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RESEARCH |
1 Reproduction, Genes and Development Group, Royal Veterinary College, Hawshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UK, 1 Animal Production Research Centre Mellows Campus, Athenry, Co. Galway, Ireland, 2 Faculty of Agriculture, University College Dublin, Dublin, Ireland and 3 Teagasc Dairy Production Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
Correspondence should be addressed to S Llewellyn; Email: sllewellyn{at}rvc.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The metabolic hormones insulin-like growth factor-I (IGF-I) and insulin are both likely candidates for mediating the effects of NEB on reproductive function. Circulating IGF-I concentrations fall sharply in the first week after calving. The extent of the fall has been related to both the interval to first ovulation and to conception (Beam & Butler 1999, Taylor et al. 2004). In the cow, the concentration of IGF-I in the follicular fluid of a dominant follicle reflects systemic IGF-I concentrations (Funston et al. 1996), whereas IGF-II is derived locally from theca cells (Perks et al. 1999, Armstrong et al. 2000). The bovine ovary can respond to IGFs via type 1 IGF receptors (Perks et al. 1999). IGF-I enhances the ovarian action of gonadotrophins (Adashi 1998) and also stimulates follicle cell proliferation and oestradiol production directly (Gutierrez et al. 1997). The local production of IGF-II supports follicle growth (Kamada et al. 1992) and steroidogenesis (Spicer et al. 2004).
Insulin also acts directly on antral follicle growth and function (Hamilton et al. 1999), appearing a less-potent mitogen of bovine granulosa cells than IGF-I (Spicer & Echternkamp 1995), but a similarly potent stimulator of oestradiol production (Gutierrez et al. 1997). Circulating insulin concentrations may (Gong et al. 2002) or may not (Meikle et al. 2004) be related to the time of re-initiation of ovarian cyclicity after parturition, but could act indirectly to influence ovarian activity, as insulin is a primary regulator of circulating IGF-I concentrations via effects on expression of GH receptors in the liver (Butler et al. 2003).
The bioactivity of both IGF-I and IGF-II in the ovary is regulated by the IGF-binding proteins (IGFBPs). In the ovine and bovine ovary, IGFBP-2 and IGFBP-5 are synthesised locally in the follicle wall by granulosa cells, and IGFBP-4 and IGFBP-6 in theca cells (Armstrong et al. 1998). Concentrations of IGFBP-2, IGFBP-4 and IGFBP-5 in follicular fluid decrease during growth and development of dominant follicles in cows (Spicer & Echternkamp 1995, Stewart et al. 1996). Dominance is associated with the inhibition of IGFBP mRNA expression (Webb et al. 1999) and post-translational modification via proteolytic degradation of both locally produced and systemically derived IGFBPs (Spicer 2004). The production of IGFBPs is thus a key factor controlling follicle growth and atresia (Bao & Garverick 1998). Expression of follicular IGF-1R, IGFBP-2 and IGFBP-4 mRNA are reported to be nutritionally sensitive (Munoz-Gutierrez et al. 2004) influencing the bioavailability of intrafollicular IGF and thus the sensitivity of follicles to gonadotrophins (Adashi 1998). We have recently shown in the liver that severe NEB specifically attenuates expression of IGF-I and IGFBP-3 to IGFBP-6 mRNA, while increasing IGFBP-2 synthesis (Fenwick et al. 2006). This profile fits a scenario of globally suppressed IGF-I availability, which likely contributes to an altered IGF environment in peripheral tissues involved in reproduction.
Development of a preovulatory follicle in the cow is estimated to take in excess of 40 days from the early antral stage to ovulation (Lussier et al. 1987). From antrum acquisition at around 300 µm, growth to 5 mm takes more than 30 days (Lussier et al. 1987) and from 5 mm follicles are recruited into a wave of gonadotrophin-dependent growth lasting 4–6 days that selects one follicle for dominance, while subordinate follicles regress (Rajakoski 1960, Fortune et al. 2001). Since the service period usually begins at about 50–60 days after calving, follicles that are due to ovulate at this time undergo their earlier stages of development during the NEB nadir. To the best of our knowledge, no previous studies have explored the effect of NEB on expression of the ovarian IGF system in lactating dairy cows. This study investigated the hypothesis that the early stages of follicular growth may be influenced by alterations to the follicular IGF system when cows are in NEB. Such changes might influence the fate of dominant follicles in the post partum period, contributing to longer anoestrus intervals seen in association with NEB. They might also affect later fertility via changes to oocyte quality (Britt 1994). The objective was to compare patterns of mRNA expression for the IGF system within pre-recruitment follicles during the time of NEB in two groups of cows managed to achieve a mild or severe negative energy balance status during early lactation.
