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a Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844
b Department of Molecular and Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66103
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We investigated LIF expression in a carnivore, the Western spotted skunk, that exhibits a 6- to 7-mo period of embryonic diapause. It has been hypothesized that the uterine environment is inadequate to support implantation during the period of arrested blastocyst development, at which time the uterus is quite small and secretes reduced levels of uterine-specific proteins [8–10]. Corpora lutea are also small and appear relatively inactive, yet sufficient steroids are secreted to maintain pregnancy [8, 11, 12]. Renewed embryonic development in the spotted skunk begins in late March to early April and is induced by increasing day length and prolactin (PRL) secretion. Blastocysts increase in diameter, renew mitosis in the inner cell mass, and exhibit increased RNA and protein synthesis [13, 14]. In synchrony with blastocyst activation, the uterus undergoes several changes including increases in weight, number of active-appearing secretory gland cells [10], synthesis of uterine secretory proteins [9], abundance of epidermal growth factor receptor (EGF-R) mRNA, and EGF-induced protein tyrosine kinase activity [15]. The temporal pattern of LIF expression in the uterus of mice, rabbits, mink, and humans and the failure of blastocysts to implant in LIF -/- mice suggest that LIF may be required for implantation [6, 16, 17]. This led us to hypothesize that embryonic diapause in the spotted skunk is due to insufficient uterine expression of LIF during delayed implantation whereas resumption of embryonic development and implantation are associated with increased LIF expression. We also investigated the effects of progesterone, estrogen, PRL, and EGF on LIF mRNA expression in the uterus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ketamine hydrochloride was obtained from Fort Dodge Laboratories (Fort Dodge, IA). Sodium pentobarbital was obtained from Abbott Laboratories (North Chicago, IL). Penicillin G was obtained from Solvay Animal Health, Inc. (Mendota Heights, MN). Recombinant human LIF (rhLIF) and anti-hLIF neutralizing antibody were obtained from R&D Systems, Inc. (Minneapolis, MN). Guanidine thiocyanate, guanidine hydrochloride, buffer-saturated phenol, and cesium chloride were obtained from Amresco (Dallas, TX). Tris-HCl, Tris base, sodium citrate, and sodium hydroxide were purchased from Fisher Scientific (Pittsburgh, PA). Specific DNA primers, Taq polymerase, Superscript II, and restriction enzymes were obtained from Gibco BRL (Gaithersburg, MD). RQ1 DNase, recombinant RNasin, and random hexamers were obtained from Promega (Madison, WI). AmpliTaq Gold polymerase was obtained from Perkin-Elmer (Branchburg, NJ). All radioactive isotopes were obtained from NEN-DuPont (Boston, MA). Normal rabbit serum and goat IgG were obtained from Vector Laboratories (Burlingame, CA). Nytran Plus membranes were purchased from Schleicher&Schuell (Keene, NH). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Animals and Experimental Treatments
Pregnant Western spotted skunks (Spilogale putorius latifrons) that had bred in the wild were obtained from a USDA-licensed dealer in Oregon. Skunks were individually housed, fed Purina (Ralston-Purina, St. Louis, MO) Ferret Chow and given water ad libitum, and maintained on a natural photoperiod. Animals were killed throughout the preimplantation stages of pregnancy by an overdose of sodium pentobarbital. Blood samples were collected into heparinized vacuum tubes prior to death by cardiac puncture. The plasma was stored at -20°C until time of hormone assay. Uteri were removed, flushed with sterile 0.05 M PBS to recover blastocysts, flash frozen in liquid propane, weighed, and stored at -80°C. Classification of the stage of pregnancy (delay, early, and late blastocyst activation) was determined by measuring embryo diameter as previously described [18].
