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a Department of Reproductive Biology, German Primate Center, 37077 Göttingen, Germany
b Institute of Chemistry, Veterinary University, Hannover, Germany
c Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that relaxin is probably the most important effector of cAMP up-regulation in endometrial stromal cells from the primate cycle [13–16] and is instrumental in inducing the decidualization necessary for successful implantation [17–21]. Altogether, there is therefore considerable indirect evidence to suggest that in primates, relaxin is involved in the hormonal control of implantation and may be a good indicator for successful implantation in the human.
Recently we have been able to clone the cDNA for relaxin from the marmoset monkey and to map its pattern of expression in female reproductive tissues from the cycle and pregnancy at the level of both the mRNA (using in situ hybridization) and its protein product (using immunohistochemistry) [22]. This New World monkey exhibits a reproductive profile very similar to that in the human [23], but additionally it offers the advantage that the ovarian cycle can be synchronized by a luteolytic application of prostaglandin (PG) F2
. In the present study, we have extended previous observations on relaxin biosynthesis by evaluating the pattern of relaxin secretion in vivo throughout the estrous cycle and early pregnancy. These results are then analyzed in relation to the detailed profiles of other hormonal parameters (LH, progesterone) and to the follicular/luteal status of the ovaries.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female marmoset monkeys (Callithrix jacchus) with normal ovarian cycles were housed in pairs with normal or castrated males under controlled conditions (24°C, 50–60% humidity, 13L:11D) in the German Primate Center, Göttingen, Germany. All females were between 2 and 7 years of age, 350–530 g body weight, nulliparous, primiparous, and multiparous. All monkeys had been successfully trained for routine blood collection prior to the commencement of the experiments. Blood samples (0.3 ml) were routinely collected for progesterone measurements using a validated immunoassay [24] twice per week from the vena saphena prior to the experiments to confirm normal cyclicity. The normal ovarian cycle of a marmoset lasts 28 days, with on average a 10-day follicular phase and an 18- day luteal phase [23]. Implantation occurs at around Day 12 of the luteal phase [25]. The marmoset monkey is sensitive toward PGF2; this allows the cycle to be regulated by PGF2-induced luteolysis [26]. Intramuscular application of a luteolytic dose of PGF2 (0.8 µg/animal Estrumate; Mallinckrodt Vet GmbH, Burgwedel, Germany) induces luteal regression, with ovulation on average 10.7 days later. This allows the precise staging of the ovarian cycle over the timing of the onset of preovulatory follicle development [27]. The experimental period for all animals started at the time point of PGF2 application, shown as cycle Day 0 in all figures.
Experimental Protocol
All experimental protocols had received approval of the local animal experiment committee. Three experimental groups were 1) females housed with intact males (n = 14) and receiving PGF2 at Day 12 luteal phase; 2) females housed with castrated males (n = 18) and receiving PGF2 at Day 12 luteal phase; and 3) females housed with castrated males (n = 3) and additionally receiving one hCG application (100 IU Ekluton; Vemie Veterinär Chemie GmbH, Kempen, Germany) per day on three subsequent days starting on Day 12 of the luteal phase. Five marmosets were excluded after preliminary study indicated relaxin levels in the cycle consistently below the limit of assay detection. Animals for the implantation study were ultrascanned and blood samples (0.3 ml) taken every third or fourth day starting on Day 3 after PGF2 application, whereas for other experimental animals, only blood samples were collected every second or third day after PGF2 application. Serum samples were stored immediately at -80°C until analysis for progesterone, bioactive LH/hCG, and relaxin. Ultrasound examination was carried out transabdominally over a period of 10 min with unsedated and unshaved animals using an ESAOTE ultrasound system fitted with mechanical probes of 7.5 and 10 MHz as previously described [28–30]. The ovaries were examined throughout the experimental period for the number of follicles and CL. In experimental group I, ultrasound was also used to confirm pregnancy as well as to detect the number of embryos. At the end of these pregnancies, the number of infants born was also documented. All ultrasound examinations were recorded on videotape, and images were printed on photographic paper.
