0013-7227/90/1265-2514$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 5 Printed in U.S.A.
Relaxin Gene Expression in the Sow Corpus Luteum during the Cycle, Pregnancy, and Lactation* CAROL A. BAGNELL, LILY TASHIMA, WALTER TSARK, SHUJATH M. ALI, AND JOHN P. McMURTRY Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96822; Nonruminant Animal Nutrition Laboratory, Livestock and Poultry Institute, USDA, Agricultural Research Service (J.P.M.), Beltsville, Maryland 20705
ABSTRACT. Relaxin (RLX) mRNA was sought in the corpus luteum of the pig during the cycle, pregnancy, and lactation using Northern analysis and in situ hybridization. Three oligonucleotide probes to regions of the preprorelaxin molecule were used for hybridization to detect RLX message and gave similar results. Northern analysis showed a single 1-kilobase RLX transcript at all three stages of the reproductive cycle studied. The intensity of the RLX hybridization signal was greatest in pregnancy and varied during the cycle. The signal in ovaries from day 3 cyclic animals increased by day 13 and declined on day 19. However, the hybridization signal at midcycle was only 2% of that during pregnancy. After parturition on day 2 of lactation, RLX message was still detected in the ovary, although at reduced
T
HE CORPUS luteum (CL) is a well recognized source of relaxin (RLX) in the pig. However, the amount of RLX in the CL varies depending on the physiological status of the animal. During the estrous cycle, low levels of bioactive and immunoactive RLX are found in extracts of pig ovaries, with maximal concentrations observed during diestrus (1). This correlates well with the RLX immunolocalization pattern in the CL during the cycle (2). During gestation, blood and tissue concentrations increase and reach peak levels in late pregnancy (3, 4). The surge of RLX into blood a few hours before parturition is associated with a decline in luteal RLX content (5). During the course of lactation, RLX tissue content declines (3), but is still detectable by immunohistochemistry through day 10 of lactation (6). In addition, episodic secretion of RLX after acute nuzzling and suckling or oxytocin administration has been reported (7, 8). Whether the presence of bioactive and immunoactive RLX in the CL during the cycle, pregnancy, and lactation represents active synthesis, unreleased residual hormone, or sequestered hormone is Received November 13,1989. Address all correspondence and requests for reprints to: Dr. Carol A. Bagnell, Department of Animal Sciences, Cook College, Rutgers University, New Brunswick, New Jersey 08903. * This work was supported by NIH Grant HD-20624.
levels compared with those during the cycle and pregnancy. In situ hybridization results showed hybridization to RLX mRNA in luteal tissue on day 13 of the cycle, but not on day 3 or 19. An increased hybridization signal was observed on days 40, 60, and 90 of pregnancy, with a decline on day 2 of lactation. Control sections incubated with labeled heterologous probe or preincubated with excess unlabeled probe did not hybridize. These results indicate a good correlation between the relative concentrations of RLX transcript and immunohistochemical results previously reported in the corpus luteum of the sow. In addition, they demonstrate that the RLX gene is expressed in luteal tissue, not only in pregnancy, but also in the cycle and early lactation. (Endocrinology 126: 2514-2520, 1990)
not clear. RLX is synthesized as a prohormone precursor, proRLX, consisting of A and B chains attached by a connecting peptide (C-peptide). The porcine RLX (pRLX) gene has been cloned and sequenced (9), and RLX mRNA has been demonstrated in extracts of CL from both pregnant (10) and cyclic pigs (11). However, there has been no study of RLX gene expression in the pig CL throughout the reproductive cycle in this species. Since the presence of RLX mRNA is a prerequisite for production of the hormone in tissue, we sought to demonstrate RLX message by Northern analysis and in situ hybridization in the CL during various reproductive stages in the pig.
Materials and Methods Animals Multiparous Duroc sows that exhibited at least two estrous cycles averaging 21 days were used for these experiments. Animals were checked for estrus in the presence of a boar, and the first day of estrus was designated day 0 of the cycle. Ovaries were collected from animals on days 3, 13, and 19 of the cycle. Ovaries from pregnant and lactating sows were obtained by mating the animals on the first day of estrus, which was designated day 0 of pregnancy. The day of parturition was
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RLX mRNA IN THE SOW CL termed day 0 of lactation. Ovaries were collected on days 40, 60, and 90 of pregnancy and day 2 of lactation. Intact uteri from cyclic animals were used as a control tissue in these studies. At the time of tissue collection, sows were transported to the abattoir, where they were stunned and exsanguinated. Tissues were frozen in liquid nitrogen within 2 min after death and stored at —80 C until use. The Northern analysis and in situ hybridization studies were performed on ovarian tissue from individual animals, with at least three animals per time point. Probes
Three 48-base oligonucleotide probes to portions of the prepro-RLX molecule were used (9). Two probes to adjacent regions of the C-peptide of porcine pro-RLX (C-peptide 1, AA 45-60; C-peptide 2, AA 61-76) were synthesized by Peninsula Laboratories (Belmont, CA). A third RLX-specific probe corresponding to a portion of the B-chain of pRLX (AA 11-26) was generously provided by Drs. W. S. Young III and M. J. Brownstein, NIMH (Bethesda, MD). To check the specificity of hybridization, a 48-base oligonucleotide to a region of the human PRL (hPRL) molecule (AA 60-75) was used as a control (courtesy of Drs. S. W. Young III and M. J. Brownstein). The sequences of the oligonucleotide probes used are shown in Table 1. The probes were 3'-end labeled with terminal-deoxynucleotidyl transferase [Bethesda Research Laboratories (BRL), Gaithersburg, MD] and either [a-32P]dATP (>3000 Ci/mmol; New England Nuclear, Boston, MA) for Northern analysis studies or [a-35S]dATP (>l000 Ci/mmol; New England Nuclear) for in situ hybridization (12). A rat cDNA probe which encodes cyclophilin (plB15) was used as a control to monitor gel loading for Northern analysis (13) (generously provided by Dr. Michael Melner, Oregon Primate Research Center, Beaverton, OR). plB15 is a gene shown to be constitutively expressed in a wide variety of tissues and conserved across species (14) and has been used in a number of laboratories as a control probe to demonstrate the quantity and integrity of the RNA present (15, 16). Isolation and Northern analysis of RNA Total RNA was prepared using guanidinium thiocyanate and phenol-chloroform extraction (17). Poly(A)+ RNA was isolated from total RNA by affinity chromatography on oligo-(dT) TABLE
cellulose (18). Purity and quantity were ascertained by spectrophotometric analysis at 260 and 280 nm. Known amounts of poly(A)+ RNA (0.2-20 ng), were denatured in 50% formamide-5.5% formaldehyde and fractionated in a 1.0% agarose gel containing 5.0% formaldehyde. The RNA was transferred to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH) overnight by the capillary method and dried at 80 C (19). A RNA ladder [0.16-1.77 kilobases (kb); BRL] was also electrophoresed in each gel as a molecular size marker. This lane was excised from the gel before transfer and stained with ethidium bromide, and migration of the RNA fragments was recorded for later comparison with autoradiograms. Hybridization was carried out using a modification of the methods described by Berent et al. (20). The dried nylon membranes were prehybridized for 5 h at 55 C in 6 X SSPE (1 X SSPE = 0.18 M NaCl; 10 mM NaPO4, pH 7.7; and 1 mM EDTA), 1 x Denhardt's solution (1 X Denhardt's = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 0.1% sodium dodecyl sulfate (SDS), 250 Mg/ml tRNA, and salmon sperm DNA. This solution was replaced with hybridization solution containing 6 x SSPE, 5 x Denhardt's solution, 0.1% SDS, 250 Mg/ml tRNA, and salmon sperm DNA containing 32 P-labeled probe (1-5 x 106 cpm/ml). After hybridization for 20 h at 55 C, filters were washed three times in 6 x SSPE-0.1% SDS at room temperature for 15 min, once in 6 X SSPE-0.1% SDS at 55 C for 1 h, and then finally in 6 X SSPE (without SDS) at room temperature for 15 min. The filters were exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) with DuPont Cronex intensifying screens (DuPont, Wilmington, DE) at -80 C for the times indicated in the figure legends. Filters were rehybridized, after removal of the RLX probe, with a 32P-labeled plB15 (cyclophilin) cDNA control probe. Autoradiograms were scanned with a Bio-Rad Scanning Densitometer (model 620, Bio-Rad, Richmond, CA), and the amount of RLX mRNA present relative to cyclophilin mRNA was determined. In situ hybridization Frozen 10-/im sections were thaw-mounted onto chromergecleaned, poly-L-lysine-coated slides and stored at -20 C until used (21). Hybridization procedures were based on methods described by Young III et al. (12). After fixation in 4% formaldehyde in PBS (0.05 M), slides were washed in PBS and
