Transcriptional Level of Cumulus-Associated GJA1, PTX3, PRSS35, and SERPINE2 Genes with Oocytes and Embryonic Development in Water Buffalo

In the present study, the potential of different groups of cumulus-oocyte complexes (COC’s) for in-vitro maturation (IVM) and embryonic development was assessed in two groups of COC’s of water buffalo. Further, the association of the expression pattern of cumulus-associated GJA1, PTX3, PRSS35, and SERPINE2 genes and their effects on embryonic development was analyzed. Slaughterhouse-derived buffalo oocytes were graded into group A and B based on surrounding cumulus rings. Out of 1000 ovaries, an equal number of 410 COC’s were taken in both the A and B groups. In-vitro maturation (IVM) was carried out using Slaughterhouse-derived buffalo epididymis. A remarkable degree of cumulus expansion was noticed in group A (92.68%) as compared to group B (81.25%) oocytes. On IVF and embryo culture, group A COC’s produced a signicantly higher rate of cleavage and blastocyst (92.682±0.7179% and 42.682±0.9683%) as compared to group B COC’s (85.365±0.7608% and 31.707±0.9688%), respectively. The transcriptional analysis of cumulus-associated GJA1, PTX3, PRSS35, and SERPINE2 genes expression by quantitative Real Time-PCR (qRT-PCR) revealed a signicantly higher expression in group A as compared to group B COC’s. Result: It was revealed that oocytes having good cumulus mass had a higher developmental potential. Based on differential gene expression of cumulus-associated genes, different quality of COC’s, and the resultant embryos after IVF, it was concluded that these genes could be used as a marker for predicting the developmental competence of the oocytes. Conclusion: It concluded from the study that morphologically good quality of COC’s had a higher developmental competence. Also, the differential expressions of cumulus-associated genes in cumulus cells and embryos, we can conclude that these genes could be used as marker genes for predicting the developmental competence of buffalo’s oocytes.


Background
In-vitro maturation (IVM), in-vitro fertilization (IVF), and gamete cryopreservation technologies provide a practical means for producing a large number of bovine embryos at a low cost for research and commercial settings [1][2][3]. Various factors have been affected the successful In-Vitro production of buffalo embryos including a low number of follicles on the ovaries, sperm mortality, in-vitro laboratory conditions. [4][5], a poor recovery rate of the oocytes [6][7], and poor in-vitro fertilization e ciency [8][9][10]. In early embryo development, de ning oocyte quality remains one of the most di cult challenges.
Follicular growth and maturation are prerequisites to oocyte fertilization and subsequent early embryo development [8]. Among all the events involved in this process, those taking place precisely at the preovulatory stage within the cumulus-oocyte-complexes (COC's) might offer new criteria for choosing embryos with the best development ability [11][12][13]. Previous studies emphasized the roles of cumulus mass in nurturing oocyte growth, gradual acquisition of oocyte developmental competence suggested that cumulus cells metabolize the bulk of glucose consumed by the COC's, supplying metabolic intermediates like pyruvate, mainly through glycolysis, to the oocyte and regulating the oocyte gene transcription [14][15][16].
In the livestock sector, in-vitro fertilization (IVF) technology can be a useful tool for fruitful output in mammalian species like cows, buffaloes, goats, and pigs. The good qualities of cumulus cells (CC's) are very important for successful in-vitro fertilization. Oocyte cumulus undoubtedly plays an important role in oocyte quality and maturation but the eject mechanism behind this is still unknown [17]. Therefore, an extensive study is required to analyze whether the developmental potential of the COC's is associated with the expression of different cumulus genes in terms of maturation, fertilization, and embryonic development. Works of literature show that genes such as GJA1, PRSS35, PTX3, and SERPINE2 play a crucial role in oocyte maturation, fertilization, and embryonic development [18][19][20][21][22][23][24]. However, literature elucidating these cumulus-associated gene expressions, their signi cance, and the effect of cumulus cell layer/mass toward the embryonic developmental process are scarce in livestock.
Cumulus-associated GJA1 gene is the major gap junction α1 protein or connexin43, essential for oocyte fertilization potential and embryo quality [18]. PRSS35 gene belongs to the trypsin class of serine proteases, which is essential for the tissue remodeling and functions of the ovary during folliculogenesis and ovulation [19]. The gene PTX3 (Pentraxin-related protein 3) is linked to extracellular matrix (ECM) hyaluronan and is a potentially reliable predictor of embryo developmental competence [20]. The SERPINE2 (serine proteases E2) gene is considered a potential pregnancy biomarker [21] and is extensively expressed in reproductive tissues [22][23][24].
Our aim was, therefore, to evaluate the effects of cumulus cell mass on oocyte competence in terms of oocytes maturation and post-fertilization developments along with the expression of GJA1, PTX3, PRSS35, and SERPINE2 genes in cumulus cells according to stages in oocyte maturation and embryonic developmental competence in slaughterhouse derived buffalo oocytes.