Hormone Assays
Progesterone immunoassay The progesterone content in the marmoset serum was detected using an enzyme immunoassay as described previously [24]. The detection limit for progesterone was 0.5 ng/ml, and the inter- and intraassay variation coefficients were below 8%.
LH bioassay Blood LH/chorionic gonadotropin (CG) concentrations were measured by an in vitro bioassay (murine Leydig cells), as described elsewhere [31], using the human standard (LER 907, NHPP, NIDDK, NIH, Bethesda, MD) for calibration. The working range of the LH bioassay was between 0.1 and 10 mIU bio-LH activity, and the intra- and interassay coefficients of variation were 7.0% and 20.9%, respectively.
Relaxin immunoassay Serum relaxin concentration was determined using a relaxin enzyme immunoassay: porcine anti-relaxin antiserum (serum 258, courtesy of Dr. O.D. Sherwood, University of Illinois) [32] and biotinylated porcine relaxin (tracer). Biotinylation of porcine relaxin was achieved as follows. Briefly, 1 mg porcine relaxin (> 90% purity; courtesy of Dr. D. Sherwood) was dissolved in 85 µl 0.2 M N-methylmorpholine-HCl buffer, pH 7.5, then mixed with 1.67 µmol biotinamidocaproate N-hydroxysuccinimide ester (B2643; Sigma Chemical Co., St. Louis; dissolved in 15 µl dimethyl formamide). The reaction solution was stirred for 2 h at room temperature and stopped with 100 µl 12% acetic acid and 50 µl dimethyl formamide. The final solution was brought to 2-ml volume by addition of 25% dimethyl formamide in PBS and then dialyzed (cut-off 1000 kDa) against the same solution overnight, followed by two changes in PBS only. The stoichiometry of biotinylation was tested using the HABA-avidin-reagent (Sigma H2153), according to the manufacturer's instructions, and estimated to be 3.0 moles biotin per mole relaxin.
The immunoassay followed a standard double-antibody, solid-phase format. The rabbit anti-relaxin polyclonal antiserum was used at a final dilution of 1:600 000 and the biotinylated porcine relaxin at 100 fmol/ml (final concentration). The incubation wells were coated with the secondary goat anti-rabbit IgG antibody. Human recombinant H2-relaxin (range 0.01–14.58 ng/ml; courtesy of Connetics Corporation, Palo Alto, CA), as well as porcine relaxin (range 0.03–14.58 ng/ml; courtesy of Prof. O.D. Sherwood), was used to generate the standard curve, including an appropriate amount of male marmoset serum in the buffer. Controls showed no change in sensitivity when 10, 25, or 50 µl of serum was included. The marmoset serum samples were applied at volumes ranging from 10 to 50 µl. After application of the primary antibody with an incubation of 6 h at room temperature in darkness, the biotinylated relaxin tracer was applied for 21 h at 4°C. This was followed by horseradish peroxidase-coupled streptavidin solution (150 ng/ml) for 30 min at 4°C in darkness, followed by several washings. Finally, the substrate solution (90 mM sodium acetate, 4.5 mM citric acid, 0.004% H2O2, 0.01% tetramethylbenzidine in 2% dimethyl sulfoxide) was added for 40 min at room temperature in the darkness, and the reaction was stopped by addition of 50 µl 0.2 M H2SO4. The yellow product was measured at 450 nm in a photometer. The lower limit of quantification was 5 pg/50-µl blood sample. The inter- and intraassay coefficients of variation were 15% and 10.5%, respectively. The cross-reactivities of the assay for insulin, transforming growth factor , and insulin-like growth factor-I were less than 1%.