1. Sequence of 48-mer oligonucleotide probes used Amino acids 11-26
DNA probe sequence 5' GGA GAC GGA GCC AC A GAT CTC CAC CCA CAG ACG GAC TAA TTC TCG GCC 3'
pRLX C-peptide 1
45-60
5' CTT TAA GAT TTC TGC ATC TTT GGT GAT GGA GGA TGG CAT GGT TTC TGC 3'
pRLX C-peptide 2
61-76
5' TGT TCC CTT CAG CTC CTG TGG CAA ATT ACG AAC AAA TTC CAA CAT CAT 3'
hPRL
60-75
5' CAT CTG TTG GGC TTG CTC CTT GTC TTC GGG GGT GGC AAG GGA AGA AGT 3'
Probe pRLX B-chain
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RLX mRNA IN THE SOW CL
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incubated in 0.25% acetic anhydride in 0.1 M triethanolamine HCl-0.9% NaCl (pH 8.0) for 10 min to reduce nonspecific binding. Slides were then dehydrated in increasing amounts of alcohol and 100% chloroform and air dried. The 35S-labeled probes (3.0 X 105 cpm/50 ix\) were dissolved in a hybridization solution containing 4 X SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 50% formamide, 1 X Denhardt's solution, 10% dextran sulfate, 500 ng/m\ sheared single stranded DNA, 250 Mg/ml yeast tRNA, and 100 mM dithiothreitol and applied to each section. After incubation in a humidified chamber at 37 C for 20 h, slides were washed 4 times (15 min each) in 2 X SSC containing 50% formamide at 40 C, followed by 2 30-min washes in 1 x SSC at room temperature. The slides were dehydrated in increasing amounts of alcohol and air dried. In some experiments to test the specificity of the hybridization reaction, tissues were preincubated with a 100-
Cyclophilin
Relaxin
fold excess of unlabeled pRLX probe for 18-24 h at 37 C (22).
After preincubation, tissues were washed in 2 X SSC (10 times) and incubated with the a labeled pRLX probe and processed as described above. To determine the appropriate exposure time for emulsion autoradiography, slides were exposed to x-ray film (Kodak XOmat with intensifying screens) overnight. Slides were subsequently coated with Kodak NTB2 liquid photographic emulsion (Eastman Kodak; diluted 1:1 with 0.6 M ammonium acetate). Slides were exposed at 4 C in boxes, with drying agent for 1 or 2 weeks. After development slides were counterstained with toluidine blue (0.5%) and photographed under both bright- and darkfield illumination.
Results Northern analysis Ovaries from sows at different reproductive states were examined by Northern analysis for the expression of RLX mRNA. In these studies similar results were obtained when any of the three oligonucleotide probes complementary to different regions of the pRLX molecule (Table 1) were used. In addition, the plB15 probe, to detect the constitutively expressed protein cyclophilin, was used as an internal control to monitor the concentration of mRNA in the Northern blots (13). RLX densitometric data were standardized against the level of cyclophilin mRNA in the samples in order to determine and correct for differences in the amount of poly(A)+ RNA applied in each lane. During the estrous cycle, a 1-kb RLX transcript was detected in porcine ovaries using a probe corresponding to the C-peptide 2 region of pRLX (Fig. 1, bottom panel). The relative density of the hybridization signal in ovaries from day 3 cyclic animals increased by 25% on day 13, followed by a decline in the hybridization signal by day 19 (Fig. 1). Poly(A)+ RNA from the control tissue (whole uterus from a day 13 cyclic sow) showed no evidence of RLX hybridization. When the blot was reprobed with the lpB15 probe to detect cyclophilin, a 1-kb transcript
cycle (d) FIG. 1. Northern analysis of pRLX mRNA from ovaries of sows on days 3, 13, and 19 of the cycle and of the uterus of a day 13 cycle sow (control). Twenty micrograms of poly(A)+ RNA from the tissues were subjected to electrophoresis and transferred to membranes, as described in Materials and Methods. RNAs were hybridized with a 32P-labeled probe corresponding to the C-peptide 2 region of pRLX and exposed to x-ray film for 24 h. The same blot was reprobed, after removal of the labeled RLX probe, with a 32P-labeled cyclophilin cDNA probe for 48 h to monitor gel loading and the integrity of the RNA. Mol wt markers are shown at the left.
kb 1.28 —
•
f
ft
.78 —
40
60
90
pregnancy (d) FIG. 2. Northern analysis of pRLX mRNA from ovaries of sows on days 40, 60, and 90 of pregnancy. One microgram of poly(A)+ RNA from each group was processed as described in Fig. 1. RNAs were hybridized with a 32P-labeled probe corresponding to the C-peptide 1 region of pRLX and exposed to x-ray film for 1 h. Mol wt markers are shown at the left.
was observed after 48-h exposure to film (Fig. 1, top panel). The hybridization signal in porcine tissues with the rat cyclophilin probe was considerably reduced compared with that of the RLX probes. On day 40, 60, or 90 of pregnancy, RLX transcript was detected using a probe to the C-peptide 1 region of pRLX (Fig. 2). The relative densities of the hybridization signals on days 40, 60, and 90 of pregnancy were similar (0.92, 1.1, and 0.90, respectively), with only a small increase in hybridization signal on day 60. In these studies, using 1 fig poly(A)+ RNA from sows throughout
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RLX mRNA IN THE SOW CL pregnancy for Northern analysis, there was no evidence of hybridization to the cyclophilin probe after 2-4 days of exposure to film. During the estrous cycle, pregnancy, and lactation, a single 1-kb RLX transcript was detected using a probe corresponding to a portion of the B-chain of pRLX (Fig. 3). As expected, the amount of RLX mRNA varied with reproductive state. The RLX transcript was detected after 1-h exposure with only 0.2 ng poly(A) + RNA from the ovary of pregnancy (day 90; Fig. 3). Since the nonpregnant ovary is known to contain much less RLX, in order to detect a signal from cyclic (day 13) and lactation (day 2) ovaries, 100-fold more poly(A) + RNA was loaded on the gel. On day 13 of the cycle an intense RLX hybridization signal was detected with 20 ng poly(A) + RNA (Fig. 3). A decline in RLX gene expression was observed on day 2 of lactation, since the signal generated with 20 ng poly(A) + RNA was comparable to that observed with only 0.2 /ig poly(A) + from day 90 of pregnancy. When data from pregnancy, the cycle, and lactation were normalized for the amount of RNA loaded, the results showed that the relative density of the hybridization signal on day 13 of the cycle was only 2% of that during pregnancy (day 90). In addition, the signal detected on day 2 of lactation declined to 0.1% of that observed before parturition.
p C d90 d13
L d2 Cyclophilin
Relaxin
0.2
20
20
poly (A) + RNA FlG. 3. Northern analysis of pRLX mRNA from ovaries of sows on day 90 pregnancy (P), day 13 of the cycle (C), and day 2 of lactation (L). The indicated amounts of poly(A)+ RNA were processed as described in Fig. 1. RNAs were hybridized with a 32P-labeled oligonucleotide probe corresponding to the B-chain of pRLX and exposed to xray film for 1 h. The same blot was reprobed, after removal of the labeled RLX probe, with a 32P-labeled cyclophilin cDNA probe as an internal control and exposed to film for 48 h.
