Detrimental Effects of Selenium Nanoparticles on Growth and Development of Mice Embryos: An In-Vitro Study

Nanotechnology has numerous applications in medicine, diagnostics, and science. Over the last ten years, nanotechnology has significantly influenced reproductive techniques such as in vitro maturation, fertilization, and follicular oocyte culture. Selenium nanoparticles have substantial applications in numerous fields including disease prevention and treatment, mammary glands’ development, reproduction, and immune systems. In present study, in vitro trial was performed to examine the effect of selenium nanoparticles (SeNPs) on mice embryo growth and development . For this purposes, sixty (n= 60) female mice were divided randomly into three equal groups (n= 20) including control and two treatment groups. The treatment groups received SeNPs at doses of 5 and 25 µg/ml, respectively. During the experiment period, mice embryo growth and development, cleavage rate, and blastocyst rate were analyzed. After the collection of oocytes from female mice, insemination and in vitro fertilization were done to culture embryo through in vitro study. It was found that during the embryonic stages, the treated group showed no significant (P>0.05) differences compared to control group. Moreover, the blastocyst rate exhibited no notable differences. Whereas, the cleavage rate was significantly (P<0.05) decreased compare with control group. Altogether, the current study showed that exposing mice embryos to SeNPs had detrimental effects on the cleavage rate in


INTRODUCTION
N anotechnology has numerous applications in medicine, diagnostics, and science (Ray et al., 2009;El-Dawy et al., 2023;Elbehary et al., 2023).Over the last ten years, nanotechnology has significantly influenced reproductive techniques such as in vitro maturation, fertilization, and follicular oocyte culture (Albanese et al., 2012;Arguelles, 2022).The field of nanotechnology is concerned with particles with a dimension of less than 100 nanometers.Nanomaterials are also referred to as zero-dimensional nanomaterials.They are classified according to their properties, shapes, and sizes (Joseph et al., 2023;Elbehary et al., 2023), and are made up of a variety of chemical substances including nanocrystals of semiconductor, dendrimers made from organic materials, and fullerenes of carbon (Jeng and Swanson, 2006).Scientists are developing nanomaterials for biomedical applications such as cell labeling, drug delivery, gene therapy, biosensors, and hyperthermia therapy (Jeng and Swanson, 2006).
Magnetic nanoparticle can be used to detect metastatic lesions in lymph nodes because they can exit the systemic circulation through the permeable vascular epithelium (Jeng and Swanson, 2006).Many new doors in disease pathophysiology and treatment options have been opened with nanotechnology development of over the last three decades.Despite their profound use, nanoparticles could harm the living things in three ways: they directly interact with biological membranes, disintegrate and release harmful ions, and they increase oxidative stress (Aktas et al., 2023).Humans are generally exposed to nanoparticles by inhalation, ingestion, skin contact and injection (Ajdary et al., 2018).The size of nanoscale constructs is similar to that of biological macromolecules such as enzymes, receptors, and hemoglobin, but it is smaller than human cells and organelles.Blood vessel walls can be penetrated by nanoparticles smaller than 20 nanometers.
A variety of nanostructures of metals have been used

