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RESEARCH |
Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian EH25 9RG, UK and 1 Division of Integrative Biology, Roslin Institute (Edinburgh), Midlothian EH25 9PS, UK
Correspondence should be addressed to Elaine Watson; Email: Elaine.Watson{at}ed.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Growth factors, in particular the insulin-like growth factor (IGF) system, are thought to play a key role in ovarian follicular growth and atresia (Armstrong et al. 2000). The IGFs have a variety of effects on follicular and luteal cells, including stimulation of steroidogenesis, via increased availability of steroid precursors and upregulation of steroidogenic enzyme expression and activity. Follicular concentrations of IGF-I increase in large equine follicles (Bridges et al. 2002), whereas IGF-II concentrations are not different among different sizes of follicle. It has been suggested that intrafollicular IGF-I may be involved in selection of dominant follicles in mares (Donadeu & Ginther 2002, Ginther et al. 2002).
IGF-binding proteins (IGFBPs) present within the ovary bind with IGFs, prolonging the half-life, but blocking the biological action of the IGFs (for review see Armstrong & Webb 1997). Follicular concentrations of IGFBP-2, -4 and -5 decrease during follicular growth and increase during atresia in most species, including horses (Spicer & Echternkamp 1995, Gerard & Monget 1998). In contrast, levels of IGFBP-3 remain constant (Stewart et al. 1996, Gerard & Monget 1998). In large, oestrogen-active equine follicles, the relative proportion of IGF:IGFBP was twofold greater than in other follicle types, indicating that the increase in bioavailable IGF may play a key role in promoting follicular maturation (Bridges et al. 2002).
It is known that circulating concentrations of growth hormone (GH) in mares are higher during the ovulatory season than during winter anoestrus (Aurich et al. 1999). GH mediates many of its effects through IGF-I (Spicer & Echternkamp 1995), and it is apparently via this mechanism that GH alters ovarian function. Systemic administration of GH raises plasma concentrations of IGF-I in a number of species, including horses (Owen et al. 1991, Spicer et al. 1992, Buonomo et al. 1996). We hypothesized that the bioavailability of IGF would be lower during spring transition, when follicles grow to preovulatory size but fail to ovulate, contributing to failure of normal follicular steroidogenesis and development at this time. In order to investigate this hypothesis, various components of the IGF system were studied in large dominant follicles obtained from transitional and cyclic mares.
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Materials and Methods |
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Western ligand blot
The IGFBPs in follicular fluid were analysed by one-dimensional SDS–PAGE, as described previously (Nicholas et al. 2002). Samples (15 µl) were heat denatured in Tris–glycine SDS buffer (5 min at 100 °C), and then fractionated on 4% stacking/12% resolving SDS–PAGE gels (8.6 x 6.8 cm Mini-Protean III system; Bio-Rad, Hercules, CA, USA) at a constant current (125 V) for 2 h under non-reducing conditions. The proteins were electroblotted onto a PVDF membrane for 1.5 h at room temperature. After rinsing of the membrane and initial blocking with 3% (w/v) BSA in PBS–0.1% (v/v) Tween 20, the membrane was incubated overnight at 4 °C with biotinylated IGF-II. On the next day, the membrane was washed and the solution was replaced with 20 ml of a 1:4000 dilution of extravidin-peroxidase in blocking solution. After incubation for 5 min and further washing, the signal was detected with the ECL-plus system on hyperfilm-ECL film, and developed using an automated system (X-Ograph) with an exposure time of 3 min. The band densities were quantitated using Quantity One Control and Analysis Software for a Fluor-S Scanning System (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK).
In situ hybridization
Plasmids containing cDNAs for bovine IGFBP-2, IGF-I and IGF-II were linearized as described previously (Armstrong et al. 1998). Antisense and sense [35S]UTP-labelled cRNA probes were transcribed from these templates with a transcription kit (Promega Ltd, Southampton, UK) according to the manufacturer’s instructions. Purification of the cRNA probes was by centrifugation on a Sephadex G-50 column. The bovine cDNA sequences for IGF-I and IGF-II are 93% and 90% homologous with those of the horse. Equine IGFBP-2 cDNA has not been sequenced to our knowledge, but homology is greater than 90% in all species tested to date.