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Analysis of Northern blots, shown in Fig. 3, demonstrate that after a 48-h exposure to film, a single 1-kb cyclophilin transcript was detected when 20 fig ovarian mRNA was loaded from day 13 cyclic and day 2 lactating sows. The hybridization signals on day 13 of the cycle and day 2 of lactation were similar, verifying that similar amounts of poly(A)+ RNA were loaded. However, with only 0.2 /zg ovarian mRNA from day 90 pregnant sows, cyclophilin mRNA was not detectable after a 48-h exposure to film and remained undetectable after a 4-day exposure. In situ hybridization In preliminary experiments the three probes complementary in sequence to different regions of pRLX mRNA (Table 1) were used for in situ hybridization of sections of ovaries from animals collected during the estrous cycle, pregnancy, and lactation. Similar results were obtained when the B-chain, C-peptide 1, or C-peptide 2 pRLX probes were used on these tissues. To facilitate comparison of RLX mRNA hybridization, tissues shown in the photomicrographs in Fig. 4 were all incubated with the same probe complementary to a portion of the Bchain of pRLX. The darkfield photomicrographs show that the majority of grains were restricted to the luteal tissue and localized over the cytoplasm of the luteal cells (Fig. 4, B, E, and H, middle panel). The location of cells in Fig. 4, B, E, and H, can be determined by comparison with the brightfield view of the same respective fields in Fig. 4, A, D, and G. RLX transcript was demonstrated in the cycle CL (day 13) after a 2-week exposure to emulsion (Fig. 4B). Although Northern analysis revealed a RLX mRNA band from ovarian tissue on day 3 of the cycle (Fig. 2), there was no evidence of hybridization to sections of ovarian tissue with any of the RLX probes on either day 3 or day 19 of the cycle. An intense hybridization signal was observed in the CL of the day 90 pregnant sow after 1 week of exposure to emulsion (Fig. 4E), which was similar to the hybridization signal observed on days 40 and 60 of pregnancy (data not shown). There was a decline in the hybridization signal after parturition, as illustrated by the reduced number of grains in the CL on day 2 of lactation (Fig. 4H). Negative controls used in these studies included 1) incubation of ovarian sections with a 35S-labeled 48-mer heterologous probe to hPRL which did not hybridize (Fig. 4, C, F, and I, right panel), 2) absence of hybridization of labeled RLX probes to control tissue (uterus from day 13 cyclic sow), and 3) incubation of ovarian tissue with a 100-fold excess of probe which did not hybridize.
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RLX mRNA *IN THE SOW CL
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FIG. 4. In situ localization of RLX mRNA in the porcine CL during different reproductive states. Brightfield (right panel) and darkfield (middle and left panels) photomicrographs of the CL on day 13 of the cycle (A, B, and C; exposed 2 weeks), day 90 of pregnancy (D, E, and F; exposed 1 week), and day 2 of lactation (G, H, and I; exposed 2 weeks). Tissues were hybridized with either a 36S-labeled probe corresponding to the B-chain region of pRLX (right and middle panels) or a 3SS-labeled hPRL probe (control; left panel). Identical fields are shown in A and B, D and E, and G and H. In the darkfield photomicrographs, silver grains, indicating hybridization to RLX mRNA, are seen as clusters of white grains over luteal tissue (B, E, and H). Nearby sections incubated with a control heterologous probe show an absence of grains over luteal tissue (C, F, and I). Magnification is indicated by the scale bar (microns).
Discussion In 1980, Gast et al. (23) reported that RNA isolated from CL of pregnant sows directed the synthesis of a protein with immunological and sequence identity to authentic RLX. Later, evidence for synthesis of RLX was also found in translation mixtures using RNA isolated from CL of nonpregnant sows (24). In addition, an increase in translatable levels of mRNA was reported which paralleled the rising levels of RLX in luteal tissue (24). The present study confirms and extends these earlier studies by showing that mRNA for RLX is present in the sow CL at early and midluteal phases of the cycle and increases during pregnancy. After parturition, RLX transcript declines, but is still detectable in early lactation. Northern analysis revealed the presence of RLX mRNA in ovaries on day 3 of the cycle, with an increase by day 13 and declining thereafter, with little evidence for RLX mRNA on day 19. When RLX message was localized in the CL by in situ hybridization, the results reflected the Northern analysis studies, except that we were unable to detect message in sections of day 3 cycle
ovaries. This is probably due to the small amount of mRNA present in a tissue section on day 3 of the cycle compared with extraction and concentration of mRNA from grams of whole tissue. The in situ hybridization data support the reports of bioactive and immunoactive RLX present in the sow ovary beginning around midcycle, with maximal levels during diestrus (2, 3). However, we have also localized immunoreactive RLX in the newly formed CL within hours after PMSG/hCG-induced ovulation (6). During pregnancy, an increase in RLX gene expression was observed compared with levels during the cycle; however, levels of RLX message were similar on days 40, 60, and 90 of pregnancy when studied by either Northern analysis or in situ hybridization. This was surprising, since both luteal content and systemic blood levels of RLX increase steadily during pregnancy (3,5). Anderson et al. (3) reported that between days 40 and 100 of pregnancy, the luteal RLX content increases approximately 4-fold. Our inability to detect changes in the RLX mRNA content of the ovary with advancing pregnancy may be due to the relatively high level of RLX mRNA present as early as day 40, which may be stored and not
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RLX mRNA IN THE SOW CL fully translated until later in pregnancy. When RLX gene transcription first increases in pregnancy is not known; however, it is likely to occur before day 20 of pregnancy, since luteal RLX levels rise dramatically over cyclic levels at this time (3). The possibility exists that during pregnancy, RLX message may not be the limiting factor in hormone production by the porcine CL and that posttranslational factors may play a role. Fluctuations in levels of RLX mRNA in the rat ovary have been reported in the cycle and during pregnancy. During the rat estrous cycle, although levels are 200- to 400-fold lower than in late pregnancy, ovarian RLX mRNA levels fluctuate (25) and reflect the RLX content of the ovary (1). This is similar to our findings, which show that when RLX message content is relatively low, during the cycle and lactation, a direct correlation between RLX message and hormone is observed. In the rat RLX mRNA levels increase with advancing pregnancy (26) and correspond to the RLX content of the rat ovary (1, 27). In addition, rat RLX mRNA has been localized in luteal cells of pregnancy (28). Whether RLX message is present after parturition in the rat ovary has not been investigated. Although luteal and peripheral blood RLX levels decline significantly in the postpartum pig (3, 5), we have demonstrated for the first time that RLX mRNA is present in ovarian tissue on day 2 of lactation. The presence of RLX message in the CL during early lactation indicates that the tissue retains its capacity for synthesis of the hormone. This supports previous studies that indicated a low level of RLX immunoactivity in peripheral plasma and sow luteal tissue during early lactation (5-7). Alternatively, since the half-life of the RLX message is unknown, it is possible that this low level RLX mRNA represents residual message remaining from pregnancy. Whether RLX message is translated during lactation is not certain, since the hormone localized in the CL may represent stored hormone not released at parturition. The presence of both protein and mRNA for RLX during the estrous cycle, pregnancy, and lactation in the pig ovary suggests that RLX has a variety of roles in these different physiological states. Relaxin's role in pregnancy as a systemic hormone acting to relax uterine smooth muscle and remodel the connective tissue of the cervix is well established (29, 30). During lactation, a systemic role for RLX has been proposed based on RLX's ability to inhibit the milk ejection reflex (31). In addition, RLX has been shown to induce rat pituitary secretion of both oxytocin (32) and PRL (33). The markedly lower levels of RLX message and hormone in the CL of the cycle and early lactation suggest a paracrine role for RLX as an intraovarian regulator. However, further
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studies are needed to determine the physiological role of RLX in the CL of the nonpregnant pig.