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Materials and Methods |
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Energy balance was estimated as the difference between energy intake and the sum of energy for maintenance and milk production, based on measurements of milk yield, milk composition, liveweight and feed intake. The French NE system was used, where 1 unité fourragère lait (UFL) is the net energy for lactation equivalent of 1 kg standard air-dry barley (Jarrige 1989). Milk yield (kg) was recorded daily at the morning, evening and night milkings using electronic milk meters (Dairy Master, Causeway, Co. Kerry, Ireland). Milk composition (fat, protein and lactose) was determined twice weekly from successive morning, evening and night milk samples by automated infrared absorption analysis using a Milkoscan 605 (Foss Electric, Hillerod, Denmark). Forage and concentrate intakes were recorded electronically using the Griffith Elder feeding system. Samples of grass silage offered were taken twice weekly. Concentrate samples were taken once weekly. The dry matter intake was calculated on a daily basis. Cow body weight (kg) was recorded immediately post-calving and once weekly thereafter. The dry cows were weighed before feeding in the morning and the lactating cows were weighed after morning milking, before feeding.
The diameter of the first dominant follicle was determined for each cow from day 7 post partum via daily transrectal ovarian ultrasonography (Aloka SSD-900, Aloka Ltd, Tokyo, Japan, 7.5 MHz transducer). Cows were slaughtered on day 6–7 of the first follicular wave after calving. This design was chosen to collect tissue at a fixed time in relation to the stage of ovarian activity. The ovary contralateral to that containing the dominant follicle was bisected and the two halves were then immediately frozen using isopentane and liquid nitrogen, and stored at –80 °C.
Blood samples were obtained by jugular venipuncture and collected into vials containing lithium heparin as an anticoagulant. Thrice weekly sampling took place after morning milking and before feeding. The samples were placed on ice packs and were centrifuged at 2000 g for 10 min immediately after sampling. The plasma was decanted and stored at –20 °C until analysis. Daily samples obtained from day 7 post partum until slaughter were analysed for oestradiol-17ß. Samples collected pre-slaughter were analysed for IGF-I, insulin, glucose, urea, ß-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFA).
In situ hybridisation procedure
All chemicals were purchased from Sigma-Aldrich Company Ltd (Poole, Dorset, UK) or VWR International Ltd (Poole, Dorset, UK) unless otherwise specified. Serial sections of 10 µm were cut from each ovary using a cryostat. The sections were thaw-mounted onto Super-Frost Plus or POLYSINE microscope slides, fixed in 4% w/v paraformaldehyde in 0.01 M PBS (pH 7.0) for 5 min at room temperature, then washed thrice with 0.01 M PBS (3 min each), dehydrated in 70% (5 min) and 95% (1 min) ethanol and stored at 4 °C in 95% ethanol until used for in situ hybridisation.
The method was based upon that described previously by McMullen et al.(2005). All probes used were single-stranded oligonucleotides of 45 bases in length (Table 1
). The oligonucleotide probes (5 ng) were end-labelled with [35S]dATP (Amersham Biosciences UK Ltd) using terminal deoxynucleotidyl transferase (Promega UK Ltd) at 34 °C for 1 h. The labelled probe was diluted to a final concentration of 100 000 c.p.m. 100 µl–1 in hybridisation buffer (50% v/v deionised formamide, 4x SSC, 25 mM sodium phosphate, 1 mM sodium pyrophosphate, 5x Denhardt’s solution, 0.2 mg/ml denatured salmon sperm DNA, 120 µg/ml sodium heparin, 100 µg/ml polyadenylic acid and 100 mg/ml dextran sulphate) and 100 µl was added to each slide. The slides were incubated in a humidified box overnight at 42, 45 or 52 °C (Table 1). Following incubation, the slides were washed in a solution of 1x SSC, 2 g/l sodium thiosulphate at room temperature for 30 min, then in fresh 1x SSC, 2 g/l sodium thiosulphate at 60 °C for 60 min. The slides were then rinsed successively for 1 min in solutions of 1 x SSC, 0.1x SSC, 75% ethanol and 95% ethanol and air-dried. The slides were exposed to ß-max hyperfilm (Kodak BioMax MR Film) for either 4 or 5 days (Table 1).
