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Female Reproductive Tract |
a Department of Veterinary Basic Sciences, Royal Veterinary College, North Mymms, Hatfield,Herts AL9 7TA, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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growth factors, placenta, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IGFBP-6 is the most recently characterized member of the IGFBP family, and relatively little is currently known about its role and regulation. It has a predicted molecular weight of 23 kDa and is O-glycosylated. The glycosylated form of IGFBP-6 exhibits much greater resistance to proteolysis than its nonglycosylated counterpart [6]. IGFBP-6 is distinct in its preferential affinity for IGF-II relative to IGF-I, which, unlike any other IGFBP, is 20- to 100-fold higher [7]. IGFBP-6 functions to inhibit the actions of IGF-II, with inhibition thought to result from the formation of high-affinity IGF-IGFBP complexes that prevent IGF-II from binding to the IGF receptors [7]. Several hormones, including a number of growth factors, have been shown to regulate IGFBP-6, but the manner in which they act appears to be very cell-specific, and the mechanisms at this time are not fully understood [7]. The purpose of the present study was therefore to investigate the spatial and temporal expression of IGFBP-6 in the nonpregnant and pregnant ovine uterus in an attempt to gain insights into its regulation and possible roles in utero.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nonpregnant samples Reproductive tracts were collected from 55 nonpregnant ewes at different time points throughout the estrous cycle. The stage of the estrous cycle was regulated by an i.m. injection of Estrumate (Coopers Animal Health Ltd., Crewe, U.K.), a prostaglandin (PG) analogue given in the midluteal phase to induce estrus. Following Estrumate treatment, estrus can be expected at 48 h with ovulation occurring at approximately 65 h. Estrus behavior was checked by running the ewes with a raddled vasectomized ram. The number of samples collected at each time point was as follows: 24–36 h post-PG (pPG), n = 9; 48 h pPG, n = 8; 60–65 h pPG, n = l0; Day 2, n = 7; Days 3–5, n = 5; Days 7–9, n = 8; Days 13–15, n = 8; and Day 15, n = 3. In addition, uteri were collected from three ovariectomized and four anestrous ewes.
Pregnant samples Reproductive tracts were taken from 35 pregnant ewes ranging in gestational age from 13 to 55 days pregnant. The number of samples collected at each time point was as follows: Day 13, n = 4; Day 15, n = 3; Days 16–18, n = 4; Days 21–30, n = 9; Days 31–40, n = 7; and Days 41–55, n = 8.
Transected samples Reproductive tracts were also collected on Days 16 and 17 from four ewes with unilateral pregnancies following uterine transection prior to mating. The horn ipsilateral to an active corpus luteum was sectioned, the endometrium cauterized, and the myometrium of the two ends sutured [8]. Both horns (the pregnant and nonpregnant) were therefore subject to the same endocrine environment, but were surgically isolated from each other. This technique provided an opportunity to study the effect of local regulatory factors produced by the embryo on IGFBP-6 expression.
All animals were kept and treated under the Home Office Animals (Scientific Procedures) Act 1986. Uteri were dissected transversely into segments of approximately 2–3 cm in length, wrapped in aluminium foil, and frozen in liquid nitrogen-tempered isopentane. Samples were stored at -80°C until sectioning.
Oligonucleotide Probe
The 45 base oligonucleotide antisense probe corresponded to nucleotides 292–336 of the bovine IGFBP-6 gene [9] and had the following sequence: 5'-CCG-CTT-CCG-GTA-GAA-GCC-CCT-ATG-GTC-ACA-ATT-AGG-CAC-GTA-GAG-3'. A sense probe corresponding to the antisense probe (identical in sequence to the respective mRNA target) was included as a negative control, as any signal it produced could be categorized as nonspecific. To date the ovine sequence has not been determined, so the region selected was chosen for 1) its high homology with the human (93%) and the mouse (82%) IGFBP-6 gene, and 2) low homology with other IGFBPs.