Sections (14 µm) were cut from each of the frozen tissue samples. Sections (n = 3 of each sample) were dehydrated, fixed and probed with the appropriate 35S-labelled riboprobes for IGFBP-2, IGF-I and IGF-II, by the method described by Xu et al.(1995). After the final high-stringency wash, the sections were dehydrated through a graded series of alcohols, air dried, dipped in autoradiographic K2 photographic emulsion (Ilford Ltd, Mobberley, UK) and exposed for up to 2 weeks at 4 °C. Sections were then developed (Kodak D-19; IBI Limited, Cambridge, UK) and fixed (Hypam fix, Ilford Ltd) before staining with haematoxylin and eosin. The sections were finally mounted in DPX mountant (BDH Laboratories, Poole, UK), before microscopic examination, using both light- and dark-field illumination. The antisense RNA probes were hybridized to two of the sections, and the remaining slide was hybridized with the corresponding sense RNA probes.
Quantitation of grain density
The intensity of the in situ hybridization signal was measured by an NIH-image analysis system (NIH, Bethesda, MD, USA). The number of graphic pixels occupied by silver grains (identified by a set grey threshold) within a defined area of the tissue section was counted and presented as a percentage of the total pixel number within the defined area. Background hybridization intensity, measured with the sense RNA probes, was subtracted from the measurements obtained with antisense probes to give the final hybridization signal. For each follicle, three separate fields were analysed for each probe. There was no significant difference (P > 0.05) in hybridization intensity obtained with antisense and sense RNA probes within a non-expressing region of a tissue section. Under the conditions described here, the hybridization signal was proportional to the length of time the slides were exposed to photographic emulsion (for up to 2 weeks).
Oestradiol assay
Concentrations of oestradiol-17ß were measured directly in follicular fluid by assays described previously (Glasier et al. 1989, Watson et al. 2002b). Intra- and interassay coefficients of variation were 4.6 and 7.8%. Assay sensitivity was 8 pg/ml, and displacement curves from serial dilutions of follicular fluid, and curves produced by addition of hormone to follicular fluid were parallel to the respective standard curve.
IGF-I assay
Concentrations of IGF-I were measured in follicular fluid and plasma by RIA after extraction using high-performance liquid chromatography (Gutierrez et al. 1997) to remove IGFBPs. The limit of detection of the assay was 23 pg. The intra-assay coefficient of variation was 7%. All samples were included in the same assay.
Statistical analyses
Differences in the steady-state concentrations of mRNAs between pairs of follicles (transitional versus preovulatory, and granulosa versus theca) and follicular fluid concentrations of components of the IGF system, within mares, were analysed by paired t-test. Differences in IGFBP-2 band densities were analysed using the OD figure generated by Quantity One Software. Correlations were analysed by Pearson’s correlation test. Hormone concentrations in samples collected at ovariectomy were compared by paired t-test. Differences were considered to be significant when P < 0.05. Means are quoted ± S.E.M.
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Results |
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Concentrations of oestradiol, IGF-I and IGFBP-2 in follicular fluid from preovulatory and transitional follicles
Concentrations of oestradiol were higher (P < 0.001) in follicles collected during cyclicity than during transition (Table 1
). There were no differences in oestradiol concentrations between follicles collected at second or third oestrus (1171 ± 274.5 vs 1725 ± 367.0 ng/ml respectively). Follicular IGF-I concentrations did not differ between transition and cyclicity (Table 1), and were not correlated with follicular oestradiol concentrations.
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). When IGF-II (1 µg) was added along with biotinylated IGF-II to the membranes, all the bands identified as IGFBP-2 and -3 disappeared. Concentrations of IGFBP-2 were 4.3 ± 1.12-fold higher (P < 0.001) in fluid from transitional follicles than in fluid from preovulatory follicles (Fig. 1).
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). There were no differences in plasma concentrations of IGF-1 between transition and cyclicity (Table 1). Plasma concentrations of IGF-I were not affected by age of the mare (3–20 years; Pearson correlation = –0.433; P > 0.05).
Concentrations of IGF-I were higher in plasma than in follicular fluid (P < 0.05; Table 1), and did not differ between cyclicity and transition. There was a positive correlation (r = 0.871; P < 0.001) between follicular and plasma IGF-I concentrations in cyclic mares, but not in transitional mares (r = 0.532; P = 0.062).
Expression of mRNAs encoding growth factors and binding proteins in follicles
Messenger RNA encoding IGF-I was present in the granu losa, with low expression in the theca (P < 0.001; Fig. 2) There was strong expression of IGF-II mRNA in theca cells. No mRNA encoding IGF-II was detected in the granulosa layer. Steady-state concentrations of mRNAs encoding both IGF-I and -II were higher (P < 0.01) in pre ovulatory follicles than transitional anovulatory follicles (Figs 3 and 4).