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To aid cellular localisation of hybridised probes, selected slides were subsequently coated with photographic emulsion (LM1; Amersham Biosciences UK Ltd) according to the manufacturer’s instructions and stored for 28, 30 or 42 days (Table 1
) at 4 °C in the dark. The slides were developed in 20% phenisol (ILFORD Imaging UK Ltd, Knutsford, Cheshire, England) fixed in 1.9 M sodium thiosulphate and counterstained with haematoxylin and eosin. All other slides were also stained with haematoxylin and eosin to aid follicular characterisation.
Follicular characterisation
The follicles were characterised according to their size (very small, 1–2.5 mm and small, 2.5–5 mm) and stage of atresia. No follicles examined in this study exceeded 5 mm in diameter. The stage of atresia was assessed by morphological criteria (Irving-Rodgers et al. 2001) from the haematoxylin- and eosin-stained sections. Follicles classified as non-atretic had a regular granulosa layer with cells tightly packed and ‘healthy’ as indicated by a rounded, regular cell outline. Atretic follicles were marked by a convoluting and/or ruptured basement membrane, an irregular antral surface or granulosa cells of the most basal layer separated from each other and from the basal lamina.
Optical density measurements
The relative expression of mRNA for components of the ovarian IGF system was quantified from the autoradiographs using the public domain NIH Image program (available through the NIH website – http://www.nih.gov), which calculated the average OD over the selected area of film based on a linear grey scale of 0.01–2.71. For each ovary, individual follicles were identified and using the ‘freehand selection tool’, follicle walls were measured separately for mRNA expression of the gene of interest. Autoradiographs were scanned into a computer and images projected on screen so that areas of interest were selected and the OD measured. Readings were obtained from at least two sections per tissue for each of the antisense (AS) and sense (S) probes. The background OD, from a blank area of film, was also measured and subtracted from OD measurements of both AS and S. Finally, the S values were subtracted from AS values to give an average OD value for specific hybridisation (Perks et al. 1995). The detection limit was taken as an OD value of 0.01.
Metabolite and hormone assays
Blood plasma was analysed for glucose, NEFA, BHB and urea concentrations using appropriate kits (Randox Laboratories Ltd, Crumlin, Co. Antrim, UK) and a Mira autoanalyzer (HORIBA ABX, Montpellier, France). Measurement of plasma oestradiol concentrations used the Estradiol MAIA assay kit (BioStat Diagnostic Systems, Stockport, Cheshire, UK) based on the method used by Prendiville et al.(1995). The intra-assay coefficients of variation (CV) for samples with a mean of 1.1 and 4.6 oestradiol pg/ml of plasma were 15.8 and 7.3% respectively. The inter-assay CV for the same samples were 0.6 and 5.6% respectively.
Plasma insulin was assayed by a double antibody RIA (Williams et al. 2001). Briefly, 100 µl aliquots of plasma and standard were incubated with first antibody (50 µl guinea pig anti-bovine insulin; Sigma-Aldrich Company Ltd; diluted 1:100 in assay buffer) overnight at 4 °C. Bovine insulin (Sigma-Aldrich Company Ltd) was iodinated with 125I (Amersham Biosciences UK Ltd; 100 µl, approx 17 000 c.p.m.) was added to each tube; tubes were vortexed and incubated for an additional 24 h at 4 °C. Second antibody (100 µl; anti-guinea pig; SAC-CEL; IDS Ltd, Boldon, Tyne & Wear, UK) was added to each tube, vortexed and incubated at room temperature for 30 min. Bound and free fractions were separated by centrifugation, the supernatants decanted and counts per minute in the pellets were determined with a gamma counter. The intra-assay CV for samples with a mean of 0.09 insulin ng/ml of serum was 0.11% and the sensitivity was 0.05 ng/ml.