Acknowledgments The authors would like to thank Drs. W. S. Young III and M. J. Brownstein, NIMH, for providing two of the oligonucleotide probes used, and Dr. M. Melner, Oregon Primate Research Center (Beaverton, OR), for the plBl5 cDNA probe. We acknowledge the assistance of D. Brocht in collection of the ovaries and thank Drs. G. D. BryantGreenwood and F. C. Greenwood, University of Hawaii, for helpful discussions and review of the manuscript.
References 1. Sherwood OD, Rutherford JE 1981 Relaxin immunoactivity levels in ovarian extracts obtained from rats during various reproductive states and from adult cycling pigs. Endocrinology 108:1171 2. Ali SM, McMurtry JP, Bagnell CA, Bryant-Greenwood GD 1986 Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle. Biol Reprod 34:139 3. Anderson LL, Ford JJ, Melampy RM, Cox DF 1973 Relaxin in porcine corpora lutea during pregnancy and after hysterectomy. Am J Physiol 225:1215 4. Sherwood OD, Chang CC, Be Vier GW, Dzuik PJ 1975 Radioimmunoassay of plasma relaxin levels throughout pregnancy and at parturition in the pig. Endocrinology 97:834 5. Anderson LL, Adair V, Stromer MH, McDonald WG 1983 Relaxin production and release after hysterectomy in the pig. Endocrinology 113:677 6. Bagnell CA, McMurtry JP, Baker NK, Timtim JK, Bryant-Greenwood GD 1987 Detection of relaxin by immunohistochemistry in the corpus luteum during lactation. Biol Reprod 37:1317 7. Afele S, Bryant-Greenwood GD, Chamley WA, Dax EM 1979 Plasma relaxin immunoactivity in the pig at parturition and during nuzzling and suckling. J Reprod Fertil 56:451 8. Whitely J, Willcox DL, Hartmann PE, Yamamoto SY, BryantGreenwood GD 1985 Plasma relaxin levels during suckling and oxytocin stimulation in the lactating sow. Biol Reprod 3:705 9. Haley J, Hudson P, Scanlon D, John M, Cronk M, Shine J, Tregear G, Niall H 1982 Porcine relaxin: molecular cloning and cDNA structure. DNA 1:155 10. Haley J, Crawford R, Hudson P, Scanlon D, Tregear G, Shine J, Niall H 1987 Porcine relaxin gene structure and expression. J Biol Chem 262:11940 11. Einspanier R, Pitzel L, Wutte W, Hagendorff G, Preub KD, Kardalinou E, Scheit KH 1986 Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. FEBS Lett 204:37 12. Young III WS, Bonner TI, Brann MR 1986 Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc Natl Acad Sci USA 83:9727 13. Danielson PE, Forss-Petter S, Brow MA, Calauetta L, Douglass J, Milner RJ, Sutcliffe JG 1988 P1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261 14. Koletsky AJ, Harding MW, Handschumacher RE 1986 Cyclophilin: distribution and varient properties in normal and neoplastic tissues. J Immunol 137:1054 15. McKinnon R, Danielson P, Brow MD, Bloom FE, Sutcliffe JG 1987 Expression of small cytoplasmic transcripts of the rat identifier elements in vivo and in cultured cells. Mol Cell Biol 7:2148 16. Skinner MK, Schlitz SM, Anthony CT 1989 Regulation of Sertoli cell differentiated function: testicular transferrin and androgenbinding protein expression. Endocrinology 124:3015 17. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156 18. Aviv H, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69:1408
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19. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 20. Berent SL, Mahmoudi M, Torczynski RM, Bragg PW, Bollon AP 1985 Comparison of oligonucleotide and long DNA fragments as probes in DNA and RNA dot, Southern, Northern, colony and plaque hybridizations. Biotechniques 3:208 21. Angerer LM, Angerer RC 1981 Detection of poly(A)+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res 9:2819 22. Stahler MS, Budd GC, Pansky B 1987 Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization. Biol Reprod 36:999 23. Gast MJ, Mercado-Simmen R, Niall H, Boime I 1980 Cell-free synthesis of a high molecular weight relaxin-related protein. Ann NY Acad Sci 343:148 24. Gast MJ 1982 Studies on luteal generation and processing of the high molecular weight relaxin precursor. Ann NY Acad Sci 380:111 25. Crish JF, Soloff MS, Shaw AR 1986 Changes in relaxin precursor messenger ribonucleic acid levels in ovaries of rats after hysterectomy and removal of conceptuses, and during the estrous cycle. Endocrinology 119:1222
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26. Crish JF, Soloff MS, Shaw AR 1986 Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy. J Biol Chem 261:1909 27. Sherwood OD, Crnekovic VE, Gordon WL, Rutherford JE 1980 Radioimmunoassay of relaxin throughout pregnancy and at parturition in the pig. Endocrinology 97:834 28. Hudson P, Haley J, Cronk M, Shine J, Niall H 1981 Molecular cloning and characterization of cDNA sequences coding for rat relaxin. Nature 291:127 29. Porter DG 1979 Relaxin: old hormone, new prospect. In: Finn CA, (ed) Oxford Reviews of Reproductive Biology. Clarendon Press, Oxford, vol 1:1 30. Bryant-Greenwood GD 1982 Relaxin as a new hormone. Endocr Rev 3:62 31. Summerlee AJS, O'Byrne KT, Paisley AC, Breeze MF, Porter DG Relaxin affects the central control of oxytocin release. Nature 309:372 32. Dayanith G, Cazalis M, Nordmann JJ 1987 Relaxin affects the release of oxytocin and vasopressin from the neurohypothesis. Nature 325:813 33. Sortino MA, Cronin MJ, Wise PM 1989 Relaxin stimulates prolactin secretion from anterior pituitary cells. Endocrinology 124:2013
Erratum In the article "Induction of /3-endorphin secretion by lymphocytes after subcutaneous administration of corticotropin-releasing factor," by A. Kavelaars, F. Berkenbosch, G. Croiset, R. E. Ballieux, and C. J. Heijnen {Endocrinology 126: 759-764, 1990), on page 763, Ref. 21 was inadvertently omitted and should be as follows: Kavelaars A, Ballieux RE, Heijnen CJ 1989 The role of interleukin-1 in the CRF- and AVP-induced secretion of ir-/3-endorphin by human peripheral blood mononuclear cells. J Immunol 142:2338.
