Effect of growth differentiation factor- 9 (GDF- 9) on the progression of buffalo follicles in vitrified warmed ovarian tissues

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1 Received: 4 April 2016 Accepted: 12 July 2016 DOI: /rda ORIGINAL ARTICLE Effect of growth differentiation factor- 9 (GDF- 9) on the progression of buffalo follicles in vitrified warmed ovarian tissues MA Abdel-Ghani 1,a TM El-sherry 1 HH Abdelhafeez 2 1 Department of Theriogenology, Faculty of Veterinary Medicine, Assuit University, Assuit, Egypt 2 Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assuit University, Assuit, Egypt Correspondence Abdel-Ghani MA. Department of Theriogenology, Faculty of Veterinary Medicine, Assuit University, Assuit, Egypt. Abdel-Ghani2016@outlook.com a Present address: Laboratory of Theriogenology, Department of Veterinary Clinical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan Contents To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor- 9 (GDF- 9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF- 9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF- 9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF- 9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF- 9 was detected in mural granulosa cells and theca cells of pre- antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF- 9. In corpora lutea, GDF- 9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF- 9 and cultured control that indicated the GDF- 9 treatment has no effect on the primordial to primary follicle transition. GDF- 9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre- antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF- 9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF- 9. In conclusion, treatment with GDF- 9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity). 1. INTRODUCTION The water buffalo (Bubalus bubalis) is an economically valued livestock species in Africa and Asia and so improvement of fertility, which reflected in high quantity of milk and meat production, ranks high among our agricultural research needs. The urgent need for improving the reproductive performance of water buffalo necessitates a better understanding of the mechanisms controlling ovarian follicular growth and development (Manik, Palta, Singla, & Sharma, 2002). The ovarian inactivity or lack of subsequent normal follicular development and atresia of the dominant follicle are the main causes of infertility problems in buffaloes (Peter, Vos, & Ambrose, 2009). Reprod Dom Anim 2016; 51: wileyonlinelibrary.com/journal/rda 2016 Blackwell Verlag GmbH 795

2 796 Abdel- Ghani et al. Many factors important for folliculogenesis have been identified in recent years; however, the delineation is incomplete, and there are differences between species in both the timing and synchronicity of follicle progression and in the role of signalling molecules (Bayne et al., 2015). As the actions of the hormones secreted by the hypothalamus and anterior pituitary have been well distinguished, more current researches focus on the regulatory proteins within the ovaries. The transforming growth factor- beta (TGF- β) superfamily contains over 40 members, many of which influence on many tissues and organ systems including the ovary (Knight & Glister, 2003). One of the more recent oocyte- derived family members is growth differentiation factor- 9 (GDF- 9) (Wang & Roy, 2006). Although the exact factors or mechanisms that signal somatic cell differentiation into the pregranulosa cells remain elusive, a convincing evidence has accumulated to suggest that GDF- 9 plays an important role in early folliculogenesis (Kedem et al., 2011), steroids synthesis and differentiation (Sun et al., 2010). Expression patterns and functions of GDF- 9 have been studied in human (Kedem et al., 2011), bovine (Hosoe, Kaneyama, Ushizawa, Hayashi, & Takahashi, 2011), goat (Silva, van den Hurk, van Tol, Roelen, & Figueiredo, 2005) and mice (Sun et al., 2010), but no data about the localization of GDF- 9 protein and its function have been available for buffaloes. Furthermore, the in vitro culture of ovarian tissues has made it feasible for ovarian cryobanking for woman receiving chemotherapy and radiotherapy, which are known to be gonadotoxic, and enables the remaining