Beginning in early January, 33 skunks were divided into six treatment groups. Animals were anesthetized with sodium pentobarbital, bilaterally ovariectomized, and allowed to recover. All animals received daily injections of penicillin (60 000 U). Six skunks were given daily injections of 0.5 mg PRL dispersed in 5% beeswax and sesame oil for 6 consecutive days beginning 3 days after ovariectomy. The remaining animals were anesthetized with ketamine 6 days after ovariectomy, and silicone elastomer capsules were placed s.c. in the interscapular region. Six animals received four capsules containing a 33 x 1.47-mm column of progesterone. Six skunks received one capsule containing a 2.0 x 0.05-mm column of estradiol-17ß:cholesterol in a 1:2 mixture. Six animals received a combination of four progesterone capsules and one estrogen capsule. Ovariectomized animals serving as controls (n = 6) received four empty silicone elastomer capsules and daily 0.5-ml injections of 5% beeswax in sesame oil. Steroid treatments continued for 3 days and PRL treatment for 6 days, after which the animals were killed by an overdose of sodium pentobarbital. Blood and tissue samples were collected as described previously.
Three skunks were anesthetized with ketamine and implanted with a Model 2001 Alzet mini osmotic pump (Alzet, Palo Alto, CA). The pumps delivered a constant infusion of EGF at a mean rate of 143 ng/h. The pumps were replaced twice, after 7 days. The entire duration of EGF infusion was 3 wk, after which time the animals were killed with an overdose of sodium pentobarbital. A blood sample was collected and the uterus removed and processed as previously described. Protocols for all experiments involving animals described in this paper were reviewed and approved by the University of Idaho Animal Care and Use Committee.
RNA Isolation and cDNA Preparation
Total RNA was isolated from the uterine cornua of individual animals by homogenization in guanidium thiocyanate followed by centrifugation through a cesium chloride gradient [19]. RNA was quantified using A260/A280 spectrophotometric readings. Total RNA (1 µg) from skunk uteri was prepared for reverse transcription-polymerase chain reaction (RT-PCR) by treatment with DNase [20]. After addition of 50 ng random hexamers, samples were heat denatured at 75°C for 5 min and transferred immediately to ice. RT reactions were carried out as described previously [20]. Identical uterine RNA samples were incubated in parallel but without reverse transcriptase to verify that all endogenous genomic DNA had been eliminated. A 1-µl aliquot of the resultant cDNA was mixed with specific primers and subjected to PCR (single-strength buffer, 0.2 mM deoxyribonucleotides, 3 mM MgCl2, 0.4 µM each primer, 1 U Taq polymerase) in order to amplify skunk cyclophilin and LIF sequences [20]. PCR amplification of cyclophilin yielded a 195-base pair (bp) fragment while the LIF primers amplified a 483-bp fragment. Products were separated by electrophoresis, cloned, and identified by sequencing in both directions [20]. Homologous primers that were internal to the cloning sites for LIF and that spanned an intron were selected for use in the quantitative RT-PCR assay and yielded a 398-bp PCR product from cDNA templates.
Quantitative RT-PCR
Preliminary experiments were performed to determine the appropriate cycle number for amplifying cDNA from each gene product during the exponential phase of PCR (LIF, 34 cycles; cyclophilin, 40 cycles). Serial dilutions of a plasmid standard and uterine cDNA sample were amplified in parallel for LIF and cyclophilin to verify that the plasmid standards and cDNA amplified with similar efficiencies. One uterine sample from each hormone treatment group and five samples from each stage of pregnancy were amplified for LIF, and the identity of the PCR products was verified by Southern blot analysis. Samples from the pregnancy series were also subjected to PCR for cyclophilin, and the product was verified by blot. Additionally, 5 aliquots of one uterine sample from each stage of pregnancy and treatment group were reverse transcribed and amplified to assess intraassay variability of the entire RT-PCR procedure.