Methods
All the chemicals and media were purchased from Sigma-Aldrich Chemicals Company (St. Louis, Monsanto) and the disposable plastic wares were from Nunc (Roskilde, Denmark) unless otherwise mentioned.

Oocyte Aspiration And Grading
Under a total of 30 trials (Sample size calculated by using, n=Z 2 *P (P-1)/D 2 formula), buffalo ovaries were collected from the large animal abattoir aseptically in normal saline solution (37ºC, pH 7.0), supplemented with gentamicin (50 µg/ml). The ovaries were trimmed and washed 3-4 times in Dulbecco's phosphate buffer saline (1X, DPBS). This was followed by a quick wash of 30-40 seconds with 70% ethanol and nally rinsed with DPBS. Oocytes were aspirated from 6-8 mm ovarian follicles by follicular aspiration method in a pre-warm (37ºC) DPBS medium. The oocytes were washed 3-4 times in  (washing media, Hyclone), supplemented with 7.5% (v/v) fetal bovine serum (FBS, Hyclone,) plus 50 mg/ml gentamicin. Recovered oocytes were grouped into 4 categories A, B, C, and D [25], following the morphology of cumulus mass and cytoplasmic appearance of oocytes. Group A oocytes had a 3-4 compact layer of cumulus mass with evenly granular homogenous cytoplasm, whereas group B oocytes had 2-3 layers of cumulus mass with evenly granular homogenous cytoplasm. The oocytes with complete or incomplete 1-2 layers of cumulus mass with irregular dark ooplasm, and no cumulus mass with irregular dark ooplasm, were categorized under the C and D group of oocytes, respectively (Fig. 1a).
Only group A and B COC's were taken for the gene expression study. Further, for gene expression analysis, oocytes from both groups were divided into three subsequent stages viz., cumulus cells of immature COC's, cumulus cells of matured COC's and fertilized early embryos.

In-vitro maturation (IVM)
In-vitro maturation medium containing TCM-199 Supplemented with 10µg/ml follicle-stimulating hormone (FSH), luteinizing hormone (LH), 1µg/ml estradiol, 7.5 percent (v/v) fetal bovine serum (FBS), 50 µg/ml gentamicin, and 0.8 mM/ml sodium pyruvate. It was used for in-vitro maturation of oocytes in groups of 30 oocytes per droplet. The droplets were overlaid with sterile pre-equilibrated mineral oil. The maturation process was carried out under a humidi ed atmosphere (99%) for 24 hours in a CO 2 incubator having a 5% CO 2 level at 37°C. The cumulus expansion and presence of the rst polar body were observed under the inverted zoom microscope (Nikon, 100X), to assess the in vitro maturation of oocytes.
Sperm preparation for in-vitro fertilization (IVF) and embryo culture Under 30 experimental trials, sperms were collected from abattoir-derived epididymis and processed under sterile conditions as described earlier for ovaries. Sperms were harvested from tubules of epididymis with a ne and gentle incision with a sterile surgical blade and collected in a sterile tube containing 10 ml of Bracket and Oliphant (BO) media, supplemented with 1mM caffeine sodium benzoate. The sperm concentration, morphology, and gross motility were observed under the inverted microscope (Nikon, 100X). For in-vitro fertilization, the sperms were prepared and capacitated for matured oocytes as described by Jeena et al 2018 [26]. In short, exudate sperms suspension with BO media was centrifuged at 168G for 10 min and the supernatant was discarded. This step was repeated twice. The pellet was resuspended in 5ml of BO fertilization media forti ed with 1% BSA plus 50 mg/ml heparin and centrifuged at 168 G for 5 min. The harvested pellet was re-suspended in 1ml BO fertilization media and centrifuged at 168 G for 1min. The pellet was loosened with a ne bore pasture pipette and kept inside the CO 2 incubator for 30 min for capacitation and to allow good quality sperms to swim up. At the same time 30 matured oocytes from both groups A and B were transferred into a pre-equilibrated (38.5°C) 50µl BO fertilization droplet overlaid with mineral oil. After 30 min capacitated sperms with a nal concentration of 10 6 were taken from the top layer of sperm suspension and co-incubated with oocytes at 5 percent CO 2 at 38.5°C temperature under a humidi ed atmosphere for 18 h (Fig. 1b). After 18 h of co-incubation, oocytes were denuded with 1 percent of hyaluronidase. The presumptive zygotes ( Fig. 1c and Fig. 1d) were cultured in RVCL media (Research Vitro Cleave Medium, COOK, Australia). Each group of embryos were assessed for their embryonic developmental stages as cleavage (Fig. 1e), 4 cells (Fig. 1f), compact morula (Fig. 1g), and blastocyst development (Fig. 1h).

