Concentrations of bone morphogenetic protein-15 (BMP-15) and growth differentiation factor-9 (GDF-9) in follicular cysts, mono - and polyoocyte follicles in gilts

Abstract The objective of the study was to determine the concentration of BMP-15 and GDF-9 in the fluid of follicular cysts and ovarian follicles, and to compare their concentrations in mono- and polyoocyte follicles in gilts. The study involved two experiments conducted on the ovaries collected post-slaughter from gilts (7-8 months old). The first experiment covered 31 follicular single cyst gilts (15-25 mm in diameter) and 41 gilts without cysts. Follicular fluid from follicles of 8-10 mm in diameter (n=41) and 5-8 mm in diameter (n=41), and cystic fluid (n=31) were collected for analysis. The second experiment involved collecting follicular fluid from poly- (n=19) and monooocyte (n=22) follicles. The concentration of BMP-15 and GDF-9 was then determined in the samples using specimen-specific ELISA kits. The differences in the concentration of these factors were calculated by means of analysis of variance and a posthoc test. Duncan’s multiple range test was used to verify the significance of differences at P<0.05 and P<0.01. In addition, correlations between the factors were calculated. BMP-15 and GDF-9 levels in the cystic fluid were significantly higher than those in the follicular fluid (P<0.01). However, no differences were observed between various size follicles or between mono- and polyoocyte follicles. BMP-15 and GDF-9 concentrations were found to be positively correlated (P<0.01). Differences in BMP-15 and GDF-9 concentrations in ovarian follicles and follicular cysts, as evidenced by our study, indicate that these factors may be related to folliculogenesis disorders in gilts. What is more, the number of oocytes in ovarian follicles does not influence the intrafollicular concentration of BMP-15 and GDF-9.


INTRODUCTION
Many studies involving ovarian follicle functions have focused on the role of factors which are a part of the transforming growth factor beta (TGF-β) superfamily [1][2][3]. Amongst them, the bone morphogenetic protein-15 (BMP-15) and the growth differentiation factor-9 (GDF-9) deserve particular attention, as they participate in signalling pathways which control the development of ovarian follicles [3]. Recent research suggests that the factors affect the proliferation and differentiation of follicular somatic cells, steroidogenesis, deposition of the extracellular matrix, ovulation, and luteinization [2,4,5]. At the pre-ovulatory stage, the factors have an effect on oocyte maturation and cumulus cell expansion [6,7]. It is known that as a result of maturation, the oocyte gains developmental competence, which preconditions regular fertilization and subsequent embryo growth [8]. It is believed that BMP-15 and GDF-9 participate in the hormonal regulation of the hypothalamus-pituary gland-ovary axis [9]. Some suggest that the described factors may intensify or weaken gonadotropin impact on the ovarian follicle, depending on the stage of folliculogenesis. They affect LH receptor expression in the ovarian follicle somatic cells. It may play a role in the case of ovarian follicular cysts, which might be a result of LH defi ciency [10,11]. Ovarian cysts are a major issue, which upsets pig fertility, in sows and gilts alike [11][12][13][14]. Cystic ovaries in pigs may be unilateral or bilateral; whereas the cysts themselves are either single or multiple [10]. In most cases, single ovarian cysts have no impact on pig fertility, but they do have the potential to become multiple ovarian cysts, which can affect fertility in females [14]. It is thought that the direct reason for ovarian cyst formation are hormonal disorders [15]. Thus, considering the involvement of BMP-15 and GDF-9 in hormonal regulation of the ovarian follicle, one may not rule out the possibility that these factors play a part in ovarian cyst pathogenesis. Research into the role of BMP-15 and GDF-9 in the ovarian follicle prove that the mentioned factors are secreted mainly by oocytes. The majority of ovarian follicles contains only one oocyte; however, in some mammal species, there are follicles containing a higher number of oocytes [16][17][18]. The presence of polyoocyte follicles has been recorded, amongst others, in the ovaries of the pig [18]. Determining BMP-15 and GDF-9 concentrations in polyoocyte follicle fl uid perhaps may indirectly indicate the level of oocyte secretion activity in the follicles, especially as there has been no explicit common standpoint regarding the quality of such oocytes. Therefore, the aim of this study was to determine the concentration of BMP-15 and GDF-9 in follicular cyst and ovarian follicle fl uid, and to compare the concentrations of the said factors in mono-and polyoocyte follicles in gilts.

MATERIAL AND METHODS
Two experiments involving gilts (7-8 months old), slaughtered in the local slaughterhouse, were performed as part of the research.

Experiment No. 1
The experiment was conducted to compare BMP-15 and GDF-9 concentrations in follicular cysts and ovarian follicle fl uid. The analysis covered 31 gilts, in which follicular cysts were found, and 41 gilts without cysts. All follicular cysts, from which the fl uid was collected, were single, unilateral structures fi lled with some light, clear liquid, and a diameter range of 15-25 mm. A selected image of a single cyst in the ovary of a gilt is shown in Figure 1.
In the remaining gilts, one ovary was selected from which to collect follicular fl uidfrom an 8-10 mm follicle (n=41) and a 5-8 mm follicle (n=41). Ovarian follicles were well-vascularised and had a shiny surface according to Paradis et al. [7] and Hunter et al. [19]. Both cystic and follicular fl uid was aspired with the application of a needle and a syringe, and subsequently centrifuged (500 g for 10 minutes), decanted and stored at a temperature of -20 o C until analysis.