The quantitative PCR assay was based upon a procedure developed to quantify growth factors in ovarian tissue [21]. After reverse transcription, all samples were diluted to 3.3 volumes with sterile water containing yeast RNA at a final concentration of 10 ng/µl. All subsequent dilutions of cDNA and standards included this concentration of carrier RNA. Plasmid DNA containing LIF or cyclophilin subclones were used to generate standard curves for both compounds. Each target sequence was assayed in a separate set of PCR reactions consisting of standards (n = 5) and cDNA samples (n = 6–8 per group) assayed in triplicate. Hot-start PCR was performed using 0.4 µM of each primer, 0.2 mM deoxyribonucleotides, 0.7 µCi [32P]deoxycytidine triphosphate (3000 Ci/mmol), 2.5 U AmpliTaq Gold, 3% dimethylsulfoxide, 2.5 mM MgCl2 (LIF reactions) or 2 mM MgCl2 (cyclophilin reactions), and 10 µl cDNA in 25 µl AmpliTaq Gold PCR buffer. The PCR profile consisted of an initial incubation (11 min, 96°C) to denature the cDNA and activate the polymerase; 34–40 cycles of denaturing (3 min, 94°C), annealing (20 sec; 60°C for LIF, 55°C for cyclophilin), and extending (20 sec plus 2 sec/cycle, 72°C) the products; and a final extension (6 min, 72°C). PCR products were separated on a 5% native polyacrylamide gel, dried, and quantified using a GS-525 Molecular Imager (Bio-Rad Laboratories, Hercules, CA). After comparison of sample signal intensity to its respective standard curve, LIF expression was normalized to that of cyclophilin.
Southern Blot Hybridization
PCR products were separated by electrophoresis on 1.2% agarose gels and transferred to Nytran Plus membranes by downward alkaline blotting. Random-primer labeling of gene-specific subclones was performed using the RadPrime DNA Labeling System (Gibco BRL, Gaithersburg, MD) and 50 µCi [32P]dCTP. Hybridizations were performed in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), 0.1% SDS, with 109 dpm/µg of probe at 65°C for 16 h. Membranes were washed under high stringency (0.1-strength SSC, 0.1% SDS, 55°C) and analyzed with a GS-525 Molecular Imager.
In Situ Hybridization
In situ hybridization was performed as described previously [22]. Frozen sections (10 µm) were mounted onto poly-L-lysine-coated slides and stored at -70°C until used. After removal from -70°C, the slides with uterine sections were placed on a slide warmer (37°C) for 2 min and then fixed in 4% paraformaldehyde in PBS for 15 min at 4°C. After prehybridization, uterine sections were hybridized to 35S-labeled LIF sense or antisense cRNA probes for 4 h at 45°C. After hybridization and washing, the slides were incubated with RNase A (20 µg/ml) at 37°C for 15 min. RNase A-resistant hybrids were detected by autoradiography using Kodak (Eastman Kodak, Rochester, NY) NTB-2 liquid emulsion. The slides were poststained with hematoxylin and eosin.
Immunocytochemistry
A polyclonal antibody to rhLIF was used to detect immunoreactive LIF in skunk uterine sections. This antibody (0.33 mg/ml) was preadsorbed with lyophilized skunk liver powder and used at a working dilution of 1:20. Uterine tissues were fixed in 2–4% paraformaldehyde (1–2 h, 4°C) and embedded in paraffin. Sections (6 µm) were mounted on poly-L-lysine-coated slides and dewaxed in xylene. Nonspecific background staining was reduced by microwaving the sections for two 5-min intervals in 0.05 M Tris-HCl, pH 7.6, allowing them to cool, and rinsing in PBS. The tissues were incubated for 30 min in 0.3% hydrogen peroxide in methanol, rinsed in PBS, and incubated for 20 min with normal rabbit serum (Vectastain ABC Goat kit); this was followed by endogenous avidin/biotin blocking (Vector Avidin Biotin kit). One section on each slide was exposed overnight at 4°C to primary antiserum (1:20 dilution in PBS). A negative control section on each slide was incubated with anti-LIF serum preadsorbed with an excess of rhLIF (5 µg/ml in PBS) or incubated with goat IgG (1:20 dilution). Sections were washed in PBS and incubated for 10 min with anti-goat IgG biotinylated secondary antibody (Vectastain ABC Goat kit). After washing in PBS, sections were incubated with streptavidin-peroxidase conjugate, then with substrate-chromogen reagent (Vectastain AEC Goat kit), and counterstained with Mayer's hematoxylin.
