Developmental competence of bovine oocytes in maturation media supplemented with follicular fluid exosomes #

The present study evaluated the role offollicular fluid exosomes on the developmental competence of bovine oocytes. Ovaries from slaughtered crossbred cattle were collected, all visible surface follicles aspirated, and culture-grade oocytes were subjected to further study. Exosomes were isolated from bovine follicular fluid by differential ultracentrifugation. A total of 358 oocytes selected for the study were randomly divided into two groups. Group I constituted 111 oocytes, in which normal maturation was carried. Group II constituted 247 oocytes, in which in vitro maturation (IVM) medium was supplemented with follicular fluid exosomes at 1 µL/100 µL IVM medium. Maturation was assessed after 24h of culture in a CO 2 incubator maintained at 38.5°C in 95 per cent humidified atmosphere of 5 per cent CO 2. Following IVM of oocytes for 24 h, in vitro fertilisation (IVF)was carried out by co-incubating with capacitated spermatozoa for 18 h and embryo culture was carried out subsequently. In group II oocytes supplemented with exosomes, a significantly higher maturation rate (p ≤0.01) (95.80 ±1.67 vs76.10 ± 0.95), fertilisation rate (53.68 ± 3.02vs37.85 ± 7.01) and cleavage rate (p ≤0.01) (43.66 ± 2.13vs(32.47 ± 5.23) was noticed compared to oocytes in group I without any supplementation.The present study established that supplementation of follicular fluid exosomescould improve the developmental competence of bovine oocytes.


Running title:Developmental competence of bovine oocytes in maturation media supplemented with follicular fluid exosomes
In vitro embryo production technology (IVEP) in bovines has received great attention during the last few decades. Even though IVEP is established, the efficiency of embryo productionis still limited to 30-40 per cent (Saeki et al., 1991).Since the early days of embryo culture in vitro, various modifications were done to create the best microenvironment that simulated the biological system, to attain optimum embryo yield (Leibfried-Rutledge et al., 1987). Even now, the developmental competence of invitro cultured embryos is markedly lower due to the lack of physiological pathways and essential components in culture system as the embryonic development is highly susceptible to heat stress in in vitro culture environment which disrupts the success of fertilization by affecting the anti-polyspermy system of oocytes (Sakataniet al., 2015).
Extracellular vesicles,found in the follicular fluid are nanoparticles released by cells that facilitate cell-to-cell contact in the follicle microenvironment (de Avila and da Silveira,2020). Early endosomes are first produced by endocytosis within cells. As they develop into late endosomes, some intraluminal vesicles (ILVs) are shed by endosomes to form multivesicular bodies (MVBs), which then move to the cell membrane. The MVBs release the vesicles inside the extracellular milieu as exosomes after fusing with the plasma membrane (Chuo et al., 2018). Exosomes, with the size of 30-200 nm are termed as molecular cargo as they carry lipids, proteins, mRNA, miRNA and DNA and are involved in intercellular and intracellular communication (Choi et al., 2013). They are extremely stable, may cross the blood-brain barrier, and so open up promising possibilities for diagnostics and therapeutic interventions (Aryani and Denecke, 2016).
The "gold standard" for isolating exosomes is density gradient centrifugation, which is a variation of ultracentrifugation (Iwai et al., 2016). Bovine follicular fluid exosomeswere reported to have the potential to enhance the maturation and fertilisation of oocytes and development to the blastocyst stage (Bridiet al., 2020). These extracellular vesicles have been investigated as biomarkers and supplementing tools to replicate natural circumstances during assisted reproductive techniques. In view of the above observations,the present study was concluded to evaluate the effect of supplementation of bovine follicular fluid exosomes on in vitro maturation, cleavage and fertilisation of bovine oocytes.