Plasma IGF-I was measured using human OCTEIA IGF-I kits (IDS Ltd; human and bovine IGF-I only differ by 1 amino acid) as described previously (Swali & Wathes 2006). The intra-assay CV was 2.1% and the sensitivity was 1.9 ng/ml. All samples for both insulin and IGF-I were measured in the same assays.
Statistical analysis
Statistical analyses were performed using Statistical Package for the Social Sciences version 13.0. Data for plasma metabolite and hormone concentrations at the time of tissue collection were analysed using Student’s t-test. Repeated measures ANOVA was used to determine the effect of treatment on daily UFL and plasma oestradiol concentrations. For mRNA expression in the ovary, the effects of treatment, follicle size and follicle health on the proportion of follicles expressing mRNA for each probe were analysed by the 
2-test. Levels of mRNA expression for each probe were compared between treatments, with follicle health (healthy or atretic) and size (very small (1–2.5 mm) or small (2.5–5 mm)) as factors, by linear mixed model analysis. Follicle and cow were entered as random effects. For this purpose, data from follicles in which a particular probe showed no detectable specific hybridisation (OD of < 0.01) were excluded from the analysis. Results were considered statistically significant when P < 0.05.
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Results |
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). Body condition score was unchanged. Measurements of plasma metabolite and hormone concentrations are reported in Table 2. Circulating concentrations of glucose and IGF-I were higher (P < 0.01) in the mNEB cows, whereas BHB and NEFA were lower (P < 0.01). Urea tended to be higher (P = 0.08) in the sNEB group. While the mean concentration of plasma insulin was numerically lower in sNEB cows, the difference was not significant (Table 2). At the time of tissue collection, mean circulating oestradiol concentrations (Table 2) and dominant follicle size (mNEB: 15.0 ± 0.69 mm, sNEB: 12.2 ± 1.25 mm) did not vary significantly between treatment groups.
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–4. The proportion of follicles showing detectable hybridisation for each probe (OD of AS–S value of >0.01 from the autoradiographs) is summarised in Table 3 according to follicle size and health. The only significant difference in proportion of follicles showing detectable hybridisation according to the EB treatment group was for IGFBP-6 mRNA (described below). For those follicles with detectable expression with a particular probe, the concentrations of mRNA in OD units are summarised in Table 4 according to size (very small (1–2.5 mm) or small (2.5–5 mm)), health and energy balance classifications. These data were analysed in a mixed model analysis and the significant effects of each factor and their two-way interactions are summarised in Table 5. The results are discussed below according to probe.
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) in 97% of follicles examined (Table 3). There was no effect of EB treatment on the expression of thecal IGF-II mRNA (Table 5). Levels of IGF-II mRNA expression were however affected by an interaction between follicle health and size. Expression was higher in healthy than atretic follicles (P < 0.05) and within healthy follicles, expression was higher (P < 0.01) in very small than small follicles (Fig. 5B).
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). Of 97 follicles examined, 26 showed no detectable IGF-1R mRNA expression and EB treatment did not affect the proportion of follicles expressing IGF-1R mRNA. Follicle health and size affected the proportion of follicles expressing IGF-1R mRNA when including all cows from both treatment groups in the statistical analysis (Table 3). There was a greater (P < 0.01) proportion of small than very small follicles expressing IGF-1R mRNA and it was detectable in more healthy than atretic follicles (P < 0.01). When IGF-1R mRNA was detectable, the concentrations measured were not affected by EB treatment, follicle size or health (Table 5).
Expression of IGFBP-1 and IGFBP-3 mRNA
The expression of IGFBP-1 and IGFBP-3 mRNA could not be detected in any follicles from either treatment group, regardless of follicle health or size, despite expression being detected in ovine oestrus uterus and placentome used respectively, as positive control tissues (data not shown).