RIA
RIAs for plasma progesterone, estrogen, and PRL that were validated for use in the spotted skunk were performed as described previously [23–25]. The interassay and intraassay coefficients of variation (mean ± SD) for the progesterone assay were 4.9 ± 6.1% and 4.6 ± 1.6%, respectively. Ovine (o)PRL (AFP 10789B from NIDDK-NIH, Rockville, MD) was iodinated by the chloramine-T method, and rabbit anti-oPRL (AFP C35810691 from NIDDK-NIH) was used at final tube dilution of 135 000. The intraassay variation (mean ± SD) between duplicate PRL samples was 8.1 ± 5.2%. Due to the limited quantity of plasma, estrogen was quantified in a single 1.0-ml sample from each animal. The interassay variation (mean ± SD) for the estrogen assay was 16.9 ± 8.9%.
Statistical Analysis
All data were analyzed by SPSS (SPSS Inc., Chicago, IL) or Statistica (Stat Soft, Inc., Tulsa, OK) software. Differences in uterine weight, LIF expression, and plasma hormone concentrations were analyzed by one-way ANOVA with post hoc multiple comparisons using a Bonferroni correction. Effects of hormone treatment on uterine weight and LIF expression were analyzed by Kruskal-Wallis one-way ANOVA. Comparisons of hormone-treated groups to controls were examined by post hoc Mann-Whitney U tests applying a Bonferroni correction to reduce the overall experimental type I error rate to 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of skunk LIF and cyclophilin in total uterine RNA extracts was demonstrated by RT-PCR. Specific primers yielded PCR products of the expected size: 398 bp for LIF and 195 bp for cyclophilin. Nucleotide sequence of the amplified region of skunk LIF mRNA was 86.4% identical to that of human LIF mRNA, and 87.3% similar in protein sequence. The nucleotide sequence of the cloned portion of skunk cyclophilin was 78.9% similar to that of human cyclophilin. No signal was detected in samples that lacked reverse transcriptase, whereas specific binding of LIF and cyclophilin DNA probes was detected in Southern blots of amplified samples containing reverse transcriptase (data not shown).
Parallelism between the plasmid standards for LIF and cyclophilin and uterine cDNA dilution curves was demonstrated (data not shown). These results verified that LIF and cyclophilin each amplified from the plasmid standards and cDNA samples with comparable efficiency. Sensitivity of the RT-PCR assay was below 10 ag for cyclophilin and 1 fg for LIF per 10 ng cDNA. Intraassay variation of the entire procedure (reverse transcription and PCR) was 34.8 ± 15.6%. The average intraassay coefficient of variation between duplicate cDNA samples in the PCR procedure was 8.3 ± 9.2% for the pregnancy series and 8.2 ± 5.8% for the hormone treatments.
Experiment 1: Pregnancy Series
The relative abundance of LIF mRNA, normalized to cyclophilin, increased significantly (p = 0.002) at the time of early embryo activation compared to that during diapause (Fig. 1A). The increase in uterine LIF mRNA concentration coincided with a significant increase in weight of the paired uterine cornua (p < 0.001) from animals with blastocysts 1.2–1.6 mm in diameter (onset of early blastocyst activation, Fig. 1B). Uterine concentration of LIF mRNA remained elevated approximately twofold throughout the period of renewed embryonic development.
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No cell-specific autoradiographic signals for LIF mRNA were detected in uteri of animals containing blastocysts in diapause (Fig. 2, a and b). LIF mRNA was first detectable in the glandular epithelium of uteri that contained early activated blastocysts (Fig. 2, c and d). During the final stage of blastocyst activation, LIF mRNA was also localized to the luminal epithelium of the uterus (data not shown). The intensity of the signal in the uterine glands remained relatively strong in both early (24–72 h) postattachment stage specimens examined. LIF mRNA was never localized in the myometrium. No positive signals were detected in uterine sections at any stage of pregnancy when hybridized with sense probe.