ovarian tissue to be stored for use in the future (Paris, Snow, Cox, & Shaw, 2004). This may allow the reproductive lifespan of the female to be prolonged or the tissue to be grafted at times (Oktay et al., 2004). In addition, the ovarian tissue vitrification prove to be a potentially promising method for preserving endangered and threatened animals as well as for the preservation of genetic resources (genome resource bank) (Ishijima et al., 2006). To improve the capability, and hence increase the productivity, of our native breeds to level can satisfy our needs, attention should be focused on an understanding of the factors regulating the follicular progression. Therefore, the objectives of this study were to investigate the expression patterns of GDF- 9 in buffalo ovaries using immunohistochemistry and to investigate its effect on progression of follicles enclosed in ovarian tissue. 2. MATERIALS AND METHODS All chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO) unless mentioned otherwise. 2.1 Animals Buffalo s ovaries (7 10 years) were obtained from local slaughterhouse. The age of buffaloes was estimated by examining their teeth and also by taking information from the animal owners. The ovaries from each buffalo were transported to the laboratory within 1 hr in a thermos containing physiological sterile saline at 37 C. After transportation, the fat, ligaments, and medulla were carefully trimmed off and removed. Next, they were placed in 35- mm petri dish (Falcon; NY, USA) containing Dulbecco s modified Eagle s medium (DMEM). 2.2 Immunohistochemically localization of growth differentiation factor- 9 (GDF- 9) in buffalo ovarian tissue The formalin- preserved tissues for 24 hr at 4 C were processed and embedded in paraffin. Localization of GDF- 9 was performed on serial 5- μm sections cut from different buffaloes ovaries (n = 6; 7 10 year old). These sections were mounted on slides, dried overnight at 37 C, deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was blocked with 0.3% H 2 O 2, diluted by phosphate- buffered saline (PBS; ph 7.4) for 10 min. After three rinses (3 5 min each) with PBS, sections were then incubated with 5% (w:v) BSA in PBS at room temperate (RT) for 20 min to block the non- specific binding. The primary antibody was anti- GDF9 antibody diluted 1:100 in PBS, and the sections were incubated overnight at 4 C. For determination of non- specific staining, the primary antibody was replaced by normal rabbit serum. Sections were subsequently rinsed in PBS (3 5 min) and incubated for 1 hr at RT with biotinylated secondary antibody and diluted 1:200 in PBS containing 5% BSA. Next, the sections were washed three times with PBS (5 min each). Immunoreactivity was visualized by incubating sections in the presence of 3,3 - diaminobenzidine (DAB; 0.05% DAB in 0.01 M PBS, 0.03% H 2 O 2, ph 7.2) substrate until a precipitate formed or a maximum of 10 min. Finally, the sections were counterstained with haematoxylin and overlaid with coverslips. The staining intensity for GDF- 9 immunoreactive protein expression was scored accordingly to Silva et al. (2005): absent ( ), occasionally found ( /+), expressed (+). 2.3 Cryopreservation of buffalo ovarian tissue The cryopreservation protocol used was as previously described by Ishijima et al. (2006). Briefly, ovarian cortex was isolated and tissue slices of approximately 1 mm 3 were prepared. After rinsing in HTF medium, each ovarian fragment was transferred into a 1- ml cryovial (Cryo Tube, Nunc International, Denmark) containing 5 μl of cryoprotectant 1 M dimethyl sulfoxide (DMSO) at RT, which was then placed in ice water for 5 min to allow DMSO to thoroughly bathe the thin slices of ovarian cortex (Newton et al., 1998). Subsequently, 95 μl of DAP213 solution (2 M DMSO; 1 M acetamide; 3 M propylene glycol), maintained at 0 C, was added to each cryotube. Finally, the cryotubes were kept in ice water for 5 min before being transferred to liquid nitrogen for storage until use. Warming was performed by holding the cryotubes at RT for 1 min and then diluted with 900- μl PB1 medium (37 C) containing 0.25 M sucrose. After warming, the contents of the cryotube were released into a petri dish containing PB1 medium, washed five times and transferred into DMEM.