In Situ Hybridization
The in situ hybridization procedure was performed as described previously [2]. All chemicals were purchased from Sigma Chemical Co. (Poole, Dorset, U.K.) or BDH (Poole, Dorset, U.K.) unless otherwise stated. Briefly, 10-µm cryostat sections were thaw-mounted onto poly-L-lysine coated slides, fixed in 4% (wt/vol) paraformaldehyde in 0.01 M PBS, washed in PBS, and dehydrated sequentially in 70% and 95% ethanol. The oligonucleotide probe for IGFBP-6 was 35S-labeled at the 3'-end using terminal deoxynucleotidyl transferase (Pharmacia Biotechnology, St. Albans, Herts, U.K.). Tissue sections were subsequently treated with 110 000 cpm probe in 200 µl hybridization buffer and hybridized overnight at 42°C. The following morning, slides were washed in lx saline sodium citrate (lx SSC) 0.2% (wt/vol) sodium thiosulphate pentahydrate solution for 30 min at room temperature, then at a higher stringency in 1x SSC 0.2% (wt/vol) sodium thiosulphate pentahydrate solution for 60 min at 55°C. Finally the slides were dehydrated in a gradient of ethanol, air-dried, and exposed to x-ray film, Hyperfilm-ßmax (Amersham International, Aylesbury, U.K.), for 28 days.
Photographic Emulsions
Slides previously exposed to x-ray film were coated with a photographic emulsion (LMI; Amersham) according to the manufacturer's instructions and stored at 4°C for 35 days. Slides were then developed and counterstained with hematoxylin and eosin in order to confirm microscopically the cellular localization of the radioactive signal.
Optical Density Quantification
The quantification method was performed as described previously [2]. An image analysis system (Seescan PLC, Cambridge, U.K.) was used to convert the radioactive signals to arbitrary optical density (OD) units for quantification using a linear gray scale of 0–2.1. All readings were on this part of the scale. The antisense autoradiograph was placed under the image analyzer lens, and a blank section of the film was measured. The region to be quantified was outlined, and the background reading was automatically subtracted from this to provide a reading of the average optical density over the area outlined. The corresponding section on the sense autoradiograph was measured in a similar fashion and the reading obtained subtracted from the antisense to give the final OD value for the specific hybridization. A minimum of two antisense slides, each containing at least two sections, were measured for each animal and the average OD value calculated. The detection limit was taken as an OD > 0.01. The coefficient of variation for duplicate absorbance measurements between two slides was 4%. All samples were processed together in one batch.
Statistical Analysis
Values are given as mean OD ± SEM. Statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) version 9.0. Differences between time points during the cycle or pregnancy were analyzed by one-way analysis of variance (ANOVA). LSD tests were used to determine which time points differed. A one-way ANOVA was also used to compare cyclic, anestrus, and ovariectomized ewes. Differences according to pregnancy status were analyzed by two-way ANOVA with pregnancy status and day as factors. If the data did not exhibit homogeneity of variance, then they were log-transformed. If this had no effect, a Kruskal-Wallis nonparametric one-way ANOVA was used instead. If no time-related changes were detected, all data from a particular region were pooled between time intervals. Results were considered statistically significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the cycle the highest level of IGFBP-6 mRNA (measured in arbitrary OD units) was found in the periepithelium as illustrated in the autoradiographs (Fig. 1, A and C) and emulsions (Fig. 2). There were moderate levels in the caruncles (Figs. 1A and 2A), whereas in the remaining endometrial stroma, IGFBP-6 mRNA levels were low. In all these regions, the relative levels of IGFBP-6 mRNA varied significantly according to the stage of the cycle, with a peak around estrus and ovulation that extended into the early luteal phase in the periepithelium (Fig. 3). In the myometrium IGFBP-6 OD, concentrations were low and did not change throughout the estrous cycle (OD 0.03 ± 0.002, n = 52; Fig. 1). In addition, 16 out of 25 ewes expressed IGFBP-6 mRNA at moderate concentrations in their endometrial glands between estrus and Day 2 of the cycle (OD 0.15 ± 0.010, n = 16; Fig. 1A). This glandular expression of IGFBP-6 was unique to these ewes at this stage of the estrous cycle and was not detected at any other time or in the glands of any other ewes, irrespective of their reproductive status.