Procurement of oocytes
Bovine ovaries of unknown reproductive status were collectedfromthe Corporation Slaughterhouse, Thrissur in normal saline fortified with Penicillin (100 IU/ mL).The extra ovarian ligaments as well as excess tissues adhering to the ovaries were trimmed off using scissors. Later, ovaries were washed in normal saline 8 to 10 times until they were stain-free and maintained at 36-38°C until aspiration of oocytes were completed. All visible surface follicles between 2 and 8 mm in size were aspirated, collected in a 10 mL test tube, and left undisturbed in the incubator for 10 min to settle. Later, sediment from the bottom was pipetted out, andcumulus oocyte complexes (COCs) were morphologically gradedas A, B, C, or D under a stereozoom microscope (Ceticaet al., 1999).

Isolation of exosomes
Follicular fluid aspirated from ovaries using 20 guage needle wasstored in the ultradeep freezer (-80°C) until the desired quantity is obtained for ultracentrifugation. For each batch of follicular fluid, a total of 16 mL was thawed to room temperature, diluted with an equal volume of phosphate-buffered saline (PBS), and centrifuged in a cooling centrifuge at 800g for 10 min, 2000g for 20 min and 12000g for 45 min at 4°C and the supernatant was filtered through 0.2µm syringe filter. Filtered supernatant was subjected to ultracentrifugation (Sorvall WX Ultra Series Centrifuge -Thermo Fisher Scientific) at 110000g for 3 h at 4°C using swinging bucket SW30Ti rotor (SuperspinSorvall). The pellet containing exosomes was resuspended in 200 µL of sterile PBS and was stored at -80°C.

Experimental design
A total of 358 culture-quality oocytes of Grades A and B were selected for the study. Group I consisted of 111 oocytes in which maturation was carried out by standard protocol and group II (n=247) maturation was carried out by supplementing exosomes @ 1 µL/100 µL IVM medium. Cumulus oocyte complexes were identified using stereozoom microscope and transferred to the washing medium. After serial washings culture quality oocytes (grades A and B) were transferred to maturation droplet in a ratio of 10-20 oocytes per 100 µL of maturation droplet.
The maturationmedium consisted of TCM-199 (HEPES modified) supplemented with FSH, estradiol-17β, sodium pyruvate, L-glutamine, gentamicin sulphate and FBS. The culture condition set for the study was 38.5°C in 95 per cent humidified atmosphere of 5 per cent CO 2. Maturation was assessed after 24 h of culture by cumulus cellexpansion and first polar body extrusion. Fertilisation was carried out using frozen-thawed semen. Sperm oocyte coincubation was carried out for a period of 18 h. The presumptive zygotes were then transferred to culture droplets after 18 h of incubation. The culturemedia used for the study was Vitrogen (IVC-0420220A). Cleavage and fertilisation were evaluated 48 h post-insemination under an inverted microscope at 40x magnification (Rizos, 2002).

Results and discussion
Out of the total 125 bovine ovaries of unknown reproductive statusfrom the slaughterhouse, 60 ovaries were subjected to follicular aspiration for oocyte retrieval and 65 ovaries for follicular fluid aspiration. A total of 823 visible surface follicles having 2-8mm diameter were aspirated. The meannumber of follicles aspirated per ovary in the present study was 13.71 ± 2.16. The result was comparable with that of Garcia and Salaheddine (1998) who obtained 12.4 ± 6.1 follicles per bovineovary and was lower than the observations of Fitzpatrick and Entwistle (1996) who observed 26.40 ±1.60follicles per ovary. The difference in the number of follicles couldbe due tothe presence of CL in the ovary as it exerted a negative impact on the number of follicles and oocytes recovered (Singhet al., 2001) and variations in the age of animal, breed, climatic conditions, nutritional, genetic and reproductive status of the animal at the time of slaughter (Sianturiet al., 2002).
The recovery rate of oocytes obtained in the present study was 68.73 ± 2.71 per cent. The oocyte recovery rate found in the study was in accordance with the observations made by Singh et al. (2001), who obtained a recovery rate of 67 per cent. Aryaet al. (2019) obtained a higher yield of culture-grade oocytes of 78.27 ± 2.08 per cent. The difference in the quality of oocytes obtained in the present study compared with other findings might be due to variability in size and functional status of the follicle, age, season, nutritional status, health condition of the animal and stage of the oestrous cycle at the time of slaughter (Nandi et al., 2002;Sianturiet al., 2002).
The yieldof follicular fluid per ovary in the present study was 0.82 ± 0.07mL. The result was comparable with that of Grimes and Ireland (1986) and Hazeleger (1995) who found that the volume of follicular fluid collected from each ovary ranged from 0.04 to 2.4 mL and 0.03 to 2.3 mL, respectively. The volumeof follicular fluid aspirated depends on the size of the follicles, skill of the person, pressure exerted and gauge of the needle used for aspiration.