3 Abdel- Ghani et al In vitro buffalo ovarian tissue culture The culture medium consisted of DMEM supplemented with 10% (v/v) foetal calf serum (FCS), 2% (v/v) essential amino acids, 1% (v/v) non- essential amino acids, 50 μg ml 1 of ascorbic acid, 100 IU ml 1 of penicillin, 100 μg ml 1 of streptomycin, 1% of amphotericin and FSH at 5 IU ml 1. ITS mix was added to the culture medium at 10 μg insulin ml 1, 5.5 μg transferrin ml 1 and 7 μg sodium selenite ml 1. Recombinant human GDF- 9 (200 ng ml 1 ) was added to the culture medium to investigate the effects of GDF- 9. Culture medium without GDF- 9 was used as the control. Ovarian tissue pieces were cultured at 37 C in a humidified incubator with 5% CO 2 in air for 14 days on floating filters (0.4 μm Millicell culture well inserts) fitted into 24- well plates. The inserts contained 100 μl culture medium with an additional 400 μl culture medium in the surrounding well. Culture media were removed and replenished every second day. 2.5 Histological examination of cultured buffalo ovarian tissues The formalin- preserved tissues for 24 hr at 4 C were processed and embedded in paraffin. The tissue pieces (5 μm in thickness) obtained from a rotary microtome were mounted onto plain glass slides and stained with haematoxylin and eosin for light microscopy evaluation. To avoid double count of follicles, 16 sections were discarded before the next was mounted onto the slide in a total of 5 fields of view. The follicles at the level of the nucleus of the oocyte in all serial sections were classified on the basis of morphology of granulosa cells and the number of granulosa cell layers surrounding the oocyte, as previously described by Rodgers and Irving- Rodgers (2010): (i) primordial follicle is identified histologically on the basis of a small non- growing oocyte, without a zona pellucida (ZP) and surrounded by flattened granulosa cells; (ii) primary follicle, the oocyte surrounded by a single layer of cuboidal granulosa cells with distinctive ZP; (iii) secondary follicle, the oocyte surrounded by 2 6 layers of granulosa cells; (iv) pre- antral follicle, the oocyte surrounded by several layers of granulosa cells and a space among granulosa cells or a segmented cavity with two or more compartments; (v) antral follicle, formation and expansion of the follicular antrum containing follicular fluid. 2.6 Experimental design First, the GDF- 9 expression in fresh ovaries obtained from slaughterhouse was investigated using immunohistochemistry. Second, the ovarian slices were vitrified using DAP- 213 cryotube method, and the fresh and vitrified warmed (uncultured) ovarian slices were processed for histological examination. A comparison between the fresh and TABLE 1 Expression of growth differentiation factor- 9 protein in different developmental stages of follicles and corpus luteum in buffalo ovary compared with other species Structure Buffalo Human a Rat b Mice c Cattle d Sheep e Goat f Pig g Dog h Primordial follicle Oocyte Granulosa cells Primary follicle Oocyte Granulosa cells Secondary follicle Oocyte Granulosa cells Pre- antral follicle Oocyte Cumulus cells + N/A Mural granulosa cells /+ Theca cells + N/A N/A N/A N/A /+ Antral follicle Oocyte Cumulus cells + N/A Mural granulosa cells Theca cells + N/A N/A N/A /+ N/A + Corpus luteum + + N/A N/A + +, expressed; /+, occasionally found;, absent; N/A, not available. a Oron et al. (2010). b Silva et al. (2004). c Sun et al. (2010). d Bodensteiner et al. (1999) and Hosoe et al. (2011). e Mery et al. (2007). f Silva et al. (2004). g Sun et al. (2010). h Abdel- Ghani (2012).

