The effects of supplemented sericin on in vitro maturation and preimplantation development of mouse embryos: An experimental study

Abstract Background Mouse embryo culture condition is an essential part of transgenic, reproductive and developmental biology laboratories. Mouse embryonic culture media may have a high risk of serum contamination with pathogens. Objective To investigate the effect of sericin as an embryo culture medium supplement on in vitro maturation (IVM), in vitro fertilization (IVF), and development of the preimplantation embryo in mice. Materials and Methods The effects of sericin at three concentrations (subgroups) of 0.1%, 0.5%, and 1% as a medium supplement on IVM, IVF, and in vitro development of mouse embryos were separately investigated and compared with a sericin-free (control) group. The cumulative effect of the three concentrations was evaluated for IVM + in vitro development and IVF + in vitro development as follow-up groups. Results In the IVM group, compared to the control group, the number of oocysts reaching the MII stage was significantly higher when 1% sericin was used (161/208 = 77.4%). No significant results were observed in the IVF and in vitro development groups with different concentrations of sericin compared to the control group. Among the follow-up groups, in the IVM + in vitro development group, the number of oocytes was higher after passing the IVM and IVF and reaching the blastocysts stage when 1% sericin was used, compared with other sericin subgroups. A significant difference was also noted when compared with the control group (p = 0.048). The IVF + in vitro development study group, on the other hand, did not show any significant relationship. Conclusion It can be concluded that 1% sericin can be used as a supplement in mouse embryo cultures to improve the IVM rate. Also, based on the findings, sericin appears to be an effective supplement which can have a positive effect on the development of embryos derived from IVM.


Introduction
In vitro culture of murine embryos has traditionally been a two-step process of retrieval and cultivation, requiring appropriate unique media for culture conditions. Embryos are routinely harvested from oviducts and placed into an embryo culture medium. In the next step, the embryos are transferred into a bicarbonate-based buffered medium placed in a culture incubator (1).
Different types of primary culture media used for in vitro embryo culture are often provided with sera such as fetal bovine serum or bovine serum albumin for the preparation of growth factors, amino acids, hormones, and proteins which are useful for embryonic development (2). The high risk of sera contamination with pathogens, prions, viruses, and bovine spongiform encephalopathy, etc. has prompted researchers to try to find alternative supplements that can be used instead of serum during in vitro embryo culture (3,4). Dash and colleagues found that sericin had an antioxidant effect on skin fibroblast cells exposed to hydrogen peroxide. They showed the potential to inhibit the production of intracellular hydrogen peroxide in ultraviolet-treated keratinocytes (5).
Sericin is a water-soluble silk protein extracted from cocoons and it is the second major protein component (besides fibroin) of silk used in biological materials due to its anti-UV and antibacterial properties. Sericin is composed of 18 types of amino acids as well as polar side groups such as carboxyl, hydroxyl, and amino groups and it is rich in serine and aspartic acid (6).
Izobe et al. found that culturing two-cell embryos separately for seven days in an environment with 0.5% sericin resulted in the highest rate of blastocyst formation and development into expanded blastocysts. It has been shown that preimplantation development and the quality of cultured bovine embryos increased following the addition of sericin to an in vitro medium, which prevented oxidative stress (7). It was reported that supplementation of a medium with 0.1% sericin during in vitro maturation (IVM) improved the nuclear maturation and fertilizability of sheep oocytes. Also, because of its antibacterial and UVresistant properties, as a biomaterial, it may replace bovine serum albumin in chemical environments without risk of disease transmission. Several studies have suggested the benefits of using sericin as a protein replacement (8). Also, some studies have suggested that sericin can have a role in cryoprotection in the freezing of mice, human, and buffalo sperm (9)(10)(11).
The present study is the first to investigate the effect of 0.1%, 0.5%, and 1% sericin as a medium supplement on IVM, the rate of in vitro fertilization (IVF), and in vitro development of mouse embryos.

Collection of MII oocytes and IVF
IVF was performed as described previously with minor modifications (12). PMSG was injected into female mice that were 4-6 wk old. After 48 hr, human Chorionic Gonadotropin (hCG) was injected, and oocytes were collected from mouse oviducts 14 hr post the hCG injection.
Then, the zygotes were monitored by a reverse microscope and the percentage of two-cell formation was recorded to assess the rate of fertilization (Table I).

Collection of zygotes and in vitro development
After the PMSG and hCG injections, the injected female mice mated with male mice.

Follow-up from IVM to the blastocyst stage
For assessing the cumulative effect of sericin as an embryo culture supplement, we used the

Follow-up from IVF to the blastocyst stage
To

Statistical analysis
In this experimental study, the IVM and IVF   (Table II).

The effect of sericin as an embryo culture supplement on mice embryo culture from GV oocytes to the blastocyst stage (IVM + in vitro development)
In this study, to assess the cumulative effect of sericin as an embryo culture supplement, the effect of 0.1%, 0.5%, and 1% sericin on the development of MII oocytes from the IVM study group to two-cell embryos (after IVF), and finally to the blastocyst stage was evaluated. The one-way ANOVA results revealed a significant relationship between HTF in which 1% sericin was added as a supplement and the follow-up of GV oocytes to blastocysts. Importantly, 0.1% and 0.5% sericin did not have significant effects (Table II).

The effect of sericin as an embryo culture supplement on IVF of MII oocytes to the blastocyst stage (IVF + in vitro development rate)
The cumulative effect of sericin as an embryo culture supplement on IVM and then on the development to blastocysts was examined in the IVF + in vitro development study group.
It was found that the rate of in vitro-fertilized MII oocytes reaching the blastocyst stage was not significantly different in the 0.1%, 0.5%, or 1% sericin subgroups than in the control group (Table II  In another study, adding 0.5% sericin to the culture medium increased the blastocyst rate compared to the control group without sericin and acted as an alternative protein supplement for IVM of bovine oocytes and zygotes (4). In addition, some researchers have found that adding 0.5% sericin to an in vitro culture medium improves oxidative stress, pre-implantation progression, and the quality of embryos (7). In another study, it was found that supplementation with sericin during

Conclusion
It can be concluded that the medium supplemented with 1% sericin improved the IVM and development of MII oocyte-derived embryos to blastocysts in the mouse model. To ensure optimal concentration, more investigations are needed.