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Immunopositive staining for LIF protein was nondetectable in the endometrium in four of five animals examined during embryonic diapause (Fig. 3A) and was only faintly detected in the basal portion of the uterine glands in the fifth skunk. LIF protein was first consistently detected in the early activation stage of pregnancy, at which time strong staining was observed in luminal and glandular epithelium (Fig. 3B). Positive LIF staining remained in the luminal and glandular epithelium of the uterus in all three animals examined when blastocysts were completing their preattachment development (Fig. 3D). LIF staining was weak and was restricted to the apical cytoplasm of the luminal and glandular epithelium and to luminal fluid at the time of trophoblast attachment and initial intrusion into the luminal epithelium (Fig. 3, E and G). LIF protein was not detected in the stroma of any animal but was consistently present in the myometrium of all animals during all stages of pregnancy examined. All immunopositive staining was eliminated when the anti-rhLIF antibody was preabsorbed with rhLIF (Fig. 3C) or when sections were incubated with normal goat serum in place of primary antiserum (Fig. 3, F and H).
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Experiment 2: Hormone Treatments
Ovariectomy significantly (p < 0.05) reduced the plasma concentration of progesterone (1.5 ± 0.8 ng/ml) to below that found during embryonic diapause. Silicone elastomer capsules containing progesterone significantly (p < 0.05) increased plasma progesterone to 22.5 ± 9.3 ng/ml and to 26.3 ± 9.8 ng/ml in the group treated with progesterone plus estradiol. Estrogen was nondetectable in all ovariectomized control animals. Silicone elastomer capsules containing estradiol elevated plasma estrogen concentration to 6.3 ± 3.5 pg/ml in the estradiol-treated animals and to 4.7 ± 1.2 pg/ml in the group treated with progesterone plus estradiol. PRL concentration in the diluent-treated control group averaged 17.6 ng/ml. Daily injections of oPRL elevated plasma PRL concentration to 117.1 ± 38.5 ng/ml.
Treatment with either of the steroid hormones significantly increased (p < 0.01) uterine weight (Fig. 4A). Animals treated with estradiol, as well as those treated with estradiol plus progesterone, exhibited a twofold increase in uterine weight over that of ovariectomized controls. Daily injections of PRL or constant infusion of EGF had no significant effect on uterine weight or on blastocyst diameter compared to these values in control animals.
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Messenger RNA for LIF was detected in all animals and in every treatment group. Uterine LIF expression in ovariectomized control animals was elevated compared to that in uteri of intact animals containing blastocysts in diapause; however, this difference was not statistically significant. There were no significant changes in LIF mRNA abundance in response to PRL or steroid treatments when compared to values in ovariectomized controls (Fig. 4B). LIF mRNA concentration was lowest in the PRL-treated group and highest in the EGF-treated group (data not shown). Although the average LIF/cyclophilin ratio in the EGF-treated animals was identical to that in uteri containing activated blastocysts, the mean value (0.147) was not statistically different from that during delayed implantation, due to the wide range in ratios and small sample size (n = 3) in the EGF-treated group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The earliest indicators of renewed embryonic development in the skunk are increased uterine weight and increased blastocyst diameter. The rapid and marked change in uterine weight was clearly evident even in the uterine sample containing embryos earliest in activation; weight of the paired uterine cornua from this animal was 2.4 times greater than the mean uterine weight during embryonic diapause. Changes in blastocyst diameter occur slowly over the several days of embryo activation. During this period, blastocysts increase in diameter from 1.2 to 1.6 mm; there is then rapid enlargement from 1.7 to > 2.0 mm in diameter 1–2 days before implantation. The strong correlation between blastocyst diameter and developmental state of the embryo [13, 14, 18], in combination with changes in uterine weight, makes it possible to accurately classify uterine samples and establish the onset and stage of renewed embryonic development in this species.