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Vol. 126, No. 5 Printed in U.S.A.
Relaxin Gene Expression in the Sow Corpus Luteum during the Cycle, Pregnancy, and Lactation* CAROL A. BAGNELL, LILY TASHIMA, WALTER TSARK, SHUJATH M. ALI, AND JOHN P. McMURTRY Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96822; Nonruminant Animal Nutrition Laboratory, Livestock and Poultry Institute, USDA, Agricultural Research Service (J.P.M.), Beltsville, Maryland 20705
ABSTRACT. Relaxin (RLX) mRNA was sought in the corpus luteum of the pig during the cycle, pregnancy, and lactation using Northern analysis and in situ hybridization. Three oligonucleotide probes to regions of the preprorelaxin molecule were used for hybridization to detect RLX message and gave similar results. Northern analysis showed a single 1-kilobase RLX transcript at all three stages of the reproductive cycle studied. The intensity of the RLX hybridization signal was greatest in pregnancy and varied during the cycle. The signal in ovaries from day 3 cyclic animals increased by day 13 and declined on day 19. However, the hybridization signal at midcycle was only 2% of that during pregnancy. After parturition on day 2 of lactation, RLX message was still detected in the ovary, although at reduced
T
HE CORPUS luteum (CL) is a well recognized source of relaxin (RLX) in the pig. However, the amount of RLX in the CL varies depending on the physiological status of the animal. During the estrous cycle, low levels of bioactive and immunoactive RLX are found in extracts of pig ovaries, with maximal concentrations observed during diestrus (1). This correlates well with the RLX immunolocalization pattern in the CL during the cycle (2). During gestation, blood and tissue concentrations increase and reach peak levels in late pregnancy (3, 4). The surge of RLX into blood a few hours before parturition is associated with a decline in luteal RLX content (5). During the course of lactation, RLX tissue content declines (3), but is still detectable by immunohistochemistry through day 10 of lactation (6). In addition, episodic secretion of RLX after acute nuzzling and suckling or oxytocin administration has been reported (7, 8). Whether the presence of bioactive and immunoactive RLX in the CL during the cycle, pregnancy, and lactation represents active synthesis, unreleased residual hormone, or sequestered hormone is Received November 13,1989. Address all correspondence and requests for reprints to: Dr. Carol A. Bagnell, Department of Animal Sciences, Cook College, Rutgers University, New Brunswick, New Jersey 08903. * This work was supported by NIH Grant HD-20624.
levels compared with those during the cycle and pregnancy. In situ hybridization results showed hybridization to RLX mRNA in luteal tissue on day 13 of the cycle, but not on day 3 or 19. An increased hybridization signal was observed on days 40, 60, and 90 of pregnancy, with a decline on day 2 of lactation. Control sections incubated with labeled heterologous probe or preincubated with excess unlabeled probe did not hybridize. These results indicate a good correlation between the relative concentrations of RLX transcript and immunohistochemical results previously reported in the corpus luteum of the sow. In addition, they demonstrate that the RLX gene is expressed in luteal tissue, not only in pregnancy, but also in the cycle and early lactation. (Endocrinology 126: 2514-2520, 1990)
not clear. RLX is synthesized as a prohormone precursor, proRLX, consisting of A and B chains attached by a connecting peptide (C-peptide). The porcine RLX (pRLX) gene has been cloned and sequenced (9), and RLX mRNA has been demonstrated in extracts of CL from both pregnant (10) and cyclic pigs (11). However, there has been no study of RLX gene expression in the pig CL throughout the reproductive cycle in this species. Since the presence of RLX mRNA is a prerequisite for production of the hormone in tissue, we sought to demonstrate RLX message by Northern analysis and in situ hybridization in the CL during various reproductive stages in the pig.
Materials and Methods Animals Multiparous Duroc sows that exhibited at least two estrous cycles averaging 21 days were used for these experiments. Animals were checked for estrus in the presence of a boar, and the first day of estrus was designated day 0 of the cycle. Ovaries were collected from animals on days 3, 13, and 19 of the cycle. Ovaries from pregnant and lactating sows were obtained by mating the animals on the first day of estrus, which was designated day 0 of pregnancy. The day of parturition was
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RLX mRNA IN THE SOW CL termed day 0 of lactation. Ovaries were collected on days 40, 60, and 90 of pregnancy and day 2 of lactation. Intact uteri from cyclic animals were used as a control tissue in these studies. At the time of tissue collection, sows were transported to the abattoir, where they were stunned and exsanguinated. Tissues were frozen in liquid nitrogen within 2 min after death and stored at —80 C until use. The Northern analysis and in situ hybridization studies were performed on ovarian tissue from individual animals, with at least three animals per time point. Probes
Three 48-base oligonucleotide probes to portions of the prepro-RLX molecule were used (9). Two probes to adjacent regions of the C-peptide of porcine pro-RLX (C-peptide 1, AA 45-60; C-peptide 2, AA 61-76) were synthesized by Peninsula Laboratories (Belmont, CA). A third RLX-specific probe corresponding to a portion of the B-chain of pRLX (AA 11-26) was generously provided by Drs. W. S. Young III and M. J. Brownstein, NIMH (Bethesda, MD). To check the specificity of hybridization, a 48-base oligonucleotide to a region of the human PRL (hPRL) molecule (AA 60-75) was used as a control (courtesy of Drs. S. W. Young III and M. J. Brownstein). The sequences of the oligonucleotide probes used are shown in Table 1. The probes were 3'-end labeled with terminal-deoxynucleotidyl transferase [Bethesda Research Laboratories (BRL), Gaithersburg, MD] and either [a-32P]dATP (>3000 Ci/mmol; New England Nuclear, Boston, MA) for Northern analysis studies or [a-35S]dATP (>l000 Ci/mmol; New England Nuclear) for in situ hybridization (12). A rat cDNA probe which encodes cyclophilin (plB15) was used as a control to monitor gel loading for Northern analysis (13) (generously provided by Dr. Michael Melner, Oregon Primate Research Center, Beaverton, OR). plB15 is a gene shown to be constitutively expressed in a wide variety of tissues and conserved across species (14) and has been used in a number of laboratories as a control probe to demonstrate the quantity and integrity of the RNA present (15, 16). Isolation and Northern analysis of RNA Total RNA was prepared using guanidinium thiocyanate and phenol-chloroform extraction (17). Poly(A)+ RNA was isolated from total RNA by affinity chromatography on oligo-(dT) TABLE
cellulose (18). Purity and quantity were ascertained by spectrophotometric analysis at 260 and 280 nm. Known amounts of poly(A)+ RNA (0.2-20 ng), were denatured in 50% formamide-5.5% formaldehyde and fractionated in a 1.0% agarose gel containing 5.0% formaldehyde. The RNA was transferred to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH) overnight by the capillary method and dried at 80 C (19). A RNA ladder [0.16-1.77 kilobases (kb); BRL] was also electrophoresed in each gel as a molecular size marker. This lane was excised from the gel before transfer and stained with ethidium bromide, and migration of the RNA fragments was recorded for later comparison with autoradiograms. Hybridization was carried out using a modification of the methods described by Berent et al. (20). The dried nylon membranes were prehybridized for 5 h at 55 C in 6 X SSPE (1 X SSPE = 0.18 M NaCl; 10 mM NaPO4, pH 7.7; and 1 mM EDTA), 1 x Denhardt's solution (1 X Denhardt's = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 0.1% sodium dodecyl sulfate (SDS), 250 Mg/ml tRNA, and salmon sperm DNA. This solution was replaced with hybridization solution containing 6 x SSPE, 5 x Denhardt's solution, 0.1% SDS, 250 Mg/ml tRNA, and salmon sperm DNA containing 32 P-labeled probe (1-5 x 106 cpm/ml). After hybridization for 20 h at 55 C, filters were washed three times in 6 x SSPE-0.1% SDS at room temperature for 15 min, once in 6 X SSPE-0.1% SDS at 55 C for 1 h, and then finally in 6 X SSPE (without SDS) at room temperature for 15 min. The filters were exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) with DuPont Cronex intensifying screens (DuPont, Wilmington, DE) at -80 C for the times indicated in the figure legends. Filters were rehybridized, after removal of the RLX probe, with a 32P-labeled plB15 (cyclophilin) cDNA control probe. Autoradiograms were scanned with a Bio-Rad Scanning Densitometer (model 620, Bio-Rad, Richmond, CA), and the amount of RLX mRNA present relative to cyclophilin mRNA was determined. In situ hybridization Frozen 10-/im sections were thaw-mounted onto chromergecleaned, poly-L-lysine-coated slides and stored at -20 C until used (21). Hybridization procedures were based on methods described by Young III et al. (12). After fixation in 4% formaldehyde in PBS (0.05 M), slides were washed in PBS and