Statistics and Analysis of Data
Immunoreactive hormone concentrations were calculated after logit-log transformation of their respective standard curves. Results from one experimental animal group were combined, and the first point was set at Day 0 with the PGF2 application. Statistical analysis was performed by Student's t-test and ANOVA. A value of p < 0.05 was taken as the level of significance. The characterization of preovulatory follicles and CL by ultrasound throughout one complete cycle in one animal was analyzed as described by Nubbemeyer and colleagues [30]. Further analysis of relaxin was performed with respect to the age, weight, and number of offspring at the time the experiments started.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The anti-porcine relaxin antibody used had already been validated in the context of immunohistochemistry and RIA [32]. Here we extended this validation to a new ELISA procedure. Porcine relaxin and recombinant human relaxin standards made up in an appropriate amount of male marmoset monkey serum (10 µl) showed identical standard curves and confirmed parallelism (Fig. 1). Serial dilution of serum from midpregnant marmoset monkeys and early-pregnant women also showed parallel standard curves (Fig. 1). In order to check further for the accuracy and specificity of the assay, the pregnancies of three marmoset monkeys were assessed for serum relaxin content (Fig. 2); they showed relaxin concentrations and profiles similar to those described previously by Steinetz and colleagues [33] using an independent RIA system.
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The relaxin profile for these 3 marmosets throughout pregnancy (Fig. 2) shows that the serum relaxin concentrations increased in the second trimester of pregnancy, at a time when progesterone production is declining and there is a shift from ovarian to placental steroidogenesis. The relaxin levels stayed elevated at about 15 ng/ml for 5 wk and then declined through the third trimester of pregnancy to low levels of < 2 ng/ml several days before parturition. During this time of decreasing relaxin concentrations, progesterone increased again and remained at a high level until immediately before parturition.
Relaxin in the Nonconceptive Cycle of the Marmoset
The endocrine hormone profiles from successive, PGF2 nonconceptive cycles of three individual monkeys are shown in Figure 3. In general, the relaxin concentration was low in the follicular phase (Days 0–10), increased after ovulation (around Day 13/14), and remained elevated during the luteal phase, essentially following the pattern for progesterone. However, there were considerable individual differences in relaxin production from one animal to another, which could persist over successive cycles. This means that there appears to be an individually specific relaxin concentration, whereas for progesterone such marked individual variation was not apparent. Although relaxin levels remained generally low during the follicular phase, a brief increase could be detected in some animals (e.g., w223 and w203, second cycles) on Day 9 just before ovulation.
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Combining data from 18 individual nonconceptive animals provides the profile of relaxin concentration shown in Figure 4, together with similar data from conceptive cycles (n = 14). As already shown for the single profiles, relaxin concentration was low during the follicular phase, increased briefly in most animals shortly before ovulation, and increased again during the luteal phase to reach maximal luteal levels of around 1.5 ng/ml. The small preovulatory relaxin rise was not detected in the samples from the conceptive cycle, probably because these were collected only at broader time intervals. However, such a peak was evident in most of the individual animal profiles discussed below (Figs. 5 and 6). The first increase above the basal levels of the follicular phase was apparent at around Day 16 after prostaglandin application (equivalent to Day 6 after LH increase) in the conceptive cycle and on Day 18 after prostaglandin application (equivalent to Day 8 after LH-increase) in the nonconceptive cycle (Fig. 4). The relaxin levels stayed high during the late luteal phase in the conceptive cycle whereas there was a decline in the nonconceptive cycle at the end of the luteal phase, corresponding to the expected time of implantation, 22 days after prostaglandin application (12 days after ovulation).
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Comparison of the conceptive cycles versus the nonconceptive cycles shows that the overall profiles were very similar; however, there are three important points. First, relaxin concentration increased significantly (p < 0.05) earlier in the conceptive cycle (Day 16 vs. Day 18); second, relaxin concentration was higher in the conceptive cycle than in the nonconceptive cycle; third, relaxin concentration in the conceptive cycles increased further in the late luteal phase, whereas in the nonconceptive cycles there was a decline by Day 24.
It should be noted that the moderately high and variable level of relaxin observed on Day 0 is probably, in part at least, attributable to the action of the injected PGF2, known to be a potent luteal secretagogue, given 2 h prior to blood collection.