4 798 Abdel- Ghani et al. vitrified warmed (uncultured) ovarian tissues was investigated. Then, the vitrified warmed ovarian slices were cultured for 14 days in presence (cultured plus GDF- 9) or absence (cultured control) of GDF Statistical analysis All data were expressed as mean ± SD. Comparisons in the mean number of follicles were performed by Tukey s test (p <.05). Nonparametric data were assessed by Kruskal Wallis test. All statistics were calculated with the help of JMP v5.0.1 (SAS campus drive, Cary, NC, USA). Differences of p <.05 were regarded as significant. All experiments were carried out in accordance with the guidelines for the care and use of the animals approved by the Veterinary Teaching Hospital s Animal Care and Use Committee. 3. RESULTS 3.1 Expression of GDF- 9 protein The results of GDF- 9 protein expression in ovarian sections containing follicles and corpora lutea investigated using immunohistochemistry compared with other species are presented in Table 1. The results showed that GDF- 9 protein was expressed in buffalo oocytes from primordial follicle stage on onward (Fig. 1) and in granulosa cells of primordial, primary, secondary, pre- antral and antral follicles (Fig. 1a c). In addition, GDF- 9 was expressed in mural granulosa cells and theca cells of pre- antral follicles (Fig. 1d). In antral follicles, cumulus cells and theca cells displayed positive expression of GDF- 9 (Fig. 1e, f). In the corpora lutea, GDF- 9 was strongly expressed in both granulosa and theca lutein cells (Fig. 1g). FIGURE 1 Immunoreactivity in the different structures found within buffalo ovaries before vitrification. (a) primordial follicle (arrow); (b) primary follicle (arrow); (c) secondary follicle (arrow); (d) pre- antral follicle (arrow); (e) antral follicle; O, oocyte; CC, cumulus cells; (f) MGC, mural granulosa cells; T, theca cells; (g) corpus luteum. Expression is stained with brown; the nucleus is stained with blue

5 Abdel- Ghani et al. 799 The expressions of GDF- 9 in vitrified (uncultured), vitrified (cultured control) and vitrified (cultured plus GDF- 9) are shown Table 2. In vitrified uncultured group, GDF- 9 was detected in oocytes from primordial until the antral follicles. Occasionally, in antral follicles, cumulus, mural granulosa cells and theca cells showed a weak reaction for GDF- 9 (Fig. 2e, f). In vitrified (cultured control) secondary and preantral follicles, the oocyte and granulosa cells had week reaction for GDF- 9 (Fig. 3c, d), whereas in vitrified (cultured with GDF- 9), they had strong reaction for GDF- 9 (Fig. 4c, d). GDF9 was present occasionally in mural granulosa cells of pre- antral follicles. In antral follicles of vitrified (cultured control), the oocyte, cumulus cells, mural granulosa cells and theca cells occasionally showed reaction for GDF- 9 (Fig. 3e, f); however, the reaction was week. In contrast, in antral follicles of cultured with GDF- 9, they showed strong reaction for GDF- 9 (Fig. 4e, f). 3.2 Vitrification of buffalo ovaries In vitrified (uncultured) ovarian tissues, there was a reduction (p <.05) in the number of all stages of follicles, except the primordial follicles, compared with fresh ovarian tissues (Table 3). 3.3 In vitro growth of follicles after culture The results of follicular progression in experimental groups were set out in Table 3. There was no difference (p >.05) in the number of TABLE 2 Showing the comparison of growth differentiation factor- 9 (GDF- 9) expression in different developmental stages of follicles in fresh, vitrified (uncultured), vitrified (cultured control) and vitrified (cultured plus GDF- 9) Structure Fresh Vitrified (uncultured) Vitrified (cultured control) Primordial follicle Oocyte Granulosa cells + + /+ /+ Primary follicle Oocyte Granulosa cells + + /+ /+ Secondary follicle Oocyte Granulosa cells + + /+ /+ Pre- antral follicle Oocyte + + /+ + Cumulus cells + + /+ /+ Mural granulosa cells + /+ /+ /+ Theca cells + + /+ + Antral follicle Oocyte + + /+ + Cumulus cells + /+ /+ + Mural granulosa cells + /+ /+ + Theca cells + /+ /+ + +, expressed; /+, occasionally found;, absent; N/A, not available. Vitrified (cultured plus GDF- 9) FIGURE 2 Immunoreactivity in the different structures of vitrified (uncultured) buffalo ovarian tissues. (a) Primordial follicle, (b) primary follicle, (c) secondary follicle, (d) pre- antral follicle (e) and (f) antral follicle.. Expression is stained with brown; the nucleus is stained with blue

