Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

Abstract

Background: Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.

Methods: Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry.

Results: The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively.

Conclusion: Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.

Figure 1

17beta-estradiol concentration in the fluid of canine follicles. Canine follicular fluid was collected at the pre-LH stage (grey, n = 72) and at the post-LH stage (black, n = 73). Number of follicular fluids assayed is indicated within round brackets

Figure 2

Correlation between 17beta-estradiol concentration in plasma and in follicular fluid. Correlations were calculated at the pre-LH stage (crosses) and at the post-LH stage (black rhombs)

Figure 3

Progesterone concentration in the fluid of canine follicles. Canine follicular fluid was collected at the pre-LH stage (grey, n = 68) and at the post-LH stage (black, n = 73)

Figure 4

Correlation between progesterone concentration in plasma and in follicular fluid. Correlations were calculated at the pre-LH stage (crosses) and at the post-LH stage (black rhombs)

Figure 5

Silver stained 2D-PAGE profile of crude canine follicular fluid. 100 μg of proteins samples were applied to a non-linear pI 3-10 IPG strip in the first dimension and separated on a 10% SDS-PAGE gel in the second dimension (Molecular weight 200-10 kDa range). The positions of the high-abundant proteins albumin (ALB) and immunoglobulin heavy chain (HV01 and HV02), are indicated by dark full and dotted rectangles. Additional albumin fragments are indicated by dark circles (see Additional file 1, Table S1). All other identified proteins are named on the figure and their characteristics are shown in Table 1. NI: non-identified proteins

Figure 6

Computerized 2D-PAGE pattern analysis and comparison of pre-LH and post-LH canine follicular fluid. (A) 2D-PAGE profile of follicular fluid at the pre-LH stage and position of differential protein spots with the post-LH stage (dark rectangles). (B) Magnified and 3D images of differentially expressed protein spots of boxes 1, 2 and 3 (indicated by dark rectangles in A). (C) Bar charts indicates the relative spot volume (Mean +/- SEM) and fold change between both stages. Spots of box 1 have been identified as complement factor B

Figure 7

Computerized 2D-PAGE pattern analysis and comparison of canine follicular fluid and corresponding plasma samples. (A) Matching image of the 2D-PAGE profiles of plasma (in purple) and follicular fluid (in green). The positions of differentially expressed spots are indicated by dark rectangles. (B) Magnified and 3D images of differentially expressed protein spots of boxes 1 and 2 (indicated by dark rectangles in A). (C) Bar charts indicates the relative spot volume (Mean +/- SEM) and fold change between both biological fluids. Spots of box 1 and 2 have been identified as gelsolin and clusterin, respectively