Effect of Vitrification on Folliculogenesis-Related Genes in Ovarian Follicles of Bubalus bubalis

Diverse advancements have been made to improve infertility, as current iatrogenic treatments are damaging reproductive systems. To preserve the fertility of affected population, cryopreservation can be used as notable option. Cryobiology in reproduction has revolutionized applications to preserve the genetic material of rare breeds, endangered species or transgenic animals. Additionally, bovine are serving as the best model to study human folliculogenesis, due to their similar ovarian physiology than rodent’s model (Sirard, 2017).
       
Various advancements have been made in cryostorage of oocytes/embryos and sperms, but still their survival rate are a major task to be sort. Alternative to cryopreservation, of oocytes/embryos, ovarian follicles are more resistant to cryoinjury due to fewer support cells around the small oocyte without zona pellucida, low metabolic rate and a small amount of cold-sensitive intra cytoplasmic lipid. Another important fact to this cryostorage is that the primordial pool is arrested at the prophase of meiosis I with fewer modifications, which makes them less vulnerable as compared to matured oocytes. Advantage to this cryopreservation, follicles present are large in number as well as they are available in gonads at all ages, allowing their in vitro activation and development to ovulatory stage. As each species has its own particulars in terms of cryopreservation protocol; insufficient data available regarding cryopreservation of buffalo ovarian follicles hinders to standardize a single protocol. Since decades, storage of female genetic sources has emerged by the means of conventional freezing. Major hurdle to this cryopreservation is the formation of ice crystallization at low temperature, resulting in the deformation of cells (Lee et al., 2019). Vitrification is an alternative to this method, highly recommended, which includes rapid cooling and glassy transformation eliminating ice crystallization with use of appropriate concentration of cryoprotectants (CPAs).
       
Beside numerous advantages of cryopreservation, there are some inevitable side effects which could result in genetic variations leading to the impairment of cellular activity after thawing. Fig 1 indicates the number of genes responsible for the normal functioning of folliculogenesis, includes FOXO3, WNT4, NLRP5, SF1, VEGFA, HAS2 (Ernst et al., 2018). FOXO3 is important to maintain dormancy factors and also expressed in granulosa cells at different stage of follicular development which is crucial for oocyte maturation (Yamamoto et al., 2017). WNT4 is required to maintain the antral growth and development of oocyte by regulating follicular cells (Boyer et al., 2010). NLRP5 is a maternal gene specific to oocyte and follicular cells and essential for normal early embryonic development (Sena et al., 2009). SF1 is a key transcriptional regulator gene for growing follicles (Logan et al., 2002). VEGFA and HAS2 interact in development of cumulus-oocyte complex (Cadenas et al., 2017).
 

       
As ovarian follicles consist of many cells, a synchronized communication between oocyte and its surrounding follicular cell is responsible for expression of various important activities, which maintain the morphology and function of follicles at different stages of growth. To study these activities, the present study was designed to focus on the gene expression, evaluating the variation of vitrification individually in primary, secondary and tertiary follicular stages. The present study, also measure the physical and chemical properties, includes histo-architecture and cytotoxicity analysis produced through vitrification in follicular cells estimated through MTT assay.

Chemicals used in the present study were acquired from Sigma Chemicals (St Louis, USA) if not otherwise stated. Plastic wares were purchased from Nunc (Roskilde, Denmark). Buffalo ovaries were obtained from the Delhi abbatoir (according to institutional ethical guidelines). The experiments were performed at Embryo Biotechnology Lab, Animal Biotechnology Centre, ICAR-National Dairy Research, Karnal, Haryana.
 
Vitrification and thawing protocol for ovarian cortical tissues
 
The buffalo ovarian cortical pieces of 1 mm thick were divided into two groups: control and vitrified. The ovarian cortical tissues were cryopreserved in 1.8 ml cryovials (5-7 pieces per vial). Two vitrification solutions (VS), both based on holding medium (HM) consisting of tissue culture media 199 (TCM-199) supplemented with 20% fetal bovine serum (FBS). In first vitrification solution (VS1), contained 8.5% dimethyl sulfoxide (DMSO) and 8.5% ethylene glycol (EG), incubated for 10 min at room temperature. The second vitrification solution (VS2), included 16.5% DMSO, 16.5% EG and 0.1 M Sucrose, incubated for 10 min and then finally stored in liquid nitrogen (LN₂) along with VS2 in cryovials.
       