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as nanoparticles (Khurana et al., 2019), but selenium nanoparticles (SeNPs) are attracting much attention (Bhattacharjee et al., 2014).SeNPs can be produced by physical, chemical and biological techniques (Tan et al., 2018).Most of selenium compounds play a biological role by reducing oxidative stress in living cells (Jeng and Swanson, 2006).The antioxidant activity and redox regulation of selenium are carried out through selenoproteins (Chandramohan et al., 2018).In the body, biologically synthesized selenoproteins can exert reductive and oxidative effects on immune, reproductive, cardiovascular, and endocrine systems.Human bodies contain the greatest concentrations of selenium in the thyroid (Tan et al., 2018).
Selenium is a cancer-preventive chemotherapeutic agent.It also lowers the risk of heart disease and other cardiovascular conditions, slows the aging process, and prevents virus growth (He et al., 2014;Zafar et al., 2021;Nossier et al., 2022).Selenium, as an antioxidant, protects cells and tissues from damage by influencing the activity of seleno-enzymes such as glutathione peroxidase (Skalickova et al., 2017).Current research on the possible negative effects of SeNPs is largely debatable.According to some studies, SeNPs have strong antioxidant properties that inhibit oxidative stress and DNA damage (Bhattacharjee et al., 2014;Karami et al., 2018).SeNPs have also been shown to act as a chemo-preventive agent, reducing toxicity (Ray et al., 2009).Several studies, on the other hand, have revealed the genotoxic and cytotoxic effects and abnormal effects on embryonic development following SeNPs exposure (Ray et al., 2009).In female mice, maternal dietary exposure to SeNPs can result in offspring malformations (Hou and Zhu, 2017).Another study found that after exposing zebrafish oocytes to SeNPs only 50% of the embryos were viable (Shi et al., 2018).
In contrast, several studies have shown that SeNPs have beneficial effects on embryos.For instance, a recent study found that in buffalo SeNPs improve oocytes' nuclear maturation, regulate antioxidant defense genes, and reduced apoptosis (Hassan et al., 2023).In another study, it reduced the oxidative stress and DNA damage caused by Cyclophosphamide (CP) in mice's peripheral blood and bone marrow cells (Bhattacharjee et al., 2014).Furthermore, pretreatment with SeNPs was reported to alleviate the negative effects of gamma radiation that induces nephropathy in mice (Karami et al., 2018).The aim of this study was to determine the effects of SeNPs exposure on the in vitro mice embryos development, cleavage rate, and blastocyst rate.

Chemicals and reagents
Most of the chemicals were purchased from Merck (St. Louis, MO, USA).A dark powder SeNP, with a size of < 80 nm and 99.9% purity (CAS No. 7782-49-2) was obtained from Nanoshel company (West Valley City, Utah, USA).

Solutions and media preparation
Human tubular fluid (HTF) medium used for gametes collection, and fertilization was prepared freshly through two steps; the stock solution and the working solution.The stock solution was prepared by dissolving 594 mg NaCl, 35 mg KCl, 5 mg MgSO 4 •7H 2 O, 210 mg NaHCO 3 , 5 mg KH 2 PO 4 , and 60 mg CaCl 2 .2H 2 O in 100 mL of Milli-Q water.The working medium was prepared a day before the experiment by adding 370 μL D-glucose anhydrous, 68.4 μL Na-Lactate, 68 μL Na-pyruvate, 20 μl penicillin, 20 μL streptomycin, and 80 mg bovine serum albumin to 20 mL of the stock solution.The final step was to optimize the pH for the media at 7-7.5 then the medium was filtered by 0.22 µm Syringe filter into a 15 ml sterile centrifuge tube and the media was stored for two weeks at 4 °C and used only in this period of time (Arroyo-Salvo et al., 2019).KSOM medium used for embryos culture was freshly prepared.The stock solution was prepared by dissolving 560 mg NaCl, 19 mg KCl, 5 mg KH 2 PO 4 , 5 mg MgSO 4 .7H 2 O, 210 mg NaHCO 3 , and 25 mg CaCl 2 .2H 2 O) in 100 mL of Milli-Q water.The working medium was prepared by adding 20 μl Na-pyruvate, 10 μL L-glutamine, 10 μl EDTA, 100 μl Ess.AA50X, 50 μL NonEss.AA100X, 8.4 μl penicillin, 10 μL streptomycin, 10 mg bovine serum albumin, 17.4 μl Nalactate, and 13.3 μL D-glucose anhydrous to 10 ml of the stock solution (ALRashd et al., 2023).

Characterization and preparation of selenium nanoparticles
To prepare the aqueous solution of nanocrystalline selenium, the nano-powder was dissolved in Milli Q water to get a stock concentration of 1 mg/ml.To avoid nanoparticle agglomeration, the suspension was sonicated using an ultrasonic for 15 min at 40 W before exposure to the gamete cells.After the sonication process, a suitable volume of the stock aqueous nanocrystalline selenium was added to the sperm, in vitro fertilization (IVF), and in vitro culture (IVC) dishes (El-Naby et al., 2020).Nanoparticle was described and characterized by using various microscopic and spectroscopic methods (KSU, Saudi Arabia).The particle size, morphology and structure were determined by using X-Ray diffraction (Bruker, Karlsruhe, Germany) and Scanning Electron Microscopy (JEOL, Tokyo, Japan)

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in conjunction with Energy Dispersive X-ray spectroscopy (EDX) according to previous study (Mourdikoudis et al., 2018;Samy et al., 2022;Liaqat et al., 2023).