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). In transitional follicles, IGFBP-2 mRNA expression was similar in theca and gran ulosa cells. There was an increase (P < 0.001) in thecal expression in preovulatory follicles compared with transitional follicles, whereas expression in granulosa remained unchanged (Fig. 6).
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Discussion |
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In a previous study, pony mares first ovulated at a median date of 3 May (E D Watson, unpublished data); therefore, the first ovary of these mares was removed at least 1 month before this. The low concentrations of oestradiol in follicular fluid from the follicles collected in transition, and in plasma from transitional mares, confirm that these follicles were correctly identified and that potential preovulatory follicles were not removed in error. The mares in the present study were hemi-ovariectomized prior to onset of ovarian cyclicity. In a previous study, we confirmed that removal of one ovary during transition did not affect circulating concentrations of luteinizing hormone, follicle-stimulating hormone or oestradiol, or follicle growth during the ovulatory season compared with mares with two ovaries (Watson et al. 2004). Therefore, it is unlikely that removal of one ovary in the present study had any influence on follicular function in the remaining ovary.
The spatial distribution and expression of mRNAs encoding IGF-I and -II in follicles appears to be species-specific (Armstrong & Webb 1997). We have shown that mRNA encoding IGF-I is present in equine granulosa cells and, to a lesser extent, theca cells. In this respect, the horse may differ from other species. In rodents, pigs and women, the expression of mRNA encoding IGF-I is confined to granulosa cells (Armstrong & Webb 1997), and is only detectable in very low amounts in ruminant follicles (Perks et al. 1999, Armstrong et al. 2000). Messenger RNA encoding IGF-II was detected only in theca cells in the present study, in a spatial distribution similar to that reported in sheep, rats, pigs and cows (Armstrong & Webb 1997, Armstrong et al. 2000).
Intrafollicular concentrations of IGF-I, but not IGF-II, have been positively correlated with follicle size in mares (Spicer et al. 1991, Bridges et al. 2002), and concentrations of IGF-I are higher in follicles that assume dominance (Ginther et al. 2002). However, in other species, the presence of a correlation between follicle development and intrafollicular IGF-I has been less clear (for review see Monget et al. 2002), and this is thought to be due to differences in the efficiency of extraction of IGF-I. In the present study, IGF-I concentrations were measured in follicular fluid after acid gel filtration. Traditional extraction methods, such as solvent extraction, fail to remove all of the IGFBPs (Gutierrez et al. 1997); therefore, this is the first report of the measurement of total IGF-I concentrations in equine follicular fluid in which possible inaccuracies due to the presence of IGFBPs in the assay have been eliminated. However, in previous studies, the differences in IGF-I levels in different follicle types were small. Despite the identification of IGF-I mRNA in equine follicle walls in the present study, expression was low. Our results concur with those of Spicer and co-workers (1991), who reported that the main source of IGF-I in equine follicles is serum. This was confirmed in the present study by the higher plasma concentrations, and the highly significant correlation between plasma and follicular concentrations in cyclic mares. The lack of correlation in transitional mares probably reflects the lower vascularity of transitional follicles (Watson & Al-zi’abi 2002). We conclude therefore that the changes seen in expression of IGF-I mRNA in transitional and cyclic follicles may have little effect on its intrafollicular concentrations, because of the high levels present in serum. By contrast, the high level of expression of IGF-II mRNA in equine follicles in the present study, along with the observation that equine granulosa cells produce 10–50-fold more IGF-II than IGF-I in culture (Davidson et al. 2002), indicates that IGF-II is the major intrafollicular form of IGF ligand in the mare. There is discrepancy between the absence of mRNA encoding IGF-II from granulosa cells in the present study, and the production of IGF-II by cultured equine granulosa cells (Davidson et al. 2002). However, it is possible that some minor thecal contamination of the cultures may have accounted for IGF-II synthesis.
GH is a key regulator of circulating IGF-I concentrations and influences hepatic expression of IGF-I mRNA (O’Sullivan et al. 2002). Plasma IGF-I concentrations were increased in mares treated with equine somatotrophin, showing that a similar relationship exists in the horse (Cochran et al. 1999). GH concentrations are reported to be higher in mares during the breeding season than during winter anoestrus (Aurich et al. 1999), a finding which might suggest that IGF-I concentrations should be stimulated during the breeding season. However, this latter study did not differentiate between deep winter anoestrus and spring transition. It is possible that increases in GH and IGF-I concentrations precede spring transition, and are the stimulus for growth of the large transitional anovulatory follicles. The similarity in plasma IGF-I concentrations in transitional and cyclic mares suggests that changes in the somatotrophic axis are not involved in the transition from anovulatory to ovulatory cycles. A direct effect of GH on the ovary cannot, however, be excluded, and it is possible that the decreased expression of IGF-I mRNA in dominant follicles in transitional mares results from inadequate GH stimulation.