For thawing, the cryovials were taken out of LN₂ and plunged into water at 37°C. Further cryovials were directly emptied into VS2, VS1 and HM sequentially for 10 min before isolation of follicles through mechanical dissection.
 
Isolation of ovarian follicles
 
Ovarian follicles were isolated mechanically from cortical pieces of control and vitrified buffalo ovaries. Primary follicles (0.1 mm-0.2 mm) with single layer of cuboidal follicular cells surrounding oocyte (Fig 2A), secondary follicles (0.2 mm-0.5 mm) with two or more layer of cuboidal follicular cells around oocyte (Fig 2B) and tertiary follicles (0.5 mm-1 mm) with circular follicular cells and prominent oocyte (Fig 2C) were isolated in isolation medium (tissue culture medium 199 (TCM-199) supplemented with 0.3% bovine serum albumin (BSA), 2 mM L-glutamine, 50 µg/ml gentamicin sulfate). Isolated follicles were stained with neutral red (15 µg/ml) for 30 min, which have taken red color were considered to be viable and survivability rate was evaluated (Fig 2D).
 

 
Histological and morphologic assessment
 
Control and vitrified buffalo ovaries (n=7 each) were dehydrated in ethanol and washed in xylene. Sequentially, tissues were fixed using paraffin wax and sectioned at 5 µm thickness. Sections were rehydrated and stained with hematoxylin and eosin. Digital images were captured using an inverted microscope (Nikon Eclipse-Ti, Japan). Follicular density was measured comparing the control and vitrified group.
 
RNA extraction
 
The total RNA was isolated from primary, secondary and tertiary follicular structures of vitrified and control group. Initially, follicles were crushed in LN₂ and subjected to lysis in TRIzol reagent for 10 min at RT. Then chloroform was added for phase separation. Aqueous phase was extracted in another eppendorf and mixed with 100% ethanol. Further, washing, elution and DNase treatment steps were carried out using the RNAqueous-Micro Kit (Ambion, USA) instructions. The total RNA absorbance was measured at 260 nm and 280 nm using UV spectrophotometer (NanoQuant infinite M200, Tecan, Austria).

For cDNA synthesis, equal concentration of RNA was used in all groups. The ‘Superscript III first strand synthesis Kit’ (Invitrogen, USA) was used, according to the manufacturer’s instructions. The cDNA synthesized was confirmed by amplification of housekeeping genes i.e. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-Actin. The cDNA prepared was stocked up at -20°C till further use.
 
Quantification of relative mRNA abundance by qPCR (qRT-PCR)
 
Quantitative real time PCR was carried out according to the protocol mentioned by Dua et al., (2019) to study the relative expression of six genes involved during folliculogenesis. The CFX96 (BioRad, USA) instrument was used to perform the various reactions with different annealing temperatures as stated in Table 1. Endogenous controls used in the study were GAPDH and β-Actin.
 

 
Determination of relative cell cytotoxicity
 
For determining the metabolic activity by MTT assay, the follicular cells collected, were mechanically obtained from different follicular structures of vitrified and control group, grown at density of ~2000 cells per well on a 96-well plate for 72 hrs. Then these cells were incubated with 5 mg/ml MTT at 37°C for 2 h after which 100 µl DMSO was added. The optical density of dissolved formazan was measured at 570 nm using a Nano Quant reader (Infinite M200 Pro, Tecan, Austria).
 
Experimental design
 
Primary, secondary and tertiary follicles (n= 30 each) were isolated in triplicate, from the control and vitrified cortical tissues, to identify the expression of genes.
 
Statistical analysis
 
Analysis in the present study was performed using GraphPad Prism software (San Diego, USA) by ANOVA and t-test to compare the means of different groups in the gene expression of different follicles isolated from fresh and cryopreserved experiments. The datasets were analyzed after arcsine transformation of percentage values. Significance was determined at P<0.05. Data were represented as Mean ± SEM.

Morphology of primary, secondary and tertiary follicles in both vitrified and control group is shown in Fig 3. Number of follicles recovered per ovary and the percentage of survival rate of follicles distributed in parenchyma for vitrified and control samples have been mentioned in Table 2. They showed a non significant decrease in vitrified group as compared to the control. This indicates that, the procedure used for vitrification in the present study, has very less impact on morphology of ovarian tissues. The potentiality of protocol used, possibly due to the use of combination of CPAs including DMSO, EG and Sucrose (Amoushahi et al., 2017). DMSO prevents intra- and extra-cellular crystallization by penetrating into the cell, leading to an increase in the permeability of other CPAs combined with it. EG is a low molecular weight CPA, that rapidly diffuse into the cell. Sucrose is known to provide nutrition to the preserved cells. This combination of CPAs has reduced the risk of cryoinjury to the tissues in the present study. These similar observations can also be seen in other species (Mofarahe et al., 2017).
 