1. Sequence of 48-mer oligonucleotide probes used Amino acids 11-26
DNA probe sequence 5' GGA GAC GGA GCC AC A GAT CTC CAC CCA CAG ACG GAC TAA TTC TCG GCC 3'
pRLX C-peptide 1
45-60
5' CTT TAA GAT TTC TGC ATC TTT GGT GAT GGA GGA TGG CAT GGT TTC TGC 3'
pRLX C-peptide 2
61-76
5' TGT TCC CTT CAG CTC CTG TGG CAA ATT ACG AAC AAA TTC CAA CAT CAT 3'
hPRL
60-75
5' CAT CTG TTG GGC TTG CTC CTT GTC TTC GGG GGT GGC AAG GGA AGA AGT 3'
Probe pRLX B-chain
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RLX mRNA IN THE SOW CL
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incubated in 0.25% acetic anhydride in 0.1 M triethanolamine HCl-0.9% NaCl (pH 8.0) for 10 min to reduce nonspecific binding. Slides were then dehydrated in increasing amounts of alcohol and 100% chloroform and air dried. The 35S-labeled probes (3.0 X 105 cpm/50 ix\) were dissolved in a hybridization solution containing 4 X SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 50% formamide, 1 X Denhardt's solution, 10% dextran sulfate, 500 ng/m\ sheared single stranded DNA, 250 Mg/ml yeast tRNA, and 100 mM dithiothreitol and applied to each section. After incubation in a humidified chamber at 37 C for 20 h, slides were washed 4 times (15 min each) in 2 X SSC containing 50% formamide at 40 C, followed by 2 30-min washes in 1 x SSC at room temperature. The slides were dehydrated in increasing amounts of alcohol and air dried. In some experiments to test the specificity of the hybridization reaction, tissues were preincubated with a 100-
Cyclophilin
Relaxin
fold excess of unlabeled pRLX probe for 18-24 h at 37 C (22).
After preincubation, tissues were washed in 2 X SSC (10 times) and incubated with the a labeled pRLX probe and processed as described above. To determine the appropriate exposure time for emulsion autoradiography, slides were exposed to x-ray film (Kodak XOmat with intensifying screens) overnight. Slides were subsequently coated with Kodak NTB2 liquid photographic emulsion (Eastman Kodak; diluted 1:1 with 0.6 M ammonium acetate). Slides were exposed at 4 C in boxes, with drying agent for 1 or 2 weeks. After development slides were counterstained with toluidine blue (0.5%) and photographed under both bright- and darkfield illumination.
Results Northern analysis Ovaries from sows at different reproductive states were examined by Northern analysis for the expression of RLX mRNA. In these studies similar results were obtained when any of the three oligonucleotide probes complementary to different regions of the pRLX molecule (Table 1) were used. In addition, the plB15 probe, to detect the constitutively expressed protein cyclophilin, was used as an internal control to monitor the concentration of mRNA in the Northern blots (13). RLX densitometric data were standardized against the level of cyclophilin mRNA in the samples in order to determine and correct for differences in the amount of poly(A)+ RNA applied in each lane. During the estrous cycle, a 1-kb RLX transcript was detected in porcine ovaries using a probe corresponding to the C-peptide 2 region of pRLX (Fig. 1, bottom panel). The relative density of the hybridization signal in ovaries from day 3 cyclic animals increased by 25% on day 13, followed by a decline in the hybridization signal by day 19 (Fig. 1). Poly(A)+ RNA from the control tissue (whole uterus from a day 13 cyclic sow) showed no evidence of RLX hybridization. When the blot was reprobed with the lpB15 probe to detect cyclophilin, a 1-kb transcript
cycle (d) FIG. 1. Northern analysis of pRLX mRNA from ovaries of sows on days 3, 13, and 19 of the cycle and of the uterus of a day 13 cycle sow (control). Twenty micrograms of poly(A)+ RNA from the tissues were subjected to electrophoresis and transferred to membranes, as described in Materials and Methods. RNAs were hybridized with a 32P-labeled probe corresponding to the C-peptide 2 region of pRLX and exposed to x-ray film for 24 h. The same blot was reprobed, after removal of the labeled RLX probe, with a 32P-labeled cyclophilin cDNA probe for 48 h to monitor gel loading and the integrity of the RNA. Mol wt markers are shown at the left.
kb 1.28 —
•
f
ft
.78 —
40
60
90
pregnancy (d) FIG. 2. Northern analysis of pRLX mRNA from ovaries of sows on days 40, 60, and 90 of pregnancy. One microgram of poly(A)+ RNA from each group was processed as described in Fig. 1. RNAs were hybridized with a 32P-labeled probe corresponding to the C-peptide 1 region of pRLX and exposed to x-ray film for 1 h. Mol wt markers are shown at the left.
was observed after 48-h exposure to film (Fig. 1, top panel). The hybridization signal in porcine tissues with the rat cyclophilin probe was considerably reduced compared with that of the RLX probes. On day 40, 60, or 90 of pregnancy, RLX transcript was detected using a probe to the C-peptide 1 region of pRLX (Fig. 2). The relative densities of the hybridization signals on days 40, 60, and 90 of pregnancy were similar (0.92, 1.1, and 0.90, respectively), with only a small increase in hybridization signal on day 60. In these studies, using 1 fig poly(A)+ RNA from sows throughout
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RLX mRNA IN THE SOW CL pregnancy for Northern analysis, there was no evidence of hybridization to the cyclophilin probe after 2-4 days of exposure to film. During the estrous cycle, pregnancy, and lactation, a single 1-kb RLX transcript was detected using a probe corresponding to a portion of the B-chain of pRLX (Fig. 3). As expected, the amount of RLX mRNA varied with reproductive state. The RLX transcript was detected after 1-h exposure with only 0.2 ng poly(A) + RNA from the ovary of pregnancy (day 90; Fig. 3). Since the nonpregnant ovary is known to contain much less RLX, in order to detect a signal from cyclic (day 13) and lactation (day 2) ovaries, 100-fold more poly(A) + RNA was loaded on the gel. On day 13 of the cycle an intense RLX hybridization signal was detected with 20 ng poly(A) + RNA (Fig. 3). A decline in RLX gene expression was observed on day 2 of lactation, since the signal generated with 20 ng poly(A) + RNA was comparable to that observed with only 0.2 /ig poly(A) + from day 90 of pregnancy. When data from pregnancy, the cycle, and lactation were normalized for the amount of RNA loaded, the results showed that the relative density of the hybridization signal on day 13 of the cycle was only 2% of that during pregnancy (day 90). In addition, the signal detected on day 2 of lactation declined to 0.1% of that observed before parturition.
p C d90 d13
L d2 Cyclophilin
Relaxin
0.2
20
20
poly (A) + RNA FlG. 3. Northern analysis of pRLX mRNA from ovaries of sows on day 90 pregnancy (P), day 13 of the cycle (C), and day 2 of lactation (L). The indicated amounts of poly(A)+ RNA were processed as described in Fig. 1. RNAs were hybridized with a 32P-labeled oligonucleotide probe corresponding to the B-chain of pRLX and exposed to xray film for 1 h. The same blot was reprobed, after removal of the labeled RLX probe, with a 32P-labeled cyclophilin cDNA probe as an internal control and exposed to film for 48 h.