Primer designing
The Primers for β-actin, PTX3, PRSS35, and SERPINE2 were designed by using online software PRIMER EXPRESS 3.0. The GJA1 gene primer was taken as a reference primer. Primer sequence speci city was checked by an online available database from BLAST, NCBI (Tab. 1). Table 1 Details of qRT-PCR (quantitative Real Time-PCR) primers [25] GJA1 (R) ATCCCTAACACCCCCAATGAACCA F* forward primer; R* reverse primer Gene expression level of cumulus-associated GJA1, PTX3, PRSS35, and SERPINE2 genes Cumulus-oocyte complexes (COC's) were denuded in 0.1 percent hyaluronidase for 10 min with a gentle vortex to remove the cumulus cells. The total RNA was extracted by the Trizol method from cumulus cells of both groups (A and B), and also from their subsequent stages of immature cumulus cells of COC's, matured COC's, and fertilized early embryos. A total of 30 COC's from both groups were taken for RNA extraction. cDNA was prepared by using the rst-strand cDNA synthesis kit (Fermentas, K1622) from an equal amount of total RNA. qRT-PCR was performed with a total volume of 20µl containing cDNA (1µl), 1pico-mole of reverse and forward primer (Table 1), and 10µl of 2X SYBER green master mix (Applied Biosciences). All reactions were performed in triplicate. The relative quanti cation of mRNA of each gene was evaluated by qRT-PCR (Applied Biosystems, 7300) using SYBR green chemistry. The relative gene expression analysis of GJA1, PTX3, PRSS35, and SERPINE2 genes was analyzed by using Livak and Schmittgen [27] formulation i.e., comparative 2 ΔΔCt method. The housekeeping gene β-actin was used as an endogenous loading control to normalize the relative gene expression. Immature COC's of group A oocyte were taken as a referral control (1.0) for the expression analysis of cumulus-associated GJA1, PTX3, PRSS35, and SERPINE2 genes in both the groups. The purity of ampli ed products was checked by Ethidium Bromide (EtBr, 0.5µg/ml) stained with 1.8 percent agarose gel electrophoreses.

Statistical analysis
The percent data of embryonic development were transformed by arcsine transformation (log N ) and subjected to analysis of variance by Hierarchical analysis of variance design, and the mean differences were compared by Duncan's Multiple Range Test (P<0.05).