Expression of IGFBP-2 mRNA
IGFBP-2 mRNA was localised to granulosa cells (Fig. 3). Of 92 follicles examined, 47% did not express IGFBP-2 mRNA. Neither EB treatment nor follicle health affected the proportion of follicles expressing IGFBP-2 mRNA. When including all cows from both treatment groups in the statistical analysis (Table 3), a larger proportion of small than very small follicles were expressing IGFBP-2 mRNA (P = 0.01). Follicular IGFBP-2 mRNA expression was the only member of the IGF family influenced by the cow’s EB status. Concentrations of granulosa IGFBP-2 mRNA were higher (P < 0.05) in mNEB than in sNEB cows (Table 5 and Fig. 5A).
Expression of IGFBP-4 mRNA
IGFBP-4 mRNA was detected in theca cells (Fig. 3) with 27% of 92 follicles examined showing no detectable expression (Table 3). The proportion of follicles expressing IGFBP-4 mRNA was unaffected by EB treatment, follicle size or follicle health. There were similarly no significant effects of EB group, follicle size or health on the measured concentrations of IGFBP-4 mRNA (Table 5).
Expression of IGFBP-5 mRNA
IGFBP-5 mRNA was expressed by granulosa cells (Fig. 4). Of 105 follicles examined, 50% did not express IGFBP-5 mRNA and the proportion of follicles expressing IGFBP-5 mRNA did not vary between EB treatment groups. Follicle size and health affected the proportion of follicles expressing IGFBP-5 mRNA when including all cows from both treatment groups in the statistical analysis (Table 3). There was a greater (P < 0.01) proportion of small than very small follicles expressing IGFBP-5 mRNA and it was detectable in more atretic than healthy follicles (P < 0.01). Concentrations of IGFBP-5 mRNA were not affected by EB treatment or follicle size, but there was a trend (P = 0.063) for increased expression in atretic follicles (Tables 4 and 5).
Expression of IGFBP-6 mRNA
IGFBP-6 mRNA was detected in theca cells (Fig. 4). Of 96 follicles examined, 32% showed no detectable expression of IGFBP-6 mRNA (Table 3). EB status had an effect on the proportion of follicles expressing IGFBP-6 mRNA. Within very small atretic follicles, the sNEB cows were more likely to have detectable expression than the mNEB cows (69% of 13 follicles examined compared with 28% of 18 follicles examined, P < 0.01). Overall, the proportion of healthy follicles expressing IGFBP-6 mRNA was greater (P = 0.01) than atretic follicles but follicle size had no effect (Table 3). When IGFBP-6 mRNA was detectable, there was no effect of EB treatment on its expression (Table 5). Concentrations of IGFBP-6 mRNA expression were however affected by an interaction between follicle health and size. Within healthy follicles, IGFBP-6 expression was higher (P < 0.05) in very small than small follicles (Fig. 5C).
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Discussion |
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The samples were obtained during the first follicular wave, which occurred in the second week after calving. The follicles which were examined did not exceed 5 mm in diameter, consistent with the data of Dufour & Roy (1985) who reported that the follicular population in early post partum dairy cows consisted predominantly of follicles < 4 mm in diameter. These smaller follicles, at the pre-recruitment stage of development (Fortune et al. 2001), may emerge in succeeding follicle growth waves and potentially progress to dominance before and during the service period. The prevailing follicular IGF system at the time of collection may therefore affect post partum ovarian cyclicity and follicular and/or oocyte quality, with consequences for subsequent conception rates.
IGF-II mRNA was expressed by theca cells, confirming earlier observations in both the bovine (Armstrong & Webb 1997, Perks et al. 1999) and the ovine (Perks et al. 1995) ovary. The higher concentrations of IGF-II mRNA found in healthy than atretic follicles may indicate that IGF-II is important for follicular survival. IGF-II mRNA expression also decreased with increasing follicle diameter (Perks et al. 1999). Since smaller ovarian follicles have a greater capacity to proliferate whereas larger follicles have greater steroidogenic ability (Spicer & Echternkamp 1995), the higher concentration of IGF-II mRNA in healthy follicles of 1–2.5 mm implies that IGF-II may play a greater role in follicle cell proliferation than oestradiol production. Both IGF-I and IGF-II have been shown to stimulate follicle growth in vitro (Kamada et al. 1992, Gutierrez et al. 1997) and so the lower concentration of circulating IGF-I in sNEB could detrimentally affect folliculogenesis (Lucy et al. 1992). Previous work has shown that cows with low circulating concentrations of IGF-I during the first 2 weeks post partum subsequently have poor conception rates to first service (Beam & Butler 1999, Pushpakumara et al. 2003), whereas those returning to positive energy balance by week 3 post partum have higher follicular fluid IGF-I concentrations and produce more good quality oocytes (Kendrick et al. 1999).