Oocyte recovery
Sixty adult female Balb/c mice (4-8 weeks old) were used to obtain a sufficient number of oocytes for IVF.Superovulation was induced by injecting female mice intraperitoneally with 5 IU of equine chorionic gonadotropin (eCG), followed by 5 IU of human chorionic gonadotropin (hCG) 48 h later in order to obtain mature oocytes from the oviducts.Next, 15-17 h after the hCG injection, the mice were euthanized by exposing to 80% CO 2 / 20% O 2 for 120 s in a sealed chamber, and the oviductal ampullae of each mouse were collected and placed in a petri dish containing drops of HTF medium covered by paraffin oil previously adjusted in an incubator with 5% CO 2 at 37°C.Then, the ampulla was opened and the cumulu oocytes complexes (COCs) were dragged into the HTF medium drops, which contained an appropriate volume of SeNPs stock in the two treated groups (Takeo and Nakagata, 2018).

Collection of spermatozoa
Fertile male mice (3-6 months old) were used as sperm donors in general.The mice were euthanized by exposing to 80% CO 2 , 20% O 2 for 120 s in a sealed chamber.In order to obtain sperm, each animal's caudal epididymis was severed and transferred to the sperm collection dish, which contained drops of HTF covered by paraffin oil, and previously adjusted in an incubator with 5% CO 2 at 37°C.Sperm cell concentration was adjusted to the appropriate volume and final concentration of ten million sperm /ml.Then a suitable volume of the SeNPs stock solution was added to the IVF medium to adjust the desired concentrations.After that, it was incubated for 60 min at 37°C under 5% CO 2 to induce sperm capacitation.

Insemination and in vitro fertilization
The fertilization dish was incubated at 37℃, 5% CO 2 for overnight to allow fertilization medium equilibration.Before insemination, the sperm cell concentration was determined by hemacytometer and sperms were further incubated for 60 min at 37℃ under 5% CO 2 to induce sperm capacitation.Finally, the appropriate number of sperms was transferred to the COCs drop on the fertilization dish to a final sperm cell concentration one million sperm/ml.After insemination, the fertilization dish was incubated at 37℃, 5% CO 2 in air for 6 h (Takeo and Nakagata, 2018).Finally, the fertilization process was evaluated under an inverted microscope after 4-6 h by observing the second polar body and two pronuclei.

Embryo culture
After insemination, all zygotes were transferred to the washing dish containing HTF supplied with 300 µg/ml of hyaluronidase enzyme to remove cumulus cells.After that, the zygotes were washed twice by HTF to remove excess enzyme.All the oocytes were transferred from the washing dish into the IVC dish containing KSOM media and then placed in 5% CO 2 incubator at 37°C.

Examination of the growth and development of mice embryos
The growth of the embryos was monitored during the 5 th day of culturing and the cleavage rate and blastocyst rate calculated.Then, the embryos were collected and stored in 5 µl of 0.1% PVA-PBS at −80 °C.

Statistical analysis
Statistical analysis of data from at least four replicates for each treatment was carried out by using SPSS.Data normality was first evaluated using the Shapiro-Wilk test.The means of cleavage rates, different stages of in vitro development, and blastocyst rates in all groups were compared by ANOVA.All the values are presented as Mean ± SEM, while P ≤ 0.05 is for significance.

Analysis of selenium nanoparticles using XRD, SEM, and EDX
XRD, EDX, and SEM were used to investigate the size, structure, and purity of selenium nanoparticles.The SeNPs XRD analysis was carried out in the 2 range of the spectrum, and the diffraction peaks correspond to the standard JCPDS data (JCPDS card No. 01-086-2246) (Fig. 1A).The average calculated crystalline size of SeNPs was found to be 58 nm approximately (Fig. 1B).The presence of Se with ultra-high purity was confirmed through EDX analysis (Fig. 2A).SEM image indicated the presence of only one phase, implying the purity of the Se nanoparticles (Fig. 2B).