 

       
Morphological parameter doesn’t explain the complete functionality of the vitrified group. So the present study, validate the changes that occurs through different developmental follicular stages, during vitrification in comparison with control group.
       
The relative expression levels of FOXO3, WNT4, NLRP5, SF1, VEGFA and HAS2 genes were evaluated in isolated primary, secondary and tertiary follicles from control and vitrified group (Fig 4). FOXO3 showed a decreasing pattern from primary, secondary and tertiary follicles and slightly more decrease in vitrified group (2.13±0.76 vs 1.68±0.55 vs 0.81±0.25) as compared to control (2.99±0.47 vs 1.94±0.30 vs 1.01±0.09). This pattern of expression shows that FOXO3 is activated at primary stage of follicular growth, providing a molecular entry point for studying the regulation of follicular growth. It is important to maintain dormancy factors; which act negatively to maintain the oocyte growth in early follicular stages. It is also maintained during cryopreservation and in vitro follicle culture (Ting and Zelinski, 2017).
 

       
Whereas, other genes, NLRP5, SF1, WNT4, VEGFA and HAS2, non-significant increasing pattern was observed from primary to tertiary follicular stage in both the groups, which signify the upregulation of expression of oocyte growth, with increasing number of follicular cells of increasing size. The non-differential expression of these genes between vitrified and control group, could be explained by the combination of cryoprotectants used in the study, which might have reduced the cryoinjury to the ovarian tissue (Fathi et al., 2013).
       
Moreover, the role of follicular cells is essentially required during early folliculogenesis, including regulating and suppressing activation, dormancy, growth and development of competent oocyte. These cells provide an optimal microenvironment for proper expression of various genes which is essentially required for the transition of follicles from one stage to another. Further, proliferation of granulosa cells, which is also directed by NLRP5, SF1 and WNT4 signaling pathway, maintains the follicular development. This increasing pattern in signals is due to the increased response of gonadotropins during secondary to tertiary transition stages (Ernst et al., 2018). Granulosa cells starts accumulating around the oocytes in primary follicle differentiate as cumulous cells, which help the oocytes to grow. This transition is generally associated with the acquirement of oocyte competence to resume the first meiotic division. Higher relative gene expression of VEGFA and HAS2 in cumulus cells surrounding oocytes are generally at the time of tertiary follicle or subsequent stage of ovulation. Focusing, VEGFA is a key factor that regulates angiogenesis in the ovary and HAS2 transcripts essential growth factors for cumulus expansion required during the development of oocytes. It is inferred that, cumulus cells are required for the formation of oocytes during the preantral to antral follicle transition. Thus, oogenesis and folliculogenesis are highly coordinated process requiring complex cell-to-cell communication and a myriad of growth factors that interact within the follicular microenvironment (Cadenas et al., 2017).
       
Significant (P<0.05) decrease of expression in WNT4 (5.46±0.39 vs 3.49±0.41) and HAS2 (3.73±0.87 vs 1.92±0.04) gene in vitrified group could be explained by the effect of cryoinjury in granulosa and cumulous cells at tertiary follicles which might hinder its growth. The specific role of various genes, reveals the cellular and developmental complexity, but, as of now, very little is known about the global nature of the transcriptomes after vitrification, which supports respective cellular and functional diversification.
 
Further, metabolically active follicular cells of primary (1.00±0.00 vs 0.63±0.30); secondary (1.00±0.00 vs 0.75±0.3) and tertiary (1.00±0.00 vs 0.96±0.28) stages were analyzed by MTT assay, which also showed the non-significant results of vitrified group in comparison to control. This confirms that the protocol used for vitrification does not distort the follicular structure and functionality. Subsequently, this study can be applied as the basics genes of granulosa cells as biomarkers,  to evaluate the efficiency in a culture system.
 
It is inferred that, ovarian tissue/follicle cryopreservation can be used as a notable option for fertility preservation, together with oocyte and embryo production. This technique can serve as the finest choice to regain fertility.

This work was financially supported by the National Agriculture Science Fund (NASF/GTR-7004/2018-19/79). There is no conflict of interest in this article.