Relaxin in the Conceptive Cycle of the Marmoset
Against this background, subsequent experiments concentrated upon the production of relaxin in early pregnancy around the time of conception and implantation, analyzing the levels of the various hormones, as well as the number of follicles and/or CL in the ovaries and the number of implanted fetuses. Analysis commenced in the early follicular phase of the conceptive cycle and continued up to Day 30 after PGF2 application, which is the early implantation phase. Ultrasound examination was restricted to the ovarian and uterine status of the conceptive cycles only. Each animal was ultrascanned to determine the number of follicles, CL, and/or implanted embryos. Figures 5 and 6 show the profiles of relaxin and LH/CG for individual animals. Each profile also indicates the serial number of the animal, and in parentheses, three numbers in the following order: the number of follicles detected by ultrasound, the number of CL detected by ultrasound, and the number of fetuses or infants born (as indicator of reproductive success or pregnancy loss)
Figure 5 shows profiles for eight animals in which the same number of follicles matured and ovulated and gave rise to the same number of born infants, in other words maximum reproductive success and no pregnancy loss. Figure 6 depicts six animals in which there was evident embryonic loss. Ultrasound examination of the uterine contents showed that embryonic loss always occurred during the first trimester.
Figure 5 indicates low relaxin levels during the follicular phase (approximately Days 0–10). These increased only after ovulation, which occurs at around Day 10 after PGF2 application. A preovulatory LH increase could be detected in most animals, although blood was collected only every second or third day. The exceptions were animals w33, w3, w13, and w4, in which no preovulatory increase was detected. In general, after ovulation in the conceptive cycle, both relaxin and LH/CG increased more or less in parallel during the midluteal phase, though the increase in relaxin concentration occurred earlier than for LH/CG. Although animals showed a great degree of individual variation, those with evident reduced fertility or loss (Fig. 6) appeared to exhibit hormone profiles indicating both lower hormone concentrations and a delay in the postconceptive hormone increase—the relaxin values in the early-midluteal phase being more like those observed in the nonconceptive cycle. This is best assessed by looking at the time when the serum values for relaxin and bioactive LH first persistently exceeded arbitrary basal values set at 0.6 ng/ml for relaxin (see above) and 50 mIU/ml for bioactive LH. For the animals with no reproductive loss (Fig. 5), this corresponds to Day 18 ± 2 for relaxin and to Day 22 ± 2 for bioactive LH. In contrast, for animals with reduced reproductive efficiency (Fig. 6), this is Day 22 ± 2 for relaxin and Day 25 ± 1 for bioactive LH.
In general, for both groups of animals (Figs. 5 and 6), no significant correlation could be detected between hormone concentrations and the absolute numbers of follicles, CL, or fetuses. Furthermore, an analysis including the mother's age, weight, or parity in relation to the relaxin levels also showed no significant association.
Application of Exogenous hCG
An additional experiment was carried out in order to examine further the relationship between relaxin and LH/hCG. Three nonpregnant female marmosets were injected with one exogenous hCG application (100 IU/animal) per day on three subsequent days during the mid/late luteal phase (Days 21–24 after PGF2 application, equivalent to Days 12–14 luteal phase). Implantation would have occurred normally around Day 12 in the luteal phase. Blood samples were collected 2 h after the hCG applications and then analyzed for progesterone and relaxin content. Whereas there was a significant increase in progesterone production compared to that in the preceding samples and control animals (n = 8 animals), there was no significant increase in the relaxin concentration (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A rise in relaxin in the late luteal phase of the nonconceptive (menstrual) cycle has also recently been described for the cynomolgus macaque [37]. In the human, the rise in relaxin concentration occurred approximately 6–8 days after the LH peak [34], with maximum concentrations at Day 12 after the LH peak. Also in the marmoset, maximal levels are reached 10–12 days after the LH peak of the nonconceptive cycle. In marmosets, the normal range of relaxin concentration during nonconceptive cycles appears to be quite broad and to show high between-individual variability. This is again similar to the human situation [34]. In general, the serum relaxin levels are lowest during the follicular phase, increase during the luteal phase, and remain elevated for the first day after prostaglandin application, at a time when progesterone is already declining. Also at Day 15 of pregnancy in the rat, PGF2 induces a large decrease in serum progesterone without influencing relaxin levels [38]. Furthermore, spontaneous abortion in the marmoset is accompanied by a precipitous fall in serum levels of progesterone, but only later is this followed by a decrease in serum relaxin [33].