In-vitro maturation (IVM) of abattoir-derived buffalo oocytes
Under 30 experimental trials, a total of 2070 COC's were retrieved from 1000 slaughterhouse-derived ovaries, out of which 550 were from group A, 800 from group B (Table 2), and the rest of 720 were from group C and D. Only good quality COC's of group A and B were taken for in-vitro maturation and in in-vitro fertilization. The analysis of variance of the data on embryonic development (Table 3) revealed a highly signi cant (P<0.01) between-group difference (A and B) for all the embryonic stages. However, there was no signi cant difference (P>0.01) between the day of the experiment (bull effect), indicating that epididymal semen collected on a different day did not signi cantly affect embryonic development.
Further, the embryonic development stages within the group differed signi cantly, shown in Table 3.
It was observed that the in-vitro maturation rate of group A was substantially higher as compared to group B oocytes, in terms of rst polar body extrusion (   Cumulus-associated GJA1 gene expression and oocyte integrity The relative expression of the GJA1 gene was found signi cantly (P≤0.05) higher in cumulus cells of immature COC's (1.0) compared to matured cumulus cells of COC's (0.74) and in the fertilized early embryos (0.22) of group A. The expression pattern was also signi cantly lower in group B immature cumulus cells of COC's (0.82), matured cumulus cells of COC's (0.42), and the fertilized early embryos (0.20) compared to group A (Fig. 2a). The present study revealed that the GJA1 gene was strongly associated with immature cumulus cells of COC's, and the abundance of this gene was reduced signi cantly towards the progression of developmental stages till fertilization (shown in Fig. 2a-b).  The results showed that the relative expression of the SERPINE2 gene was signi cantly higher (P≤0.05) in fertilized early embryos of both group A (1.6) and B (1.08). As shown in Fig. 5a, the SERPINE2 gene expression was signi cantly lower (P≤0.05) in matured oocytes of both groups A (0.59) and B (0.46) along with immature group B (0.79) and group A (1.0) oocytes. The expression level of the SERPINE2 gene in embryos produced by in-vitro fertilization was signi cantly higher in fertilized early embryos than the cumulus cells of immature and matured COC's ( Fig. 5a-b). The overall expression pattern of cumulus-associated GJA1, PRSS35, PTX3, and SERPINE2 genes at their subsequent developmental stages in both groups were analyzed collectively. The expressions of these genes were found signi cantly higher in group A compared to group B (Fig S1). The ampli ed products were con rmed by ampli cation plot, melting curve (Fig. 6), and agarose gel electrophoresis. The products were identi ed by their speci c band size (GJA1-425bp, SERPINE2-170bp, PRSS35-179bp, and PTX3-217bp) as shown in Fig. 2b, 3b, 4b, and Fig. 5b.

