Two other spots also identified as albumin most probably correspond to the native protein that has not correctly entered into the IEF gel indicated by dotted rectangle in Figure 5. Among them, 4 spots were identified as apolipoprotein A-1, and 2 spots as clusterin, complement factor B, inhibitor of carbonic anhydrase and haptoglobin. Most of the identified proteins are known to be secreted proteins, whereas 2 other are mostly known as cytosolic, such as actin and gelsolin, which are described as cytoskeleton proteins.
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. Computerized 2D protein pattern analysis was performed in order to compare 2D profiles at the two stages pre- and post-LH and to detect the differentially expressed proteins. We observed a high similarity in the 2D protein patterns before and after the LH peak, with only one group of spots and two singles that displayed significantly different levels of staining indicated by rectangles in Figure 6A.
The volumes of these spots are lower at the post-LH stage, by a magnitude of 2. Protein spots numbered 3, 5 and 14 corresponding to box 1 Figure 6A were identified as complement factor B, which has previously been detected in human follicular fluid [ 19 ]. B Magnified and 3D images of differentially expressed protein spots of boxes 1, 2 and 3 indicated by dark rectangles in A.
Spots of box 1 have been identified as complement factor B. Computerized comparison of 2D patterns allowed detection of differentially stained protein spots between these two biological fluids Figure 7A. One group of spots box 1 and a single spot box 2 were found differentially expressed between plasma and follicular fluid, as demonstrated by the magnified images and quantified spot volumes presented in Figures 7B and 7C , respectively. They were found at higher levels in plasma 7.
Spot number 33 box 2 was identified as a small isoform of clusterin which is in higher amount in follicular fluid than in plasma 3. Computerized 2D-PAGE pattern analysis and comparison of canine follicular fluid and corresponding plasma samples. 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. Spots of box 1 and 2 have been identified as gelsolin and clusterin, respectively.
During follicular growth and maturation, the follicular fluid is the microenvironment of follicular cells and of oocyte. It contains substances presumably implicated in cell differentiation, rupture of the follicular wall, and gamete quality. One can reasonably expect that the determination of its steroid content and protein composition will contribute to a better understanding of ovarian physiology and of regulation of follicular growth and maturation.
The canine species is characterized by several ovarian activity features that are not extensively described and well known yet, such as preovulatory luteinization; oocyte ovulated at GV prophase 1 stage and poly-oocytic follicles. The present study was designed to characterize the steroid and, for the first time, the proteomic content of canine follicular fluid and plasma. These may help in the future to explain and to better understand the species specificities that are described in dogs. In our investigation, 2D-PAGE study of canine follicular fluid revealed few differentially expressed proteins between follicular fluid and plasma gelsolin, clusterin , and between follicular fluid collected before or after the endogenous LH surge complement factor B and some unidentified proteins.
As previously mentioned, one of the main peculiarities of the canine ovarian physiology is the preovulatory luteinization [ 3 ], which is clearly observed in our study, with a significant increase in plasmatic progesterone nearly 2. The plasmatic levels of progesterone, but also of 17beta-estradiol, found in our study are similar to the ones previously reported by others [ 20 , 21 ] and by our own group [ 4 ].
Almost no published data are available regarding the steroid content of canine follicular fluid. To our knowledge, a single study has been carried out earlier Metcalfe, unpublished data [ 22 ] in Labrador bitches at the post-LH stage, with no precise description of punctured follicle sizes from 5 to 11 mm in diameter.
Our study is the first describing of intrafollicular levels of 17beta-estradiol and progesterone in parallel to blood concentrations with precise characterization of follicular diameter and stage pre- and post-LH stages in the canine species. As recorded previously in several mammalian species, concentrations of 17beta-estradiol and progesterone were found to be dramatically lower in blood than in follicular fluid.