LIF transcripts were significantly more abundant in uteri containing embryos 1.2–1.6 mm in diameter than in those containing blastocysts in diapause. The concentration of LIF mRNA in the uterine sample containing embryos earliest in activation (1.05–1.2 mm) was 1.6 times greater than the mean uterine LIF concentration during embryonic diapause. The concurrent increase in LIF mRNA at the onset of renewed embryonic development is consistent with the pattern of uterine LIF expression in the mink [17], and upon termination of facultative delayed implantation in the mouse [3]. The observation that blastocysts appear to enter diapause upon arrival in the uterus of LIF -/- mice further emphasizes the relationship between elevated LIF expression and implantation [6].
Quantitative comparisons of LIF mRNA concentration were possible by normalizing to the constitutively expressed gene cyclophilin [27]. Increased LIF mRNA concentration relative to that of cyclophilin suggests that LIF is transcriptionally regulated in the skunk uterus. However, in the present study it was not possible to determine whether the change in LIF transcript abundance is due to changes in mRNA synthesis or rate of degradation. Regardless of the mechanism of regulation, the increase in uterine concentration of LIF mRNA at the time of early embryo activation was concurrent with the detection of increased LIF protein. Our RT-PCR assay does not discriminate between the matrix-associated and diffusible forms of LIF described by Rathjen and coworkers [28]. Consequently, it is not known which forms of LIF are produced by the skunk uterus during the periimplantation period. During the early stages of embryo attachment, LIF protein was localized primarily to the lumen of the uterus, indicating that much of it is being secreted. Secretion of LIF has also been demonstrated in human endometrial and stromal cell cultures and in uterine flushings of the human and pig [29–33]. Once in the uterine lumen, LIF may bind to receptors (LIFRß) located on the blastocyst and on the apical surface of the luminal epithelium in the mouse and human [5, 33–35].
Endocrine regulation of LIF has been investigated in vitro in a few species and in vivo in the sheep and mouse, but no work has been published on the effects of hormones on LIF expression in a carnivore. In the skunk, PRL is the primary pituitary hormone responsible for initiating increased luteal activity and induction of blastocyst implantation [23]. In our experiments, blood PRL concentration exceeded the normal physiologic range during the periimplantation period. The dose employed was equivalent to that used previously to induce implantation without adverse effects; however, the dose had no significant effect on the abundance of LIF mRNA when compared to that in the ovariectomized controls or in intact females whose uteri contained blastocysts in diapause. The duration of PRL treatment used in this study was less than half that required to renew blastocyst development and induce early implantation [23]. Consequently, we cannot rule out the possibility that a more prolonged exposure to elevated PRL could affect LIF expression.
Ovarian steroids are required for some changes in the skunk uterus that occur during the periimplantation period. Estrogen is required for development of tall columnar luminal epithelium, and progesterone stimulates uterine protein synthesis [9, 36]. However, elevated plasma estrogen concentration inhibits uterine protein synthesis in this species [9]. Blood levels of estrogen are high and fluctuate considerably during embryonic diapause, but they decline during resumption of blastocyst development [24]. Progesterone concentration is highly correlated with that of PRL and increases at the time of renewed blastocyst development [23]. Treatment of ovariectomized skunks with progesterone maintains embryo viability but fails to induce implantation [12]. In the present study, uterine LIF mRNA expression increased at early embryo activation, during which progesterone is the predominant ovarian steroid, suggesting that it might be responsible for the up-regulation of LIF expression. Constant administration of progesterone to ovariectomized skunks significantly increased uterine weight but failed to alter uterine LIF expression. LIF mRNA or protein is most abundant in the human, mink, and rabbit endometrium when progesterone concentration is elevated [17, 30, 34, 37–39]. Administration of progesterone results in increased staining for LIF protein in the uterus of unmated rabbits [39], whereas in the ewe, progesterone results in decreased staining for LIF in the luminal epithelium and increased staining in the glandular epithelium and stroma of the uterus [38].