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Analysis of Northern blots, shown in Fig. 3, demonstrate that after a 48-h exposure to film, a single 1-kb cyclophilin transcript was detected when 20 fig ovarian mRNA was loaded from day 13 cyclic and day 2 lactating sows. The hybridization signals on day 13 of the cycle and day 2 of lactation were similar, verifying that similar amounts of poly(A)+ RNA were loaded. However, with only 0.2 /zg ovarian mRNA from day 90 pregnant sows, cyclophilin mRNA was not detectable after a 48-h exposure to film and remained undetectable after a 4-day exposure. In situ hybridization In preliminary experiments the three probes complementary in sequence to different regions of pRLX mRNA (Table 1) were used for in situ hybridization of sections of ovaries from animals collected during the estrous cycle, pregnancy, and lactation. Similar results were obtained when the B-chain, C-peptide 1, or C-peptide 2 pRLX probes were used on these tissues. To facilitate comparison of RLX mRNA hybridization, tissues shown in the photomicrographs in Fig. 4 were all incubated with the same probe complementary to a portion of the Bchain of pRLX. The darkfield photomicrographs show that the majority of grains were restricted to the luteal tissue and localized over the cytoplasm of the luteal cells (Fig. 4, B, E, and H, middle panel). The location of cells in Fig. 4, B, E, and H, can be determined by comparison with the brightfield view of the same respective fields in Fig. 4, A, D, and G. RLX transcript was demonstrated in the cycle CL (day 13) after a 2-week exposure to emulsion (Fig. 4B). Although Northern analysis revealed a RLX mRNA band from ovarian tissue on day 3 of the cycle (Fig. 2), there was no evidence of hybridization to sections of ovarian tissue with any of the RLX probes on either day 3 or day 19 of the cycle. An intense hybridization signal was observed in the CL of the day 90 pregnant sow after 1 week of exposure to emulsion (Fig. 4E), which was similar to the hybridization signal observed on days 40 and 60 of pregnancy (data not shown). There was a decline in the hybridization signal after parturition, as illustrated by the reduced number of grains in the CL on day 2 of lactation (Fig. 4H). Negative controls used in these studies included 1) incubation of ovarian sections with a 35S-labeled 48-mer heterologous probe to hPRL which did not hybridize (Fig. 4, C, F, and I, right panel), 2) absence of hybridization of labeled RLX probes to control tissue (uterus from day 13 cyclic sow), and 3) incubation of ovarian tissue with a 100-fold excess of probe which did not hybridize.
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RLX mRNA *IN THE SOW CL
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FIG. 4. In situ localization of RLX mRNA in the porcine CL during different reproductive states. Brightfield (right panel) and darkfield (middle and left panels) photomicrographs of the CL on day 13 of the cycle (A, B, and C; exposed 2 weeks), day 90 of pregnancy (D, E, and F; exposed 1 week), and day 2 of lactation (G, H, and I; exposed 2 weeks). Tissues were hybridized with either a 36S-labeled probe corresponding to the B-chain region of pRLX (right and middle panels) or a 3SS-labeled hPRL probe (control; left panel). Identical fields are shown in A and B, D and E, and G and H. In the darkfield photomicrographs, silver grains, indicating hybridization to RLX mRNA, are seen as clusters of white grains over luteal tissue (B, E, and H). Nearby sections incubated with a control heterologous probe show an absence of grains over luteal tissue (C, F, and I). Magnification is indicated by the scale bar (microns).
Discussion In 1980, Gast et al. (23) reported that RNA isolated from CL of pregnant sows directed the synthesis of a protein with immunological and sequence identity to authentic RLX. Later, evidence for synthesis of RLX was also found in translation mixtures using RNA isolated from CL of nonpregnant sows (24). In addition, an increase in translatable levels of mRNA was reported which paralleled the rising levels of RLX in luteal tissue (24). The present study confirms and extends these earlier studies by showing that mRNA for RLX is present in the sow CL at early and midluteal phases of the cycle and increases during pregnancy. After parturition, RLX transcript declines, but is still detectable in early lactation. Northern analysis revealed the presence of RLX mRNA in ovaries on day 3 of the cycle, with an increase by day 13 and declining thereafter, with little evidence for RLX mRNA on day 19. When RLX message was localized in the CL by in situ hybridization, the results reflected the Northern analysis studies, except that we were unable to detect message in sections of day 3 cycle
ovaries. This is probably due to the small amount of mRNA present in a tissue section on day 3 of the cycle compared with extraction and concentration of mRNA from grams of whole tissue. The in situ hybridization data support the reports of bioactive and immunoactive RLX present in the sow ovary beginning around midcycle, with maximal levels during diestrus (2, 3). However, we have also localized immunoreactive RLX in the newly formed CL within hours after PMSG/hCG-induced ovulation (6). During pregnancy, an increase in RLX gene expression was observed compared with levels during the cycle; however, levels of RLX message were similar on days 40, 60, and 90 of pregnancy when studied by either Northern analysis or in situ hybridization. This was surprising, since both luteal content and systemic blood levels of RLX increase steadily during pregnancy (3,5). Anderson et al. (3) reported that between days 40 and 100 of pregnancy, the luteal RLX content increases approximately 4-fold. Our inability to detect changes in the RLX mRNA content of the ovary with advancing pregnancy may be due to the relatively high level of RLX mRNA present as early as day 40, which may be stored and not
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RLX mRNA IN THE SOW CL fully translated until later in pregnancy. When RLX gene transcription first increases in pregnancy is not known; however, it is likely to occur before day 20 of pregnancy, since luteal RLX levels rise dramatically over cyclic levels at this time (3). The possibility exists that during pregnancy, RLX message may not be the limiting factor in hormone production by the porcine CL and that posttranslational factors may play a role. Fluctuations in levels of RLX mRNA in the rat ovary have been reported in the cycle and during pregnancy. During the rat estrous cycle, although levels are 200- to 400-fold lower than in late pregnancy, ovarian RLX mRNA levels fluctuate (25) and reflect the RLX content of the ovary (1). This is similar to our findings, which show that when RLX message content is relatively low, during the cycle and lactation, a direct correlation between RLX message and hormone is observed. In the rat RLX mRNA levels increase with advancing pregnancy (26) and correspond to the RLX content of the rat ovary (1, 27). In addition, rat RLX mRNA has been localized in luteal cells of pregnancy (28). Whether RLX message is present after parturition in the rat ovary has not been investigated. Although luteal and peripheral blood RLX levels decline significantly in the postpartum pig (3, 5), we have demonstrated for the first time that RLX mRNA is present in ovarian tissue on day 2 of lactation. The presence of RLX message in the CL during early lactation indicates that the tissue retains its capacity for synthesis of the hormone. This supports previous studies that indicated a low level of RLX immunoactivity in peripheral plasma and sow luteal tissue during early lactation (5-7). Alternatively, since the half-life of the RLX message is unknown, it is possible that this low level RLX mRNA represents residual message remaining from pregnancy. Whether RLX message is translated during lactation is not certain, since the hormone localized in the CL may represent stored hormone not released at parturition. The presence of both protein and mRNA for RLX during the estrous cycle, pregnancy, and lactation in the pig ovary suggests that RLX has a variety of roles in these different physiological states. Relaxin's role in pregnancy as a systemic hormone acting to relax uterine smooth muscle and remodel the connective tissue of the cervix is well established (29, 30). During lactation, a systemic role for RLX has been proposed based on RLX's ability to inhibit the milk ejection reflex (31). In addition, RLX has been shown to induce rat pituitary secretion of both oxytocin (32) and PRL (33). The markedly lower levels of RLX message and hormone in the CL of the cycle and early lactation suggest a paracrine role for RLX as an intraovarian regulator. However, further
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studies are needed to determine the physiological role of RLX in the CL of the nonpregnant pig.