Experiment No. 2
Experiment no. 2 was carried out to compare the intrafollicular concentration of BMP-15 and GDF-9 between follicles classifi ed according to the number oocytes. Follicular fl uid was aspired from same-sized follicles (7-10 mm in diameter). To determine the number of oocytes, follicular fl uid was aspired with the use of a separate syringe with a fi tted needle, and then transferred into individual wells, according to the method by Stankiewicz et al. [18]. In order to minimise errors related to the possibility of oocyte loss, after each aspiration of a single follicle the syringe was rinsed out several time and its contents were transferred to another well. The content was evaluated in both wells. Moreover, the needle was used to gently scratch the follicular wall during aspiration. As described by Fouladi-Nashta et al. [20] this technique can be helpful in isolating oocytes from follicular wall compartments. After calculation of the number oocytes under the stereomicroscope, the follicular fl uid samples ,both the polyoocyte (n=19) and monooocyte (n=22) follicles, were centrifuged, separated and stored at -20 0 C until analysis.

Statistical analysis
The obtained results were further submitted for statistical analysis. The resulting data are presented as the mean ± SEM, marked on the graph, and tabulated. The differences in the concentration of the studied factors were calculated by analysis of variance and the posthoc test. Duncan's multiple range test was used to verify the signifi cance of differences at P<0.05 and P<0.01. In addition, correlations between the studied factors were calculated with the Spearman rank correlation coeffi cient. Statistical analysis was conducted using the STATISTICA version 7.1, Stat Soft, Poland.

RESULTS
Mean BMP-15 concentration in the fl uid of follicles with a diameter of 8-10 mm (n=41) was 0.63±0.02 ng/ml, and it was higher than in follicles with a diameter < 8 mm (0.53±0.02 ng/ml). Nevertheless, the differences between the groups were not statistically signifi cant (Fig. 2). Whereas, the concentration of BMP-15 in ovarian cysts was signifi cantly higher than in both groups of ovarian follicles, and was determined as 3.02±0.52 pg/ml (Fig. 2). As presented in Figure 3, differences in GDF-9 concentrations were distributed similarly. The concentration of GDF-9 in cysts (12.17±0.89 pg/ml) was signifi cantly higher than in follicles with a diameter of 8-11 mm (3.80±0.04 pg/ml) and in follicles with a diameter of 5-8 mm (3.46±0.08 pg/ml) (Fig. 3).  On the another hand, no statistically signifi cant differences were observed between concentrations of the examined factors in the mono-and polyoocytes follicle fl uid ( Table 1).
The BMP-15 and GDF-9 concentrations were found to be positively correlated (r=0.69; P<0.01), with the most pronounced correlation observed in the fl uid of ovarian cysts (Fig. 4).

DISCUSSION
Until recently, an oocyte in a developing follicle had been perceived as a passive structure, having no effect on the ovarian follicle development. However, it has been shown that the communication between an oocyte and follicular somatic cells is bidirectional, and the oocyte itself is capable of secretion [21]. Amongst factors secreted by oocytes are BMP-15 and GDF-9. Current research focuses on the interrelation between an oocyte and follicular somatic cells [2][3][4][5]22]. The positive correlation between BMP-15 and GDF-9 demonstrated herein confi rms a synergistic activity of the aforementioned factors [1,6]. This study demonstrates the presence of BMP-15 and GDF-9 in the ovulatory follicular fl uid of gilts, which proves the fact that they are components of the follicular microenvironment [2,4,23]. However, BMP-15 and GDF-9 concentrations in  follicular cysts were signifi cantly higher than those in the ovarian follicles. Such high concentrations in the cysts might have been a consequence of BMP-15 and GDF-9 synthesis via somatic cells.It has been demonstrated that the expression of GDF-9 mRNA in pigs occurs not only in oocytes but also in the granulosa cells [23]. Thus, it cannot rule out intercellular interaction disorders, which may result in high levels of BMP-15 and GDF-9. Sun et al. [24] indicates that BMP-15 and GDF-9 affect the balance between follicular cell proliferation and apoptosis. This balance disturbance is in turn one of the reasons of ovarian cyst formation [25]. Namely, the transition of the follicle from the preantral to early antral stage is the "penultimate" stage of development in terms of gonadotropin dependence and follicle destiny [growth versus atresia] [26]. Follicles selected for further development are thought to receive precise gonadotropic and intra-ovarian regulatory signals for survival, whereas follicular atresia is a consequence of inadequate growth support [27]. Since ovarian dysfunctions are the consequence of this transitional stage-specifi c dysregulated follicle growth, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral early antral transition may provide important insight into the pathophysiology of these conditions [28]. Another possible relation between BMP-15 and GDF-9 and cysts is demonstrated by their infl uence on the change in the direction of steroidogenesis. GDF-9 in rat intensifi es androgen synthesis via theca cells of the ovarian follicle [32]. However, as Pache et al. [30] described, androgens play an important role in the genesis of ovarian cysts. Furthermore, BMP-15 and GDF-9 gene mutations impact fertility and ovulation [31][32][33].
As the main source of BMP-15 and GDF-9 in the ovarian follicle are oocytes, the present study comprises an additional analysis of the concentration of these factors in the fl uid of mono-and polyoocyte follicles. One might have presumed that an increased oocyte count in the follicle would lead to higher concentration of both factors in the follicular fl uid. However, the concentration of BMP-15 and GDF-9 in polyoocyte follicles was only slightly higher than in the monooocyte ones. This might be the result of a diversifi ed secretory activity of oocytes from polyooocyte follicles. This suggestion found its confi rmation in the studies conducted by Stankiewicz et al. [18], who demonstrated the varied quality of oocytes from polyoocyte follicles. On the other hand, regardless of the number of oocytes and their secretory activity within a follicle, there might be mechanisms responsible for the maintenance of an optimal BMP-15 and GDF-9 concentration to provide for folliculogenesis.