During delayed implantation in the mouse, an injection of estrogen induces implantation and is associated with a significant rise in LIF mRNA [3]. LIF protein increases in response to an injection of estrogen, whereas injection of progesterone does not stimulate LIF protein expression in ovariectomized mice [39]. Treatment of ovariectomized sheep with estrogen maintains or reduces immunoreactive LIF in the endometrium [38]. In our study, constant administration of physiologic concentrations of estradiol increased uterine weight in ovariectomized skunks to levels similar to those observed during blastocyst activation, but failed to alter uterine LIF mRNA concentrations compared to those in ovariectomized controls.
The time between ovariectomy and initiation of the hormone treatments (6 days) should have been sufficient for steroid-dependent mRNA levels to decline [40–43]. The apparent lack of LIF mRNA down-regulation after ovariectomy, together with the absence of any inducement or inhibition of LIF expression by steroid treatments in comparison with values in the ovariectomized controls, suggests that uterine LIF is not regulated by either progesterone or estradiol. However, we did not examine uterine LIF expression at varying time points throughout any of the hormone treatments. Therefore, we cannot rule out the possibility of a transient change in LIF mRNA abundance in response to these steroids or PRL. Yet the process of blastocyst activation occurs over several days, and uterine LIF mRNA appears to remain elevated for several days throughout this period. Therefore, it is likely that continuous administration of the appropriate compound would induce a prolonged rather than transient increase in mRNA concentration in the uterus.
Mean uterine LIF mRNA concentration in ovariectomized (control) skunks was about 1.7 times greater than that in intact animals with blastocysts in diapause. The difference between the means of these two groups approached statistical significance (p = 0.059). Since these results were obtained in separate assays, it is possible that interassay variation might have obscured a physiological effect of ovariectomy, which in turn may have hindered our ability to detect an effect of one or more of the steroid treatments on LIF abundance. If one assumes the latter to be true, one would interpret the results as indicating that ovariectomy induced an increase in uterine LIF concentration. It should also be noted that LIF expression in uteri of ovariectomized or ovariectomized steroid-treated skunks was only slightly lower and that it did not significantly differ from the level observed in pregnant females whose uteri contained early activated blastocysts.
Interleukin-1, tumor necrosis factor, platelet-derived growth factor, transforming growth factor ß, and EGF alter LIF expression in the human uterus [30]. EGF-R mRNA and EGF-induced protein tyrosine kinase activity increase at the time of renewed embryonic development [15]. EGF treatment of skunks with delayed implanting blastocysts seemingly elevated uterine LIF mRNA concentrations to values equivalent to those observed in intact skunks with activated blastocysts. However, the small sample size and disparate LIF/cyclophilin ratios (0.24, 0.13, 0.07) prevent one from drawing conclusions from this result. Moreover, prolonged infusion of intact pregnant skunks with EGF failed to induce blastocyst activation or implantation.
Data presented in this study suggest a relationship between uterine LIF expression, embryo development, and implantation in the skunk that is consistent with that observed in the mouse, rabbit, mink, and human. However, the essentiality of LIF in implantation has been directly demonstrated only in the mouse. Constant infusion of recombinant mouse LIF at a rate of 790 pg/min per milliliter (assuming an average body weight of 30 g and blood volume of 2.1 ml) for 72 h resulted in embryo implantation in LIF -/- female mice and induced cachexia, which suggests that the dose used was pharmacological [6]. Preliminary experiments involving constant infusion of rhLIF (120 pg/min per milliliter; average body weight of 370 g and assumed blood volume of 28 ml) into intact skunks with delayed implanting blastocysts for 7 days failed to increase uterine weight or blastocyst diameter or to induce implantation or cachexia. Consequently, the importance of LIF in renewing blastocyst development or implantation in the skunk remains unclear. In other species, addition of recombinant LIF to embryos results in an increased rate of blastocyst formation and survival in vitro [44–46]. If the function of LIF is similar in the skunk, then uterine LIF concentration might be expected to correlate with the proportion of viable embryos present in the uterus. In our study, dead or dying blastocysts were recovered from all ovariectomized progesterone-treated animals, from 57% of the estradiol-treated skunks, from 29% of those treated with progesterone and estrogen, and from 43% of the ovariectomized control animals receiving only diluent. None of the ovariectomized PRL-treated animals contained dead or dying blastocysts. Despite these differences in blastocyst viability, LIF mRNA concentration did not significantly differ between the hormone treatment groups. This lack of correlation between blastocyst viability and uterine LIF concentration suggests that for the spotted skunk, embryo viability in vivo is dependent upon PRL and ovarian hormones rather than uterine LIF expression.