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A B  Data between parenthesis indicated percentage (%) and the mean number of embryos replicates ± SEM.Superscripts that were similar indicated that the difference was not statistically significant.Different superscripts mean significant differences as indicated by (P ≤ 0.05).

In vitro fertilization and culture
To determine the toxicity of SeNPs, mice embryos were exposed to 58 nm SeNPs at two different concentrations (5 and 25 µg/ml).Data regarding the effect of selenium nanoparticles on the embryonic development of mice embryos was presented in Tables I, II and Figure 3.The experiment recorded neither significant differences in blastocyst rate nor means of embryonic development stages of mouse oocytes across groups.As shown in Table I and Figure 4A-D, the results indicated that the embryonic stages include both the early stages and the late stages exhibited no significant differences

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Detrimental Effects of Selenium Nanoparticles Data between parenthesis indicated percentage (%) and the mean number of embryos replicates ±SEM.Superscripts that were similar indicated that the difference was not statistically significant.Different superscripts mean significant differences as indicated by (P ≤ 0.05).between treated groups with concentrations of 5 µg /ml and 25 µg /ml compared with control group.As shown in Table II and Figure 4E, the blastocyst rate did not differ significantly in 5 µg /ml (0.72 ± 0.037) and 25 µg /ml (0.78 ± 0.036) concentrations in comparison to the control group (0.72 ± 0.067).Our results also showed that there was a significant difference (P<0.05) in cleavage rate between treatment groups and the control group.In SeNPs treated groups, the cleavage rates were lower than in the control group.Table II, and Figure 4F revealed that the cleavage rate was significantly (P<0.05)reduced in the groups that were exposed to 5 µg /ml (0.525 ± 0.024) and the 25 µg/ ml group (0.45 ± 0.044) compared with the control group (0.655 ± 0.041).