Cumulus cells refer to a group of cells that surround and nourish the oocytes.
Cumulus expansion mainly occursas a result of extracellular matrix synthesis of  In the present study, cumulus expansion was found to be greater in exosome supplemented group compared to the control group. Also, there is a significant difference noticed in the IVM rate. It clearly depicts that exosome supplementation in IVM medium is having a positive influence in increasing the cumulus cell expansion and maturation rate. During in vivo, at the time of oestrus, the surge of luteinizing hormone triggered ovulation and increased the expression of genes that support cumulus cell expansion. They areprostaglandinendoperoxide synthase 2 (Ptgs2), pentraxinrelated protein 3 (Ptx3), and tumour necrosis factor alpha-induced protein 6 (Tnfaip6) (Hung et al., 2015). The theca and mural granulosa of antral follicles contain the majority of LH receptors, whereas early cumulus cells do not. It is known that bidirectional communication takes place across the antral follicular fluid between the mural granulosa and the cumulus-oocyte complex (COC). Accordingly, the luteinizing hormone (LH) surge promotes the release of mural granulosa epidermal growth factor (EGF) ligands, which must then pass through the follicular fluid to activate the cumulus cells in order to cause the COC to expand and the typical alterations in gene expression. Thus, when bovine oocytes are supplemented with follicular fluid exosomes, it resulted in increased cumulus cell expansion and maturation rate (Hung et al., 2015).An increased maturation rate of significance in exosome supplemented oocytes compared to the oocytes in the nonsupplemented medium corroborates to the findings of da Silvieraet al. (2017) and Lopez et al.(2019) who observed that supplementation of exosomes enhanced cumulus cell expansion and in vitro maturation rate of bovine oocytes.
The fertilisation rateand cleavage rateof exosome supplemented oocytes in the present study showed a significant difference incomparison with the control group. Extracellular vesicles (EVs) have a significant role in fertilisation and the early stages of embryonic development and are found in the oviductal and uterine fluid. Small EVs from the oviduct and endometrium can be taken up by embryonic cells, and EVs from the embryo can alter ovarian and uterine function (Bridiet al., 2020). According to Quet al. (2017) supplementation of exosomes to the culture medium significantly improved blastocyst formation and quality as well as their growth using bovine somatic cell nuclear transfer (SCNT) embryos as a model system. They found that during in vitro culture, SCNT embryos are capable of secreting exosomes into the culture medium which is essential for an increase in blastocyst formation rate, total cell numbers of the blastocyst and ratio of inner cell mass/ trophectoderm. Exosomes secreted by earlystage embryos canincrease the expression of early pleuripotent genes like Oct4, Sox2, Klf4, c-Myc, and Nanog(de Avila and da Silveira., 2020).
The results of the present study signify that exosome termed molecular cargo, contain mRNA, miRNA, lipids, proteins and DNA involved in both intercellular and intracellular communication. This communication of exosomes within and between cells could have resulted in increased cumulus cell expansion, maturation, cleavage and fertilisation rate (Bridiet al., 2020).

conclusion
From the above findings, it can be concluded that the supplementation of exosomes in the IVM mediumhas a positive effect on the developmental competence of bovine oocytesas signified by increase in the cleavage and fertilisation rate in the exosome-supplemented group. Hence,the presentstudy reportsthat the efficiency of IVEP could be increased by incorporating follicular fluid exosomes in the IVM medium of bovine oocytes.

acknowledgements
The authors are thankful to the Dean, College of Veterinary and Animal Sciences, Mannuthy, Kerala Veterinary and Animal Sciences University for providing the facilities needed for carrying out this research work.