Discussion
Cumulus cells (CC's) originating from undifferentiated granulosa cells (GC's) differentiate in mural granulosa cells (MGC's) and CC's during antrum formation in the follicle by the distribution of location. [5][6]. GJA1 is the major isoform of connexins between GCs (granulosa cells): MGCs-MGCs (mural granulosa cells) and MGCs-CCs (cumulus cells) [12]. Gap junctions transmit nutrients and small molecules such as ions, metabolites, amino acids, and intracellular signaling molecules from GCs to oocytes via CCs [18][19]. We observed the expression level of the GJA1gene in immature cumulus cells of COC's was signi cantly higher as compared to matured cumulus cells of COC's and fertilized oocytes of both groups (A & B, Fig. 2a-b). A similar type of study was reported by Edry and workers, they found GJA1-mediated gap junctional communication regulates oocyte meiosis resumption, and lower levels of GJA1 in cumulus cells are bene cial for oocyte maturation [28]. While Feuerstein and Mishra reported contrasting results wherein relative expression of the GJA1gene was signi cantly lower in immature cumulus cells of COC's [29][30]. However, GJA1expression in cumulus cells surrounding the matured oocytes did not show any difference in developing embryos with good or poor morphology [29]. Hasegawa reported a signi cantly lower expression of GJA1 for embryos with good morphology [12]. Similarly, we revealed signi cantly lower expression of the GJA1gene was observed in fertilized oocytes and matured cumulus cells of COC's.
PRSS35 belongs to the trypsin class of serine proteases, essential for follicular growth, ovulation, as well as for luteal formation and regression. PRSS35 gene expression was localized in theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory, ovulatory follicles, and developing corpus luteum [15,19,31]. Whereas, the PTX3 gene is localized in the cumulus matrix and plays a crucial role in cumulus expansion. Various cumulus proteins linked to extracellular matrix hyaluronan, are required for regulating cumulus integrity, which ensure cumulus expansion and oocyte maturation [14-15, 18, 20, 32-35]. A previous study reported PRSS35 and PTX3 mRNA levels are associated with oocyte interiority and fertilization potential [15].
Our data revealed that PRSS35 and PTX3 mRNA levels are associated with the oocyte maturation potential (Fig. 2). The higher expression of PRSS35 and PTX3 genes were observed in cumulus cells of COC's of matured oocytes of both groups (A & B, Fig. 3a-b & Fig. 4a-b). However, it was found signi cantly lower in cumulus cells of immature COC's and fertilized oocytes. Li et al [15] reported a relationship between PRSS35 expression and oocyte competence. While the expression of PTX3 has been reported to be signi cantly lower in cumulus cells from immature oocytes than in those from mature oocytes, which was found similar to our study. Similarly, Huang [32] demonstrated that the expression level of the PRSS35 gene plays an important role in oocyte nuclear maturation and gaining developmental competence. According to Miyakoshi the PRSS35 mRNA level increased at the time of ovulation and remained elevated in the developing corpus luteum [36]. Although the scienti c literature regarding the association of the PRSS35 and PTX3 genes with the oocyte and embryo developments is less in livestock. However, some studies have been reported in a few species like mouse and human oocytes and embryos.
Salustri and co-workers [34] reported the infertility of PTX3 null mice was associated with severe abnormalities of the cumulus ionophores and failure of in-vivo, but not in-vitro oocyte fertilization. Zhang [20] reported that the expression of the PTX3 gene in cumulus cells was indicative of oocyte and embryo quality. However, Diao and co-workers reported the PRSS35-null mice to have no defects in female fertility, suggesting that the gene is non-functional for murine fertility as well as in embryonic development. Also, he did not detect any compensatory up-regulation of other proteases reported in the uterus; but, the expression of other protease-related genes cannot be ruled out [31]. However, this remains unclear in the case of mammalians. We found the higher expression of PRSS35 and PTX3 genes in cumulus cells of matured COC's had a bene cial effect on oocyte competence and embryonic development.
Hamel et al, reported the SERPINE2 mRNA levels in granulosa cells have been suggested to be a potential pregnancy biomarker [21].
Our data revealed that SERPINE2 mRNA levels are associated with oocyte fertilization potential.
We found the expression of this gene was higher in fertilized oocytes of both groups (A & B, Fig. 5a-b). However, the expression of the SERPINE2 gene was lower in cumulus cells of immature and matured COC's (Fig. 5a). Hamel and co-workers [13] also reported the SERPINE2 gene to be considered as a potential pregnancy biomarker and extensively expressed in reproductive tissues, the placenta, and the uterus [22,31]. Sadeesh et al, reported the silencing of SERPINE2 expression using small interfering RNAs or blockage of SERPINE2 protein using a speci c antibody did not affect oocyte maturation [2]. However, in a mouse, higher levels of SERPINE2 were demonstrated to impair cumulus expansion and oocyte maturation [19,23,[37][38]. Li and coworkers reported the higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes [15]. According to our data, we found the higher expression of the SERPINE2 gene in fertilized oocytes suggested its signi cant role in fertilization and embryonic development.
The results of the present study showed that in all the cases, the expressions of cumulus-associated genes in group A were signi cantly up-regulated as compared to group B (supplementary Figure S1). The higher expression of these genes in group A COC's can be related to higher embryonic development compared to group B COC's showing down-regulation of all these genes compared to group A COC's. It might be concluded that the signi cantly higher expression of the GJA1 gene in cumulus cells of immature COC's along with PRSS35 and PTX3 genes in cumulus cells of matured COC's have a positive impact on oocyte developments. Similarly, a higher expression of the SERPINE2 gene in fertilized early embryos may have bene cial effects on oocyte fertilization and embryonic development in buffalo.
We found some contradictory results from a previous study which was in human embryos, where the study reported the GJA1 and SERPINE2 represent potential gene markers associated with oocyte maturation and PRSS35 may be correlated with oocyte fertilization potential [15,39]. However, our study model was buffalo's embryos.

Conclusion
The selection of embryos with higher developmental potential has been one of the major factors in assisted reproductive technology (ART). Because, cumulus cells play a very important role in oocyte integrity and embryonic development. Although cumulus gene expression may represent a promising method compared with the currently used morphology-based method, more investigations are warranted. Thus, a prospective larger cohort study or the use of SET cumulus samples remains necessary to clarify the effectiveness. However, it was concluded from our study the morphologically good quality of COC's had a higher developmental potential in terms of maturation, fertilization, and embryonic development as compared to oocytes having poor quality of COC's (in both A & B groups). Based on molecular analysis of differential expressions of cumulus-associated genes we found the GJA1 gene associated with immature oocyte integrity, PTX3 and PRSS35 represent gene markers potentially associated with oocyte maturation, and SEPINE2 may be correlated with oocyte fertilization potential. In summary, GJA1, PRSS35, PTX3, and SERPINE2 in cumulus cells of different quality of oocytes and their resultant embryos after IVF, we can