INRA, UMR 6175 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France.
Reprod Biol Endocrinol. 2010 Nov 1;8:132. doi: 10.1186/1477-7827-8-132.
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.
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.
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.
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.
卵泡液中含有参与卵泡活动、细胞分化和卵母细胞成熟的物质。对其成分的研究可能有助于更好地理解卵泡发育和卵母细胞质量的机制。犬种的特点是存在一些卵巢活动特征，这些特征尚未得到广泛描述，例如排卵前黄体化、GV 期（前期 1 期）排出的卵母细胞和多卵母细胞卵泡。在这项研究中，我们假设排卵前 LH 激增与排卵前卵泡液中类固醇和蛋白质含量的变化有关。
在排卵前阶段从犬卵巢中采集卵泡液样本，在 LH 激增之前（预 LH；n = 16 只母犬）和之后（后 LH；n = 16 只）。同时采集血液。类固醇通过放射免疫测定法进行测定，蛋白质组学分析通过 2D-PAGE 和质谱法进行。
在预 LH 阶段，卵泡液中 17β-雌二醇和孕酮的浓度分别为 737.2 ± 43.5 ng/ml 和 2630.1 ± 287.2 ng/ml，而血浆中分别为 53 ± 4.1 pg/ml 和 3.9 ± 0.3 ng/ml。在该阶段，记录到卵泡大小与卵泡内类固醇浓度之间存在显著正相关。在 LH 峰后，卵泡内 17β-雌二醇的浓度显著下降（48.3 ± 4.4 ng/ml；p < 0.001），而孕酮的浓度增加（11690.2 ± 693.6 ng/ml；p < 0.001）。LH 峰后，血浆中 17β-雌二醇的浓度没有变化（49 ± 9.6 pg/ml），但孕酮的浓度显著增加（9.8 ± 0.63 ng/ml）。犬卵泡液的蛋白质组学分析鉴定出 38 个蛋白斑点，对应 21 种蛋白质，其中一些已知在卵巢生理学中发挥作用。比较预 LH 和 LH 后阶段的卵泡液 2D-PAGE 图谱显示 3 个单独染色的斑点或斑点群。其中一个被鉴定为补体因子 B。比较卵泡液和血浆蛋白图谱显示，有一组 4 个斑点在血浆中的浓度高于卵泡液，而一个仅在卵泡液中存在的斑点。这些蛋白质分别被鉴定为凝胶蛋白和簇蛋白。
我们的结果首次证明了犬卵泡液中与 LH 激增相关的类固醇浓度与卵泡大小有关。2D 蛋白图谱分析鉴定出几种可能在排卵前阶段发挥作用的卵泡生理学和卵巢活动的蛋白质。这有助于未来解释和更好地理解在犬中描述的种特异性。