The IGF-1R was localised to follicle granulosa cells, confirming earlier observations in the cow (Perks et al. 1999), whereas in the ovine ovary we have detected expression in both granulosa and theca layers (Munoz-Gutierrez et al. 2004). IGF-1R mRNA expression did not vary with follicle size (Stewart et al. 1996, Perks et al. 1999), which also contrasts with the ewe, where concentrations decreased with increasing follicle diameter (Perks et al. 1995). We found a clear reduction in the proportion of atretic follicles expressing IGF-1R mRNA compared with healthy follicles. A reduction in the number of IGF-1R in the follicle wall would be likely to reduce IGF-I and IGF-II stimulated follicle growth and function in atretic follicles (Monget et al. 1993). sNEB did not down-regulate IGF-1R mRNA expression, implying that IGF-stimulated ovarian function is not arrested despite concurrent body catabolism. Hamilton et al.(1999) similarly found that granulosa cells from nutritionally induced anoestrus cows retained the capacity to respond to IGF-I in vitro.
The expression of IGFBP-2 mRNA was confined to granulosa cells (Armstrong et al. 1998, Munoz-Gutierrez et al. 2004). Whilst there was no effect of EB on the proportion of follicles expressing IGFBP-2 mRNA, EB status was the main factor influencing the concentration, with higher expression in the mNEB group. Munoz-Gutierrez et al.(2004) also found that granulosa IGFBP-2 mRNA was nutritionally regulated in the ewe, with concentrations increasing in response to glucose infusion. Work in vitro showed that the production of IGFBP-2 by cultured ovine and bovine granulosa cells was dependent on and stimulated by the presence of IGF-I in the culture medium (Armstrong et al. 1996, Voge et al. 2004). The reduced expression of granulosa IGFBP-2 mRNA in the sNEB group may therefore have been caused by the lower glucose and/or IGF-I concentrations found in these cows. Neither follicle health nor size affected the expression of IGFBP-2 mRNA. Armstrong et al.(1998) also found no difference in granulosa IGFBP-2 mRNA expression in 1–8 mm healthy bovine follicles, but reported that levels of gene expression decreased with atresia. In contrast, de la Sota et al.(1996) found increased levels of IGFBP-2 in large atretic follicles collected in the mid-luteal phase. As IGFBP-2 is generally considered to inhibit the action of the IGFs (Monget et al. 2003), then assuming the reduced expression of IGFBP-2, mRNA leads to lower intrafollicular IGFBP-2 in sNEB, the availability of free IGF-I and IGF-II may be increased for IGF-stimulated follicle growth and oestradiol production in small follicles (Monget et al. 1993). Indeed, levels of free IGF must increase as follicle size increases (Monget et al. 2003), in order to enhance follicular response to follicle-stimulating hormone (FSH; Mihm et al. 2000) and increase follicular oestradiol secretion (Gutierrez et al. 1997). This might represent a mechanism to support the continued growth of antral follicles despite NEB or severe chronic undernutrition (Rhodes et al. 1995).
IGFBP-4 mRNA localisation in theca cells is in agreement with other studies (Armstrong et al. 1998). Treatment did not affect concentrations of IGFBP-4 mRNA, implying that transcription of this IGF-binding protein is resistant/insensitive to severe negative energy balance. Levels of expression did not vary with follicle size, concurring with work by Armstrong et al.(1998) in the cow. We also found no effect of follicle health on concentrations of IGFBP-4 mRNA, which is dissimilar to other studies in the cow and ewe that report lower IGFBP-4 mRNA in atretic follicles when data is combined from small, medium and large follicle sizes (Armstrong et al. 1998, Hastie & Haresign 2006). The survival of growing follicles becomes increasingly dependent upon luteinizing hormone (LH; Mihm & Bleach 2003), and thereby expression of the LH receptor (LHR) and thecal IGFBP-4 production in vitro is also stimulated by LH (Armstrong et al. 1996, 1998). It may be that expression of IGFBP-4 mRNA and thecal LHR mRNA (Stewart et al. 1996) are associated.