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Uteri from Anestrous and Ovariectomized Ewes
In ovariectomized ewes, IGFBP-6 mRNA was detected at a similar level to that seen in the mid to late luteal phase of the cycle (Fig. 3). In contrast, levels were higher in anestrous ewes, with OD measurements in most tissues equivalent to the peak concentrations recorded around ovulation (Fig. 3).
Pregnant Uteri
During early pregnancy (13–55 days), IGFBP-6 mRNA was expressed in similar regions and at similar OD concentrations to those recorded during the estrous cycle, although it was absent from the glands (Fig. 2D and Fig. 4, A and C). In the endometrial stroma and myometrium, IGFBP-6 mRNA levels remained low throughout the period of gestation studied (OD 0.05 ± 0.004, n = 33, and OD 0.05 ± 0.003, n = 28, respectively; Fig. 4). In the periepithelium and caruncular stroma, IGFBP-6 mRNA OD concentrations measured during pregnancy were equivalent to those at ovulation (Table 1). A comparison was made between samples collected from nonpregnant and pregnant ewes over the period of 13–17 days of the cycle/pregnancy. There was no change in time over this period, so values were pooled. In both the periepithelium and caruncular stroma, OD concentrations were significantly higher in pregnant than nonpregnant ewes (Fig. 5). Following fetal allantochorion invasion and placentome formation, moderate levels of IGFBP-6 mRNA were found in the placentome capsule (Fig. 4E). Analysis of the photographic emulsions also revealed an intense localization of IGFBP-6 mRNA to immune cells, possibly monocytes (Fig. 2, E–G). There was no detectable hybridization to any fetal placental tissue (Fig. 2D).
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Transected Uteri
There was no significant difference in IGFBP-6 mRNA levels between nonpregnant and pregnant uterine horns collected on Days 16–17 of gestation for any uterine region (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the estrous cycle, IGFBP-6 mRNA was abundant in the periepithelium, caruncles, and glands, with lower levels found in the endometrial stroma and myometrium. A previous study in the rat reported IGFBP-6 mRNA localized to the myometrium and stroma [10], whereas in the human localization was restricted to the stroma [11]. Detailed studies are, however, lacking, and the significance of these species differences awaits further investigation. In the ewe we have now shown that in all regions, with the exception of the myometrium, IGFBP-6 mRNA showed cyclic variations in relative levels, with a marked increase around the time of ovulation and early luteal phase followed by a decline in the mid to late luteal phase. Particularly intriguing was the rapid and substantial increase of IGFBP-6 mRNA measured in the glands, which was initiated at the onset of estrus and terminated by Day 3 of the cycle. Interestingly, this time frame would be coincident with the transport of spermatozoa through the ovine uterus. In humans IGFBP-6 mRNA is more dominant in the late luteal phase [11], whereas by comparison in the rat IGFBP-6 mRNA predominates on the morning of estrus and is low or absent at other times of the cycle [10].
The distribution pattern for IGFBP-6 mRNA throughout gestation was similar to that of a nonpregnant cyclic ewe but lacked glandular expression. In the periepithelium and caruncular stroma the relative levels of IGFBP-6 mRNA in early pregnancy were higher than those found during the mid to late luteal phase of nonpregnant ewes, indicating a pregnancy effect. In the transected uteri, however, no differences in concentration were found between the nonpregnant and pregnant horns during Days 16–17. Information on uterine IGFBP-6 during pregnancy in other species is sparse. In the rat, expression in the myometrium was found to increase with gestational age [12], and IGFBP-6 mRNA increased moderately throughout the uterus between Gestational Day 20 and Postnatal Day 24 [13]. Conversely, in the human and rhesus monkey there was no reported increase in IGFBP-6 mRNA with time, and localization was confined to the maternal and fetal decidua [14, 15]. It should be noted that in the present study there was no evidence for hybridization of IGFBP-6 to any fetal placental tissue. These discrepancies in the spatial-temporal expression of IGFBP-6 between species presumably reflect differences in the types of placentation.