This may be due to the fact that in dogs like other species, the follicle is a major site of steroid synthesis, and that steroids are mostly secreted and concentrated in follicular fluid, before entering blood flow. Nevertheless, we found no data regarding the ovarian localisation and regulation of steroid synthesis and secretion in the canine species.
We observed that the concentration of 17beta-estradiol in follicular fluid dropped significantly after the LH peak whereas that of progesterone increased significantly, which is consistent with findings in other mammals sheep: [ 23 ]; pig: [ 24 ]; cow: [ 25 ]; human: [ 26 ]; horse: [ 27 ]. For example the decrease in intrafollicular 17beta-estradiol after LH may be due to a decrease in aromatase level or activity, or to a decrease in androgen availability.
Additionally, the increase in intrafollicular 17beta-estradiol and progesterone concentrations coincident with the increase in follicle size that we observed before the LH surge, may also be the result from steroidogenic enzyme regulation within each follicle size category. Progesterone levels recorded in the present study in canine follicular fluid are much higher than those found in sheep [ 28 ], pig [ 24 ], cow [ 25 ] and human [ 26 ].
This is probably linked to preovulatory luteinization observed in the female dog [ 3 ]. This result suggests that, in view of mimicking in vivo conditions prior to in vitro maturation of canine oocytes, one should use high level of progesterone in the culture medium [ 29 ]. Finally, the high rate of poly-oocytic follicles is a peculiarity of the ovarian physiology in dogs [ 5 , 6 , 30 ]. In our study, no relationship could be demonstrated between the presence of several oocytes within a follicle and 17beta-estradiol or progesterone intrafollicular levels, but the number of follicles examined was very limited.
The second objective of our study was to describe the protein composition of the canine follicular fluid during the preovulatory phase, with special emphasis on the potential effects of LH. First, we used 2D-PAGE to characterize the global protein profile of canine follicular fluid, and mass spectrometry to identify some of the proteins present in follicular fluid. By combining these two approaches, we identified 38 protein spots in the canine follicular fluid, corresponding to 21 different proteins.
Our identification data are in agreement with earlier studies performed in human [ 31 ], bovine [ 32 ] and porcine [ 33 ] follicular fluids. Most of the identified proteins have previously been reported in plasma of various species [ 34 , 35 ], but in the present study, only follicular fluid free from blood contamination were used for the proteomic analysis.
Indeed, the blood-follicular barrier is known to allow the free diffusion of molecules under kDa [ 36 ]. Thus, the presence of such proteins in follicular fluid may be due to the vascular permeability of ovarian vessels during follicular growth and, at least for some of them, to local synthesis and secretion by follicular cells. In this study, we were able to identify 11 of the protein spots in the canine follicular fluid as albumin or its fragments, and 2 as heavy chains of immunoglobulin. These two types of proteins albumin and immunoglobulin were reported earlier in human [ 31 ] and bovine [ 32 ] follicular fluids proteome studies.
The existence of multiple forms of albumin with different isoelectric points could be due to chemical modifications of amino acid side chains [ 37 ]. Furthermore, this protein is known to function as carrier and transporter of proteins within blood and to bind physiologically important species such as lipid soluble hormones steroid hormones , free fatty acids apoprotein , calcium, ions transferrin , and cytokines [ 38 , 39 ].
Intrafollicular albumin may participate in the transport of metabolites involved in follicular growth or in the transfer to the general circulation of some follicle specific products like steroids.
Metcalfe SS: Assisted reproduction in the bitch. Progesterone levels recorded in the present study in canine follicular fluid are much higher than those found in sheep [ 28 ], pig [ 24 ], cow [ 25 ] and human [ 26 ]. ISBN Radioimmunoassays of arginine vasopressin and atrial natriuretic peptide: application of a common protocol for plasma extraction using Sep-Pak C18 cartridges. Internal Quality Control. Concepts worked out by years of clinical observations seemed possible of more definite proof or disproof.