The expression of follicular IGFBP-5 mRNA was localised to granulosa cells, supporting the detection of bovine granulosa IGFBP-5 mRNA with RT-PCR (Schams et al. 1999). The proportion expressing IGFBP-5 mRNA was significantly greater in atretic than healthy follicles. In addition, concentrations of IGFBP-5 mRNA also tended to be greater in atretic follicles, as reported for the ovine ovary (Hastie & Haresign 2006). Assuming IGFBP-5 message leads to IGFBP-5 protein that reduces IGF ligand bioavailability, the subsequent decrease in IGF-stimulated follicular development is probably a factor in follicular atresia. Other studies similarly report higher IGFBP-5 protein in oestrogen-inactive, atretic and subordinate follicles compared with oestrogen-active, healthy and dominant follicles (de la Sota et al. 1996, Santiago et al. 2005).
The localisation of IGFBP-6 mRNA in the ovary is poorly documented, but this study detected IGFBP-6 mRNA in theca cells, which agrees with Schams et al.(1999). IGFBP-6 has a markedly higher affinity for IGF-II (Baxter 2000). The higher IGFBP-6 expression in healthy follicles 1–2.5 mm in diameter than follicles 2.5–5 mm in diameter parallels that of IGF-II mRNA and may thus contribute to the control of IGF-II-stimulated folliculogenesis.
IGF-binding proteins probably act to inhibit the activities of IGFs, but they may also enhance IGF action by storing IGFs intracellularly (Blum et al. 1989) or facilitating transport of IGFs into their target tissues (Hall et al. 1987). IGFBP-2 and IGFBP-4 have been found within the extracellular matrix surrounding granulosa cells (Glazyrin et al. 1996), where they may act as a reservoir to sustain controlled delivery of IGFs to the IGF-1R (Clemmons 1998). NEB may thus influence the storage of IGFs within the ovary by influencing the association of IGFBPs with the extracellular matrix. Another possibility, which we did not examine, is that EB status might alter the action of specific IGFBP proteases that are needed to access the extracellular store of IGFs (Hill et al. 1989), thus influencing the bioavailability of IGF for follicle growth and function (Armstrong et al. 1998). The ability of the low molecular weight binding proteins to cross the endothelial barrier (Bar et al. 1990) would suggest that follicular IGFBPs may transport IGFs from the vasculature into theca and granulosa cells, further increasing the intraovarian concentration of IGFs. It seems then that the lower concentration of IGFBP-2 in sNEB, whilst increasing the bioavailability of IGFs within follicle cells, could impact negatively upon the transport and storage of IGFs within the ovary. There is also increasing evidence that some of the IGFBPs may exert IGF-independent effects on target cells (Rajaram et al. 1997); IGFBP-3, for example, has been shown to inhibit proliferation of breast cancer cells (Oh et al. 1993), while IGFBP-5 possesses transcription-regulatory activity (Xu et al. 2004) and may promote cell proliferation in osteoblasts (Andress & Birnbaum 1992, Mohan et al. 1995). In the ovary, IGFBPs may thus act directly to inhibit granulosa steroidogenesis (Bicsak et al. 1990).
In summary, we have shown that the concentration of mRNA for IGFBP-2 and the pattern of IGFBP-6 mRNA expression in pre-recruitment ovarian follicles are influenced by EB status in the post partum dairy cow. Expression of mRNA for IGF-1R, IGF-II, IGFBP-4 and IGFBP-5 did not follow the same trend, indicating that regulation of the ovarian IGF system is mediated by more than one mechanism. Future studies should develop a better understanding of the mechanisms underlying the effects of NEB on the IGF system, which in turn are likely to influence the timing of the return to ovarian cyclicity after parturition.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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) = milked once daily on ad libitum grass silage with 8 kg/day concentrate; sNEB (
) = milked thrice daily on 25 kg/day silage with 4 kg/day concentrate. Values are mean ± S.E.M., n = 6 cows per group. Over the 2-week treatment period, there was a significant (P = 0.02) difference in mean EB between treatments. UFL, unité fourragère lait.
very small, VS;