There is currently no direct information on the factors controlling IGFBP-6 expression in the uterus. Potential regulatory cis elements, including a putative estrogen receptor binding site, have been documented in the promoter region of the rat IGFBP-6 gene [16]. Furthermore, in vitro estradiol increased IGFBP-6 production in MCF-7 breast cancer cells [17], and a recent study measuring plasma levels in humans reported significant differences between genders [18]. A functional retinoid response element has been identified in the human IGFBP-6 promoter [19]. There is also an increasing body of evidence linking high levels of IGF-II to the concomitant appearance of IGFBP-6. Examples from in vitro studies include IGF-II overexpressing NIH 3T3 cells [20], and rat phaeochromocytoma and human neuroblastoma cells cultured with exogenous IGF-II [21, 22]. In vivo indications come from non-islet-cell tumor-induced hypoglycemia attributable to excessive production of IGF-II; patients with this condition have a marked elevation of IGFBP-6 in their serum [23].
Our data showing 1) higher IGFBP-6 mRNA concentrations around ovulation, 2) higher relative levels in anestrus than in ovariectomized ewes, and 3) increased caruncular concentrations in early pregnancy all support a role for estradiol. Estradiol concentrations in plasma increase during the follicular phase of the cycle [24] and were higher in uterine flushings from pregnant compared with nonpregnant ewes on Days 9–15 after estrus [25]. Estradiol receptor concentrations in both caruncular stroma and endometrial glands also peak at estrus [26], are highly expressed in anestrus ewes [26], and are detectable in the caruncles from Day 13 of gestation [27]. Another candidate regulator of uterine IGFBP-6 expression could be IGF-II. We have previously reported high expression in caruncules in nonpregnant ewes [28], and concentrations also increase in the adjacent fetal mesoderm during early placental development [2]. Cooperation between estradiol and IGF-II could help to explain the higher relative levels of IGFBP-6 mRNA in caruncules compared with myometrium, as these two tissues have similar estradiol receptor concentrations [26] but IGF-II expression was undetectable in the myometrium [2, 28]. In the transected uteri no differences were found between the nonpregnant and pregnant horns on Days 16–17. This finding rules out any possibility of an early embryonic factor such as the antiluteolytic hormone, interferon tau, which is secreted from the conceptus between Days 10 and 21 of gestation, functioning to regulate IGFBP-6 expression via a paracrine mechanism [29].
A definitive role for IGFBP-6 has not yet been determined. The consensus view is that IGFBP-6 is involved in the attenuation of IGF-II stimulated cell proliferation. To date, IGFBP-6 has been shown to arrest the division of myoblasts [30], osteoblasts [31, 32], neuroblastoma [33], and breast cancer cells [17]. Furthermore, its association with quiescent or nonproliferating in vitro systems could explain its almost ubiquitous high level of expression in the anestrous ewe. With regard to a role during the cycle and pregnancy, we speculate that IGFBP-6 functions to regulate IGF-II-driven endometrial proliferation. During gestation IGFBP-6 production in the caruncular stroma of the maternal villi and periepithelium may serve as a barrier blocking the transport of fetal IGF-II across the fetal-maternal interface into the endometrium (Fig. 6).
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In summary, our data have shown that IGFBP-6 mRNA is present in the ovine uterus and placenta. The relative level of mRNA measured in different regions is dependent on the stage of the estrous cycle, is maximal around ovulation, and is also high in anestrous animals. During gestation high levels of IGFBP-6 mRNA are present in the periepithelium and caruncular stroma. We conclude that IGFBP-6 may be functionally important in the ovine uterus by virtue of its ability to regulate IGF-II mediated actions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D.C. Wathes, Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, U.K. FAX: 01707 666371; dcwathes{at}rvc.ac.uk
Received: 24 May 2000.
First decision: 22 June 2000.
Accepted: 25 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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