Another group of proteins that we identified in the canine follicular fluid is made up of acute phase proteins including fibrinogen gamma, haptoglobin, complement factors, apolipoprotein A-I, alphaHS-glycoprotein, transferrin and retinol-binding protein 4, which are involved in inflammatory events [ 40 , 41 ]. Kim et al.
Moreover, the impact of haptoglobin on women's fertility has been shown by Bottini et al. The transport of this protein into the antrum depends on the integrity of the blood-follicle barrier and might be associated with oocyte quality, possibly by interfering with the role of apolipoprotein A1 in cholesterol or vitamin E exchange between high-density lipoproteins and granulosa cells [ 44 ]. Jarkovska et al. These proteins are the major protein component of high density lipoprotein HDL.
Balestrieri et al. These two proteins have already been observed in human follicular fluid [ 31 ]. Retinol and retinoids have been suggested to be essential for reproduction and to be involved in ovarian steroidogenesis, oocyte maturation and early embryonic development [ 47 , 48 ]. In the present study, a single isoform of retinol-binding protein was found in canine follicular fluid. This result is in accord with earlier observations by Anahory et al. In canine follicular fluid, other isoforms of retinol-binding protein may be present but either spots have not been stained and collected, or spot analysis did not permit identification.
Its presence in follicular fluid may be due to either passive filtration from serum across the blood follicular barrier into the follicular fluid, or to local synthesis [ 50 ]. In the present study, paraoxonase and transferrin were identified in canine follicular fluid. Both proteins are synthesized in the liver. Paraoxonase is bound and transported in plasma along with HDL.
It functions as an antioxidant by preventing the oxidation of LDL Low-density lipoproteins. Its presence has already been shown in human follicular fluid [ 31 ]. Transferrin is known as a circulating iron carrier protein, but is also produced in the testis and the ovary, where it acts as a growth factor, in addition to its role in iron endocytosis. The expression of transferrin was demonstrated in granulosa cells in human and mouse follicles [ 51 ]. In humans, the level of transferrin in the follicular fluid is highly correlated with the circulating level, suggesting that the contribution of local synthesis by granulosa cells during follicle maturation may be important.
There is plenty of evidence supporting the role of the LH surge in regulating numerous proteins implicated in ovulation and luteinization [ 52 ]. Although most of these proteins display a cellular localization, some have been localized in follicular fluid such as extracellular matrix glycoproteins [ 53 , 54 ], proteinases [ 55 , 56 ] and their inhibitors [ 57 , 58 ]. In the present investigation, we attempted to visualize and identify proteins in canine follicular fluid that may be modulated in response to the increase in circulating LH level.
Computerized protein pattern analysis and comparison of pre-LH and post-LH canine follicular fluids demonstrated the presence of higher levels of complement factor B at the pre-LH stage. The reason why its expression decreases at post-LH stages in dog is not clear, and such a decrease has never been described previously. According to previous studies in other species, the activation of the complement system may cause a deficiency of free vascular endothelial growth factor VEGF. Two other protein spots that were demonstrated in our study with a lower expression at the post-LH stage could not be identified by mass spectrometry.
This could be due to the low quantity of protein within spots, to the small number of cleavage sites for trypsin, which results in small number of peptides for identification.
The presence of formaldehyde in the coloration method may also interfere with amine functions and modify the protein mass. Because of the high permeability of the blood-follicle barrier, the follicular fluid content resembles that of plasma. In the present study, we hypothesize that comparison of 2D-PAGE proteins profiles of follicular fluid and plasma may reveal some proteins specific to the ovary. In this view, we demonstrated in the present study a higher level of clusterin in canine follicular fluid than in plasma.
Our result is in accordance with that of Jarkovska et al. Clusterin is a complement regulatory protein which plays an active role in inhibition of complement-mediated cell damage [ 62 ] and may also play a protective role in reproduction [ 63 ].