DISCUSSION
In the past few years, the need for in vitro fertilization has been considerably increased.Despite their beneficial applications, there are some factors that limit the success of in vitro fertilization.The use of selenium as nanoparticles instead of selenium is due to its physical and chemical properties, which is distinguished from material properties in its large size.As a result, it has played a significant role in a variety of fields that benefit humans.Therefore, there is a noticeable increase in the use of this technique in the field of research.It has been shown that inflammation, necrosis, ROS, and apoptosis play key roles in NPs' toxicity mechanisms.The dose of NPs through intravenously injection are more toxic than those administered to the skin (Bhattacharjee et al., 2014;Ullah et al., 2022Ullah et al., , 2023)).SeNPs provide improved biological availability with an additional feature of low toxicity (Khurana et al., 2019).This study was motivated by the need for comprehensive scientific data on the role of selenium nanoparticles on the growth and development of mice embryos in vitro.
The SeNPs were thoroughly characterized to validate their purity and structure using different analytical techniques, including XRD, EDX, and SEM.The characterization results indicated the SeNPs' ultra-high purity.In addition, the size of SeNPs was found to be 58 nm, which is consistent with the previous study on SeNPs with sizes around 20-80 nm (Alagesan and Venugopal, 2019).For the first time, the current study evaluated the putative effect of adding the SeNPs in the culture media of mouse embryos in vitro.The experiment did not record any significant changes in blastocyst rate and means of embryonic development stages of mice oocytes in different groups.Our findings also demonstrated that a significant difference was observed in cleavage rate between treatment groups compared with the control group, where the cleavage rate was lower in the SeNPs treated groups than the control group, which indicated that the 58 nm selenium nanoparticles have a cytotoxic effect in the culture media of mice embryos.Our data is in line with previous studies that clearly indicated that when fish were exposed to SeNPs (5-25 mg/mL) at size 100-200nm and sodium selenite (5-25 mg/mL) before 48 h post-fertilization, SeNPs seemed more toxic than sodium selenite (Bano et al., 2022).The most common malformations observed were pericardial edema and tail malformations when embryos were incubated with SeNPs at 5-25 mg/mL.It was found that only half of the zebrafish embryos were viable after treatment with 25 mg/ ml of SeNPs (Kalishwaralal et al., 2016).The results of the present work are also consistent with previous findings (He et al., 2014), who showed that several abnormalities were observed in zebrafish embryos after treatment with Hollow selenium nanoparticles (hSeNPs) of various concentrations ranging from 10-50 μg/ml, including abnormal heartbeat, oedema of the embryo sac, oedema of the eye, and swelling of the head.The results agree with previous studies on SeNP antioxidant activity, high concentrations of hSeNPs showed high antioxidant activity (Torres et al., 2012).In previous study (Shi et al., 2018), a comparison was made between elemental selenium nanoparticles (SeNPs), selenite, and selenomethionine (Se-Met) and the results showed that maternal dietary exposure to SeNP was associated with significant malformations in offspring along with other common Se species.These findings further support the idea of that NPs can enter the fetus via passive diffusion and cause detrimental effect in fetus (Hou and Zhu, 2017).Furthermore, treatment of embryos with 50 M nanoAg in vitro led to increased resorption and decreased fetal weight (Grumezescu, 2018).Other nanoparticles were studied on mice like ZnO-NP which caused cytotoxic on mouse ovarian germ cells based on concentration and time.Increasing ROS levels were observed when cells were exposed to ZnO-NPs, and premeiotic germ cell markers were significantly increased but meiotic and postmeiotic markers were decreased compared to untreated cells (Farroh et al., 2020).
In other study CeO 2 nanoparticles affected the testis tissue, sperm parameters in mice, (Hosseinalipour et al., 2021).On the other hand, our results differ from some published studies (Ray et al., 2009), they studied the effects of SeNPs on Swiss albino mice in vitro, and the results showed that the activity of antioxidant enzymes increased, causing less bone marrow cell death and prevented DNA damage, it might act as a potential chemo-preventive.A study has shown that the SeNPs at supra-nutritional levels were not toxic to rats and could potentially be used as cancer chemo-preventive agents (He et al., 2014), although doses greater than 2.0 mg Se/kg b.wt.caused chronic toxicity.Previous study also evaluated the effect of SeNPs and bulk selenium on buffalo oocytes maturation in vitro (El-Naby et al., 2020).This finding has been shown that Se and SeNPs (40 nm) had a positive effect on oocytes nuclear maturation rates via regulation the expression of development competence and antioxidant defense gene.It is proved that the SeNPs have been marketed as antiapoptotic effecting human lymphocytes that exposed to UVB radiation in vitro experiments, selenium nanoparticles in this case may be helpful in reducing reactive oxygen species (Prasad et al., 2013).Kalishwaralal et al. (2016) identified optimal concentrations of SeNP for potential therapeutic applications.Researchers found that SeNPs at 5-10 μg/ml may provide an economical method of treating cardiovascular diseases.

CONCLUSION
In the current study, we studied the putative impact of selenium nanoparticles on the growth and development of mice embryos in vitro.Based on the results of the present study we can say that mice embryos that supplemented by 5 µg/ml or 25 µg/ml SeNPs in size of 58 nm at concentration exhibited a reduced cleavage rate and consequently embryonic development.It led to minor toxic effects and abnormal embryo development.Eventually, SeNPs have a very narrow margin between beneficial and drawbacks effects.Moreover, studies based on both in vitro and in vitro models are needed to identify the mechanisms of nanoparticle toxicity.

Funding
The authors extend their appreciation to Researchers Supporting Project number (RSPD2023R965), King Saud University, Riyadh, Saudi Arabia for funding this research.

IRB statement and ethical approval
This research work belongs to master student which was approved by supervisory committee.Furthermore, all animal experiments took place following the ethical approval number KSU.SE-21-37 dated in 27-05-2021 from the King Saud University (KSU), Riyadh, Saudi Arabia.

Statement of conflict of interest
The authors have declared no conflict of interest.

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Detrimental Effects of Selenium Nanoparticles

Fig. 4 .
Fig. 4. Effect of selenium nanoparticle on the number of embryos in two-cell stage (A) four-cell stage (B) morula stage (C) blastocyst stage (D) blastocyst rate (E) and cleavage rate (F) development in various experimental groups.Similar letters indicated that the difference was not statistically significant.Different letters mean significant differences (P ≤ 0.05).