Although high individual variability is evident in the relaxin profiles in the conceptive cycles of the marmoset, this variance appears to be less than in the nonconceptive cycles. There appears to be an earlier and larger increase in the serum relaxin concentration in cycles in which conception occurs. In these cycles, the rise in relaxin concentration occurs 6–13 days after mating and is closely followed by a rise in LH/CG bioactivity, very similar to the findings of Stewart et al. [37] for the macaque. There thus exists a close temporal association between the rise in relaxin and CG. It is known that relaxin secretion can be stimulated by the infusion of hCG during the luteal phase of nonpregnant cycles in women [39] and in rhesus monkeys [40–42]. These results, and the observation that in primates serum relaxin generally increases following hCG application during early pregnancy [9, 37, 43], implies that relaxin secretion might be directly regulated by LH/CG. However, in other observations, relaxin secretion appears to be independent of LH/CG [11, 44]. For example, no close relationship between LH/CG and relaxin could be shown in the rhesus monkey [45]. Also in the present study we are unable to demonstrate a direct link between LH/CG and relaxin. Quagliarello and colleagues [39] showed that the effect of CG application on relaxin secretion could be demonstrated only in the late luteal phase. It is therefore possible that a specific stage of CL differentiation or CL status is a prerequisite for a concomitant rise in relaxin and CG. Nevertheless, a direct connection between the two hormones has still not been convincingly demonstrated. Indeed, the exogenous application of CG during the mid/late luteal phase in the marmoset, at a time when implantation occurs, had no affect on relaxin secretion, despite causing an increase in progesterone production. Also, at later stages of pregnancy in the marmoset, there is a lack of concordance between CG and relaxin profiles, with progesterone effectively responding to the CG stimulus [46, 47].
In luteal cell cultures from both humans and macaques, the addition of hCG resulted in a clear increase in relaxin secretion into the culture media [48–50]. However, this is not an acute effect, since exposure to hCG for 2–3 days is required to observe a response [50]. Furthermore, the addition of hCG to incubated human term CL failed to stimulate increased relaxin formation [51].
Thus, although the present study indicates a temporal association between relaxin and CG increases during early pregnancy, these do not appear to be causally linked, with relaxin increasing before CG. A possibility that should also be considered here is that the earlier rise in relaxin may be only apparent, reflecting rather the relative sensitivities of the assays used. However, two other studies [46, 47] have also shown the same LH/CG increase in the conceptive cycle at around Day 18 after ovulation as shown here. Thus, if LH/CG is playing at best an indirect role, what is directly controlling relaxin secretion?
Both hormones are acting to support implantation, and there appears to be a delay in the postfertilization increase of both LH/CG and relaxin where there is early pregnancy loss, the relaxin profiles tending toward the pattern of the nonconceptive cycle. This would imply that relaxin in primates may be involved in the luteal mechanism of maternal recognition of pregnancy, with lower relaxin levels pointing to luteal insufficiency. Also, Stewart et al. [34] showed altered relaxin concentrations in women with early pregnancy loss, though these were higher than normal and exceeded the values obtained in nonconceptive cycles [11].
In women, circulating levels of relaxin correlate with the number of fetuses, with those in twin pregnancies being higher than in singleton pregnancies [8, 9], reflecting the number of CL present during pregnancy. This could not be confirmed here for the marmoset, in which relaxin concentration failed to correlate with the number of developed CL or with the weight, age, or parity of the mothers. The inability to correlate relaxin concentration with the number of CL, although these are major sources of relaxin [22], could be due to a special feature of the marmoset ovary, namely the presence of CL accessoria [52]. These are smaller than a regular CL, though they have a typical luteal structure, produce relaxin [22], and are present throughout the cycle and early pregnancy (unpublished results). The origin of the CL accessoria is still uncertain. Wislocki [52] suggested that they arise from luteinized small follicles. However, they could also develop from fragmented luteal tissue, or by differentiation of stromal cells (e.g., from theca tissue derived from ovulatory or even atretic follicles). Such CL accessoria might have both endocrine and local effects within the ovary and thus be able to buffer any consequences of specific luteolysis.
