).