There was a wide age range in the mares in the present study, four of the mares being only 3 years old. It is thought that IGF-I and IGFBPs modulate growth in horses (Malinowski et al. 1996). However, in our study, age of the mare was not correlated with circulating IGF-I concentration, probably because adult size had almost been achieved.
In other domestic species, changes in IGFBP concentrations are more closely associated with follicle growth and regression than are changes in levels of IGF-I and -II (Spicer & Echternkamp 1995, Stewart et al. 1996), because only free IGF has biological activity. Furthermore, it has recently been shown that IGFBPs can directly inhibit steroidogenesis (Wright et al. 2002). In cyclic mares, intra-follicular levels of IGFBP-2, -4 and -5 decrease in large, oestrogenically active dominant follicles compared with subordinate follicles or large regressing follicles, which contain only low concentrations of oestrogen (Gerard & Monget 1998, Bridges et al. 2002). Therefore, it has been proposed that follicular levels of these binding proteins are closely related to the physiological status of equine follicles. Changes in these IGFBPs are probably regulated through changes in gene transcription and/or proteolysis (Rajarm et al. 1997). Levels of the IGFBPs in follicular fluid depend not only on synthesis, but also on degradation by proteases. In equine follicular fluid, there is little or no proteolysis of IGFBP-2 and -3; therefore, it was suggested that levels of these IGFBPs directly represent production (Bridges et al. 2002). However, IGFBP-4 and IGFBP-5 protease activity has been found in preovulatory equine follicles (Mazerbourg et al. 2000, Bridges et al. 2002), and therefore intrafollicular levels of these IGFBPs represent synthesis plus degradation.
It is reported that incubated granulosa cells from mares produce IGFBP-2 and -5 (Davidson et al. 2002); however, there has been no information on the presence or location of mRNA encoding the IGFBPs in equine follicles. In the present study, IGFBP-2 mRNA was located in both granulosa and theca layers, in contrast to the cow and sheep, where IGFBP-2 mRNA is confined to granulosa cells (Besnard et al. 1996, Armstrong et al. 1998), but similarly to the pig (Yuan et al. 1996) and women (Zhou & Bondy 1993).
In the present study, levels of IGFBP-2 were approximately fourfold lower in preovulatory follicles than in transitional follicles, despite the increased thecal expression of IGFBP-2 mRNA in preovulatory follicles. In contrast to the mare, in the cow and sheep the decrease in levels of IGFBP-2 during follicle growth is due largely to a loss of expression of mRNA encoding IGFBP-2 in the granulosa cells of dominant follicles (Besnard et al. 1996, Armstrong et al. 1998). One explanation of the discrepancy between message and protein in the mare would be the presence of IGFBP-2 protease activity in follicular fluid of preovulatory follicles. In the ewe and sow, there is increased IGFBP-2 proteolysis during follicle growth (Monget et al. 2002). However, earlier work has suggested that equine follicular fluid does not contain IGFBP-2 proteolytic activity (Bridges et al. 2002).
Subordinate or large regressing follicles with low concentrations of oestrogen are characterized by higher levels of IGFBP-2, -4 and -5 than are preovulatory follicles (Gerard & Monget 1998). The large transitional anovulatory follicles in the present study similarly contained higher levels of IGFBP-2 than preovulatory follicles, even though histological examination of transitional anovulatory follicles collected in the present study showed that they were healthy and did not have features associated with atresia (Kenney et al. 1979). The high levels of IGFBP-2 in transitional follicles would be expected to decrease IGF bioavailability in these follicles, and so contribute to the steroidogenic inadequacy of these follicles.
In conclusion, we have shown that expression of mRNAs encoding IGF-I and -II is greater in preovulatory follicles that are destined to ovulate than in large follicles collected during spring transition. Although expression of mRNAs encoding IGFBPs was similar, or even higher, in preovulatory follicles, intrafollicular concentrations of IGFBP-2 were lower in preovulatory follicles. The increased expression of IGF-I and -II in preovulatory follicles, coupled with decreased concentration of IGFBP-2, could increase intrafollicular IGF bioavailability in preovulatory follicles, compared with anovulatory follicles, and so contribute to the increased steroidogenesis and subsequent ovulation of these follicles.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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