In conclusion, relaxin could be measured in the circulation during most follicular and all luteal phases of the marmoset, showing marked between-individual variation. There appears to be a distinction in the relaxin profiles from the conceptive and nonconceptive cycles, both in terms of the timing and the amount of the postovulatory relaxin increase, which may be related to the maternal recognition of pregnancy. There is thus also a temporal association between relaxin and CG increase at the time of implantation. However, the results obtained in the present study suggest that the control of relaxin secretion is independent of direct CG support, though CG may modulate relaxin secretion indirectly through other factors. As in the human, and shown here in detail for the marmoset, relaxin is an important marker for the quality of early pregnancy and implantation, providing additional support for the use of the marmoset as a model species in which to study periimplantation events and early embryonic loss.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Almuth Einspanier, Department of Reproductive Biology, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany. FAX: 49 551 3851 288; aeinspa{at}gwdg.de
Accepted: March 23, 1999.
Received: January 28, 1999.
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REFERENCES |
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on relaxin and progesterone secretion during pregnancy. Biol Reprod 1995; 53:834–839.[Abstract]This article has been cited by other articles:
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  R. L. Bogan, M. J. Murphy, R. L. Stouffer, and J. D. Hennebold Systematic Determination of Differential Gene Expression in the Primate Corpus Luteum during the Luteal Phase of the Menstrual Cycle Mol. Endocrinol., May 1, 2008; 22(5): 1260 - 1273. [Abstract] [Full Text] [PDF]
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 O. BARTSCHA and R. IVELL Relaxin and Phosphodiesterases Collaborate during Decidualization Ann. N.Y. Acad. Sci., December 1, 2004; 1030(1): 479 - 492. [Abstract] [Full Text] [PDF]
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 O. D. Sherwood Relaxin's Physiological Roles and Other Diverse Actions Endocr. Rev., April 1, 2004; 25(2): 205 - 234. [Abstract] [Full Text] [PDF]
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 J. J. Luna, A. Riesewijk, J. A. Horcajadas, R. d. van Os, F. Dominguez, S. Mosselman, A. Pellicer, and C. Simon Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle Mol. Hum. Reprod., February 1, 2004; 10(2): 85 - 90. [Abstract] [Full Text] [PDF]
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 O. Bartsch, B. Bartlick, and R. Ivell Phosphodiesterase 4 Inhibition Synergizes with Relaxin Signaling to Promote Decidualization of Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 324 - 334. [Abstract] [Full Text] [PDF]
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 B. Husen, J. Adamski, A. Bruns, D. Deluca, K. Fuhrmann, G. Moller, I. Schwabe, and A. Einspanier Characterization of 17{beta}-Hydroxysteroid Dehydrogenase Type 7 in Reproductive Tissues of the Marmoset Monkey Biol Reprod, June 1, 2003; 68(6): 2092 - 2099. [Abstract] [Full Text] [PDF]
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 G. A. Maston and M. Ruvolo Chorionic Gonadotropin Has a Recent Origin Within Primates and an Evolutionary History of Selection Mol. Biol. Evol., March 1, 2002; 19(3): 320 - 335. [Abstract] [Full Text] [PDF]
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A. Einspanier, D. Muller, J. Lubberstedt, O. Bartsch, A. Jurdzinski, K. Fuhrmann, and R. Ivell Characterization of relaxin binding in the uterus of the marmoset monkey Mol. Hum. Reprod., October 1, 2001; 7(10): 963 - 970. [Abstract] [Full Text] [PDF]
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T. Klonisch, C. Froehlich, F. Tetens, B. Fischer, and S. Hombach-Klonisch Molecular Remodeling of Members of the Relaxin Family During Primate Evolution Mol. Biol. Evol., March 1, 2001; 18(3): 393 - 403. [Abstract] [Full Text]
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