In summary, LIF mRNA is present in the uterus of the spotted skunk throughout delayed implantation, increases at the time of renewed embryo development, and remains detectable in the uterine glands for up to 72 h postattachment. Immunoreactive LIF protein is up-regulated in the luminal and glandular epithelium during the early stages of embryo activation and appears in the uterine lumen during periimplantation. Although these changes in uterine LIF expression are consistent with our initial hypothesis and occur concurrently with precise changes in the endocrine environment, LIF mRNA appears to be regulated by factors other than PRL, progesterone, or estradiol. The function and regulation of LIF expression during the periimplantation period in the spotted skunk remain unknown.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 208 885 7905; rmead{at}uidaho.edu
Accepted: September 21, 1998.
Received: June 19, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 J. R. Chen, J.-G. Cheng, T. Shatzer, L. Sewell, L. Hernandez, and C. L. Stewart Leukemia Inhibitory Factor Can Substitute for Nidatory Estrogen and Is Essential to Inducing a Receptive Uterus for Implantation But Is Not Essential for Subsequent Embryogenesis Endocrinology, December 1, 2000; 141(12): 4365 - 4372. [Abstract] [Full Text] [PDF]
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H. Song, H. Lim, S. K. Das, B. C. Paria, and S. K. Dey Dysregulation of EGF Family of Growth Factors and COX-2 in the Uterus during the Preattachment and Attachment Reactions of the Blastocyst with the Luminal Epithelium Correlates with Implantation Failure in LIF- Deficient Mice Mol. Endocrinol., August 1, 2000; 14(8): 1147 - 1161. [Abstract] [Full Text]
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C. Passavant, X. Zhao, S.K. Das, S.K. Dey, and R.A. Mead Changes in Uterine Expression of Leukemia Inhibitory Factor Receptor Gene During Pregnancy and Its Up-Regulation by Prolactin in the Western Spotted Skunk Biol Reprod, July 1, 2000; 63(1): 301 - 307. [Abstract] [Full Text]
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 C. J. Auernhammer and S. Melmed Leukemia-Inhibitory Factor--Neuroimmune Modulator of Endocrine Function Endocr. Rev., June 1, 2000; 21(3): 313 - 345. [Abstract] [Full Text]
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 K. C. Reinhart, R. K. Dubey, P. J. Keller, U. Lauper, and M. Rosselli Xeno-oestrogens and phyto-oestrogens induce the synthesis of leukaemia inhibitory factor by human and bovine oviduct cells Mol. Hum. Reprod., October 1, 1999; 5(10): 899 - 907. [Abstract] [Full Text] [PDF]
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S.K. Das, J. Wang, S.K. Dey, and R.A. Mead Spatiotemporal Expression of Cyclooxygenase 1 and Cyclooxygenase 2 during Delayed Implantation and the Periimplantation Period in the Western Spotted Skunk Biol Reprod, April 1, 1999; 60(4): 893 - 899. [Abstract] [Full Text]
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J.-G. Cheng, J. R. Chen, L. Hernandez, W. G. Alvord, and C. L. Stewart Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation PNAS, July 17, 2001; 98(15): 8680 - 8685. [Abstract] [Full Text] [PDF]
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