6 800 Abdel- Ghani et al. FIGURE 3 Immunoreactivity in the different structures of vitrified (cultured control) buffalo ovarian tissues. (a) Primordial follicle, (b) primary follicl, (c) secondary follicle, (d) pre- antral follicle (e) and (f) antral follicle. Expression is stained with brown; the nucleus is stained with blue primordial follicles between vitrified (cultured plus GDF- 9), vitrified (cultured control), vitrified (uncultured) groups. The vitrified (cultured plus GDF- 9) resulted in a decrease (p <.05) in the number of primary follicles (00.01 ± 00.01) and secondary follicles (00.01 ± 00.01) compared with vitrified (cultured control) (00.02 ± and ± respectively). In contrary, vitrified (cultured plus GDF- 9) resulted in an increase (p <.05) in pre- antral follicles (00.05 ± vs ± for vitrified [cultured control] and ± vs ± for vitrified [uncultured] ovarian slices). Similarly, the presence of GDF- 9 increased the numbers of antral follicles by ± that was higher than the control ( ± 0.006) (p <.05). There was no change (p >.05) in total follicle number was observed with any of the treatments utilized, only a change in the follicle stages (Table 3). 4. DISCUSSION In the current research, the localization of GDF- 9 in buffalo ovaries was investigated. The data showed that the GDF- 9 protein was expressed in primordial, primary, secondary, pre- antral and antral follicles. These results are similar to those reported for porcine (Sun et al., 2010), ovine (Bodensteiner, Clay, Moeller, & Sawyer, 1999), bovine (Hosoe et al., 2011), caprine (Silva et al., 2005) and dog (Abdel- Ghani, 2012), where GDF- 9 was found as early as in oocytes of primordial follicles. These expressions are earlier than that found for human (Oron et al., 2010), and mice (Sun et al., 2010). In rodents with an incomplete oestrous cycle (oestrous cycle without CL formation), GDF- 9 was expressed exclusively in oocytes (Hayashi et al., 1999). However, in other species (cow, sheep, goat and pig) and buffalo with a complete oestrous cycle (oestrous cycle with CL formation), GDF- 9 was expressed in cumulus cells as well as in oocytes (Bodensteiner et al., 1999; Hosoe et al., 2011; Silva et al., 2005; Sun et al., 2010). In buffalo antral follicles, GDF- 9 protein was detected in the oocyte, cumulus and mural granulosa cells. The expression pattern of GDF- 9 in antral follicles was consistence with the results exhibited in bovine (Hosoe et al., 2011), ovine (Mery et al., 2007), caprine (Silva et al., 2005) and dog (Abdel- Ghani, 2012), but not for rodents (Sun et al., 2010). In addition, GDF- 9 protein was detected in corpora lutea that was similar to caprine (Silva et al., 2005) and human (Oron et al., 2010) and in contrast to that reported for rodents (Sun et al., 2010) porcine (Sun et al., 2010) and dog (Abdel- Ghani, 2012). The expression pattern of GDF- 9 in buffalo follicles suggested that the GDF- 9 might play an important role in the further progression of

7 Abdel- Ghani et al. 801 FIGURE 4 Immunoreactivity in the different structures of vitrified (cultured plus GDF- 9l) buffalo ovarian tissues. (a) Primordial follicle, (b) primary follicle, (c) secondary follicle, (d) pre- antral follicle (e) and (f) antral follicle. Expression is stained with brown; the nucleus is stained with blue TABLE 3 Mean (±SD) number of buffalo follicles enclosed in ovarian tissue/mm 2 in fresh, vitrified (uncultured), vitrified (cultured control) and vitrified (cultured plus growth differentiation factor- 9 [GDF- 9]) Culture (days) Treatment No. of tissues Number of follicles Primordial Primary Secondary Pre- antral 0 Fresh ± ± a ± a ± a ± a ± a 0 Vitrified (uncultured) 14 Vitrified (cultured control) 14 Vitrified (cultured plus GDF- 9) Antral ± ± b ± 0.01 b ± b ± b ± b ± ± b ±00.02 b ± b ± b ± b ± ± c ± c ± a ± c ± b a c Within a column, means without a common superscript differed (p <.05). Total follicles and antrum formation in buffalo; hence, vitrified warmed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the absence or presence of GDF- 9. After in vitro organ culture, our findings showed that treatment with GDF- 9 promoted the primary follicles transition leading to a decrease in the number of primary and secondary follicles and a