The high level of clusterin in follicular fluid might contribute to the inhibition of cytolytic activity of complement-mediated membrane attack. Finally, we showed higher level of gelsolin in plasma than in canine follicular fluid. Circulating gelsolin is the secreted isoform of cytoplasmic gelsolin, which participates in the clearance of actin from general circulation [ 64 ]. This protein was identified earlier in human follicular fluid [ 65 ], and in the mouse ovary where it is predominantly found in the theca externa and in stromal cells [ 66 ].
In the ovary, Teubner et al. Nevertheless, intrafollicular gelsolin may also be related to actin clearance that takes place in follicular fluid and its lower concentration in canine follicular fluid compared to plasma could be explained by this function [ 64 ]. Our results show that in canine as in other species, the intrafollicular steroid content is related to the follicle size, and is regulated at the time of the LH surge.
In contrast, intrafollicular progesterone levels are much higher than those found in other mammals, most probably due to intense luteinization in canine. Of note is the fact that in canine, intrafollicular steroid levels are largely higher than blood levels. Moreover, a combination of 2D-PAGE, computing image analysis and mass spectrometry led to the identification of 21 different proteins in canine follicular fluid. Most of them are functionally interesting and are expected to play an essential role in female reproduction. We demonstrated the regulation of intrafollicular complement factor B at the preovulatory stage, and the specific levels of gelsolin and clusterin in plasma and in follicular fluid.
These proteins may play a role in follicle physiology and ovarian activity at the preovulatory stage, or may be involved directly or indirectly in the regulation of the meiosis resumption.
Our observation may help in the future to explain and to better understand the species specificities that are described in dogs. Fortune JE: Ovarian follicular growth and development in mammals. Biol Reprod. Payer AF: Permeability of ovarian follicles and capillaries in mice. Am J Anat. J Reprod Fertil Suppl.
Tsutsui T: Gamete physiology and timing of ovulation and fertilization in dogs. Mol Reprod Dev. Canine and Feline Theriogenology. Fingland R: Ovariohysterectomy. Current Techniques in Small Animal Surgery.
Edited by: Bojrab M. Terqui M, Thimonier J: [New rapid radioimmunologic method for estimation of plasma progesterone. Application to early diagnosis of gestation in the ewe and goat]. Saumande J: Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH. J Steroid Biochem Mol Biol. Reprod Domest Anim. Anal Chem. J Proteome Res Metcalfe SS: Assisted reproduction in the bitch.
J Reprod Fertil. Stankiewicz T, Blaszczyk B, Udala J: A study on the occurrence of polyovular follicles in porcine ovaries with particular reference to intrafollicular hormone concentrations, quality of oocytes and their in vitro fertilization. Anat Histol Embryol. Nishimoto H, Hamano S, Hill GA, Miyamoto A, Tetsuka M: Classification of bovine follicles based on the concentrations of steroids, glucose and lactate in follicular fluid and the status of accompanying follicles.
J Reprod Dev. Cahill DJ, Wardle PG, Harlow CR, Hull MG: Effect of progestogen therapy on follicular development, related hormone concentrations and fertilization in vitro in unstimulated cycles and unexplained and endometriosis-associated infertility. Hum Reprod. Belin F, Goudet G, Duchamp G, Gerard N: Intrafollicular concentrations of steroids and steroidogenic enzymes in relation to follicular development in the mare. Telfer E, Gosden RG: A quantitative cytological study of polyovular follicles in mammalian ovaries with particular reference to the domestic bitch Canis familiaris.
Biochim Biophys Acta. J Endocrinol Invest. J Biomol Tech. Acta Endocrinol Copenh. Proteomics Clinical Applications.
The combination of GC with MS exploits the high-resolving power of gas chromatography to separate closely related molecules, and the ability of the MS to provide precise data for identification and quantification of the separated substances. GC-MS is a very powerful technique for analysis with specificity of hormones in biological fluids.
The general principles of GC-MS are described in this chapter along with some examples that illustrate specific applications of hormone analysis. Read Article at publisher's site.