Acknowledgments The authors would like to thank Drs. W. S. Young III and M. J. Brownstein, NIMH, for providing two of the oligonucleotide probes used, and Dr. M. Melner, Oregon Primate Research Center (Beaverton, OR), for the plBl5 cDNA probe. We acknowledge the assistance of D. Brocht in collection of the ovaries and thank Drs. G. D. BryantGreenwood and F. C. Greenwood, University of Hawaii, for helpful discussions and review of the manuscript.
References 1. Sherwood OD, Rutherford JE 1981 Relaxin immunoactivity levels in ovarian extracts obtained from rats during various reproductive states and from adult cycling pigs. Endocrinology 108:1171 2. Ali SM, McMurtry JP, Bagnell CA, Bryant-Greenwood GD 1986 Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle. Biol Reprod 34:139 3. Anderson LL, Ford JJ, Melampy RM, Cox DF 1973 Relaxin in porcine corpora lutea during pregnancy and after hysterectomy. Am J Physiol 225:1215 4. Sherwood OD, Chang CC, Be Vier GW, Dzuik PJ 1975 Radioimmunoassay of plasma relaxin levels throughout pregnancy and at parturition in the pig. Endocrinology 97:834 5. Anderson LL, Adair V, Stromer MH, McDonald WG 1983 Relaxin production and release after hysterectomy in the pig. Endocrinology 113:677 6. Bagnell CA, McMurtry JP, Baker NK, Timtim JK, Bryant-Greenwood GD 1987 Detection of relaxin by immunohistochemistry in the corpus luteum during lactation. Biol Reprod 37:1317 7. Afele S, Bryant-Greenwood GD, Chamley WA, Dax EM 1979 Plasma relaxin immunoactivity in the pig at parturition and during nuzzling and suckling. J Reprod Fertil 56:451 8. Whitely J, Willcox DL, Hartmann PE, Yamamoto SY, BryantGreenwood GD 1985 Plasma relaxin levels during suckling and oxytocin stimulation in the lactating sow. Biol Reprod 3:705 9. Haley J, Hudson P, Scanlon D, John M, Cronk M, Shine J, Tregear G, Niall H 1982 Porcine relaxin: molecular cloning and cDNA structure. DNA 1:155 10. Haley J, Crawford R, Hudson P, Scanlon D, Tregear G, Shine J, Niall H 1987 Porcine relaxin gene structure and expression. J Biol Chem 262:11940 11. Einspanier R, Pitzel L, Wutte W, Hagendorff G, Preub KD, Kardalinou E, Scheit KH 1986 Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. FEBS Lett 204:37 12. Young III WS, Bonner TI, Brann MR 1986 Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc Natl Acad Sci USA 83:9727 13. Danielson PE, Forss-Petter S, Brow MA, Calauetta L, Douglass J, Milner RJ, Sutcliffe JG 1988 P1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7:261 14. Koletsky AJ, Harding MW, Handschumacher RE 1986 Cyclophilin: distribution and varient properties in normal and neoplastic tissues. J Immunol 137:1054 15. McKinnon R, Danielson P, Brow MD, Bloom FE, Sutcliffe JG 1987 Expression of small cytoplasmic transcripts of the rat identifier elements in vivo and in cultured cells. Mol Cell Biol 7:2148 16. Skinner MK, Schlitz SM, Anthony CT 1989 Regulation of Sertoli cell differentiated function: testicular transferrin and androgenbinding protein expression. Endocrinology 124:3015 17. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156 18. Aviv H, Leder P 1972 Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69:1408
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RLX mRNA IN THE SOW CL
19. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 20. Berent SL, Mahmoudi M, Torczynski RM, Bragg PW, Bollon AP 1985 Comparison of oligonucleotide and long DNA fragments as probes in DNA and RNA dot, Southern, Northern, colony and plaque hybridizations. Biotechniques 3:208 21. Angerer LM, Angerer RC 1981 Detection of poly(A)+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res 9:2819 22. Stahler MS, Budd GC, Pansky B 1987 Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization. Biol Reprod 36:999 23. Gast MJ, Mercado-Simmen R, Niall H, Boime I 1980 Cell-free synthesis of a high molecular weight relaxin-related protein. Ann NY Acad Sci 343:148 24. Gast MJ 1982 Studies on luteal generation and processing of the high molecular weight relaxin precursor. Ann NY Acad Sci 380:111 25. Crish JF, Soloff MS, Shaw AR 1986 Changes in relaxin precursor messenger ribonucleic acid levels in ovaries of rats after hysterectomy and removal of conceptuses, and during the estrous cycle. Endocrinology 119:1222
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26. Crish JF, Soloff MS, Shaw AR 1986 Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy. J Biol Chem 261:1909 27. Sherwood OD, Crnekovic VE, Gordon WL, Rutherford JE 1980 Radioimmunoassay of relaxin throughout pregnancy and at parturition in the pig. Endocrinology 97:834 28. Hudson P, Haley J, Cronk M, Shine J, Niall H 1981 Molecular cloning and characterization of cDNA sequences coding for rat relaxin. Nature 291:127 29. Porter DG 1979 Relaxin: old hormone, new prospect. In: Finn CA, (ed) Oxford Reviews of Reproductive Biology. Clarendon Press, Oxford, vol 1:1 30. Bryant-Greenwood GD 1982 Relaxin as a new hormone. Endocr Rev 3:62 31. Summerlee AJS, O'Byrne KT, Paisley AC, Breeze MF, Porter DG Relaxin affects the central control of oxytocin release. Nature 309:372 32. Dayanith G, Cazalis M, Nordmann JJ 1987 Relaxin affects the release of oxytocin and vasopressin from the neurohypothesis. Nature 325:813 33. Sortino MA, Cronin MJ, Wise PM 1989 Relaxin stimulates prolactin secretion from anterior pituitary cells. Endocrinology 124:2013
Erratum In the article "Induction of /3-endorphin secretion by lymphocytes after subcutaneous administration of corticotropin-releasing factor," by A. Kavelaars, F. Berkenbosch, G. Croiset, R. E. Ballieux, and C. J. Heijnen {Endocrinology 126: 759-764, 1990), on page 763, Ref. 21 was inadvertently omitted and should be as follows: Kavelaars A, Ballieux RE, Heijnen CJ 1989 The role of interleukin-1 in the CRF- and AVP-induced secretion of ir-/3-endorphin by human peripheral blood mononuclear cells. J Immunol 142:2338.
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