concomitant increase in the number of pre- antral and antral follicles (Table 2), with clear significant progression to the pre- antral stage seen in culture containing GDF- 9 compared to vitrified uncultured and vitrified cultured groups. This indicates that the follicle growth from one stage to the next is progressive (Table 3). In corroboration of the present study, Nilsson and Skinner (2002) demonstrated that GDF- 9 treatment has promoted the growth of the primary follicles in neonatal rat ovaries in vitro, but it has no effect on the growth of primordial follicles. Yet, in our previous study, Abdel- Ghani (2012) and Vitt, McGee, Hayashi, and Hsueh (2000) found that treatment with GDF- 9 in vivo

8 802 Abdel- Ghani et al. resulted in a decrease in primordial follicles compared with control, and this would suggest that GDF- 9 promotes primordial follicle transition in vivo model system. The possible explanation for the contradictory is that some primordial follicles may form during in vitro culture, however, a significant increase in the number of primordial follicles may not observed because also GDF- 9 promotes the transition of primordial follicles to primary. The importance of GDF- 9 for theca cell development has been shown in the GDF- 9 null follicles in mice that failed to form thecal layers, and thus, the GDF- 9 is required for recruiting theca precursors to surround the follicle (Dong et al., 1996). Subsequently, that may be explain why in buffalo antral follicles, GDF- 9 protein was expressed in the oocyte, theca cells, cumulus and mural granulosa cells (Table 1). The possible mode of action by which GDF- 9 stimulates differentiation of follicles from the primary up to and throughout the preantral stages could be by acts of GDF- 9 on many functions in the ovary. Wang and Roy (2006) have shown that when FSH- induced folliculogenesis is compromised by GDF- 9, ovarian somatic cells surrounding the oocyte nests or primordial oocyte clusters undergo morphological changes that are quite distinct from that occur in untreated ovaries. In cultured granulosa cells, GDF- 9 stimulates steroidogenesis (Yamamoto, Christenson, McAllister, & Strauss, 2002), synthesis of prostaglandin E2 receptor and progesterone by granulosa cells (Silva et al., 2005). Additionally, it has been demonstrated that granulosa cells lose their follicle- forming potential when the oocytes are surgically removed (Sadeu, Adriaenssens, & Smitz, 2008). Furthermore, deletion of GDF- 9 gene in mice results in the arrest in folliculogenesis beyond the primary stage, and exogenously added GDF- 9 promotes FSH- induced growth of rat pre- antral follicles in vitro (Jaatinen et al., 1999). Moreover, the GDF- 9 stimulates the growth of granulosa cells, and the oocytes from secondary follicles when transferred to primordial follicles accelerate the growth of the pre- granulosa cells in primordial follicles (Eppig, 2001). It was reported that the mechanism of action by which oocyte- derived GDF- 9 stimulates primordial and primary follicle progression could be indirectly by inducing the expression of other growth factors such as Kit ligand (KL) that is produced by granulosa cells, which in turn act on the cells of follicles to enhance the critical proliferation and differentiation events of follicles development (Nilsson & Skinner, 2002). Additionally, it appears that vitrification/warming has no remarkable effect on the expression of GDF- 9 protein related to primordial, primary and secondary follicles. However, the culture for long period resulted in a decrease in the GDF- 9 protein. These results imply that the addition of GDF- 9 in the culture media influenced the progression of follicles and resulted in progression and improvement in the number of pre- antral and antral follicles. In conclusion, GDF- 9 through its expression pattern plays a role in follicular progression. An understanding of the factors regulating the follicular progression could give rise to interesting future studies such as improving therapies and management of most common infertility problem in buffaloes (ovarian inactivity). CONFLICT OF INTEREST None of the authors have any conflict of interest to declare. AUTHOR CONTRIBUTIONS Dr. Tymor Mohammed El- sherry involved in analysis of the data and drafted the manuscript. 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