Role of Relaxin and Estrogen in the Control of Eosinophilic Invasion and Collagen Remodeling in Rat Cervical Tissue at Term¹

^a Department of Human Physiology, Faculty of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, Santa Fe, Argentina ^b Department of Molecular and Integrative Physiology and the College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
Ripening and dilation of the rat cervix at term involves a widespread reduction in the density and organization of collagen fibers following a polymorphonuclear eosinophilic invasion. These are hormonally regulated events; however, the correlation between hormonal milieu and these morphological changes is not well understood. To investigate the role of preparturient relaxin and estradiol-17ß (E₂) in cervical collagen remodeling and eosinophilic infiltration, pregnant rats were either sham-ovariectomized (group C) or ovariectomized in the morning of Day 22. Ovariectomized rats were treated with E₂ (group OE), relaxin (group OR), E₂ plus relaxin (group OER), or hormone vehicles (group O). There were 4 or 5 animals per group. Cervices were taken from animals killed approximately 1 h before expected parturition. Five-micrometer serial sections of paraffin-embedded cervices were stained with either Sirius Red in alkaline solution to measure eosinophil infiltration or in Picrosirius to measure collagen birefringence. Ovariectomized rats treated with E₂ (group OE or OER) showed high levels of eosinophilic infiltration that did not differ from those in group C intact controls. However, the values of eosinophilic infiltration in ovariectomized rats treated with relaxin or hormone vehicles (groups OR and O) were low and far below (p < 0.01) those of groups OE and C. In ovariectomized rats treated with E₂ alone (group OE), the widespread reduction in collagen organization that occurred in group C controls was not observed. It was only in ovariectomized rats treated with relaxin or E₂ + relaxin (groups OR and OER) that the values of birefringence were low, and they were as low as in control group C. In conclusion, this study indicates that eosinophilic infiltration and collagen remodeling in the rat cervix at term are under the control of different hormones: E₂ stimulates eosinophilic invasion, and relaxin promotes a widespread reorganization of collagen fibers.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammals, including humans, rats, and pigs, cervical ripening is essential for vaginal delivery [1–3]. Cervical ripening is thought to be largely attributable to disruption of ordered collagen bundles [1–5] following a polymorphonuclear leukocyte invasion of cervical tissue in humans, rats, sheep, and guinea-pigs [6–13].

The mechanisms that control polymorphonuclear leukocyte invasion to cervical tissue and the role, if any, of polymorphonuclear leukocytes in subsequent disruption of collagen bundles are poorly understood for all mammalian species. Nevertheless, some progress has been made with studies on the rat. We reported that, in the rat, the eosinophilic infiltration and collagen disruption that normally occur at the time of delivery may be under control of different hormones [14]. We found that estradiol-17ß (E₂) stimulates eosinophilic infiltration of the rat cervix [14], and this finding is supported by the demonstration of estrogen regulation of an eosinophil chemotactic factor in the immature rat uterus [15]. In contrast, progesterone was found to inhibit eosinophilic infiltration of the cervix [14]. Neither estrogen nor progesterone alone were found to be responsible for the collagen remodeling seen at term [14].

There is reason to suspect that relaxin plays a key role in regulating the modifications that occur in the rat cervix during pregnancy. Relaxin, which is produced in the corpus luteum and endometrium of the female rat [16], is present at high levels in the peripheral circulation throughout the second half of pregnancy [17]. It is well established that relaxin plays a major role in promoting growth and softening of the rat cervix [18–20]. Whereas the mechanism(s) whereby relaxin modifies the rat cervix remains to be determined, there is limited qualitative evidence that it does so by reducing the density and organization of collagen bundles [19, 20].

The hypothesis tested in this report is that relaxin brings about its effects on rat cervical collagen remodeling, at least in part, by promoting eosinophilic invasion and degranulation. In the study reported in this paper, relaxin-induced changes in the organization of cervical collagen fibers were quantified for the first time. The Picrosirius-polarization method, a histochemical procedure that allows morphometric analysis of collagen fiber organization, was used [21, 22]. Employing this procedure, Junqueira et al. [6] observed a greater reduction in collagen fiber density than was found by measuring the amount of collagen by means of its hydroxyproline content.

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals

Female adult rats (approximately 90 days of age and 200–250 g of body weight) of a Wistar-derived strain bred in the Department of Human Physiology (Santa Fe, Argentina) were used. Animals were maintained in a controlled environment (22 ± 2°C; lights-on from 0600–2000 h), and they had free access to pellet laboratory chow (Nutric, Cordoba, Argentina) and tap water. Either bilateral ovariectomy or sham ovariectomy was performed under ether anesthesia on primiparous pregnant rats at 0600 h on Day 22 (Day 1 = the day sperm were found in the vaginal smear). In our colony, delivery in untreated control rats normally occurs on Day 23 between 1230 and 1400 h. After ovariectomy or sham operation, the animals were assigned to two sets of 5 different experimental groups.

Hormone Treatments

Both sham-ovariectomized (group C, n = 5) and ovariectomized (group O, n = 5) controls received s.c. injections of E₂ vehicle (0.1 ml sesame oil) at 0800 h on Day 22 and Day 23, and of relaxin vehicle (0.2 ml of 1% benzopurpurin [Sigma Chemical Co., St. Louis, MO] in sterile saline) at 0700 h, 1400 h, and 1800 h on Day 22 and at 0700 h on Day 23. The remaining ovariectomized rats received hormones. Group OE (n = 5) received 1.0 µg E₂ (Sigma Chemical Co.) and relaxin vehicle as described for groups C and O. Group OR (n = 5) received highly purified porcine relaxin [23] (100 µg at 0700 h on Day 22, 200 µg at 1400 h and 1800 h on Day 22, and at 0700 h on Day 23) and E₂ vehicle as described for groups C and O. Group OER (n = 4) received both E₂ and relaxin as described for groups OE and OR. The protocol for E₂ administration was chosen because it maintains serum E₂ levels comparable to those in late pregnancy [24]. The regimen of relaxin administration was chosen with the intention of reproducing the second antepartum elevation of plasma relaxin levels [25]. All rats were killed at 1200 h on Day 23.

In order to determine whether hormone replacement with E₂ and relaxin had effects that were similar to those of endogenous hormones during pregnancy, a second set of rats, treated as above, delivered pups. Birth parameters were determined in groups C (n = 8), OE (n = 5), OR (n = 4), and OER (n = 4) as previously described [26]. The birth parameters were as follows: 1) length of gestation—number of hours between the estimated time of ovulation (0200 h on Day 1) and the initiation of active labor; 2) duration of straining—time interval between the start of active labor and the appearance of first pup; 3) duration of delivery—time interval between the appearance of the first pup and the expulsion of the last pup and its placenta; 4) percentage of pups born alive&; number of pups born alive/total number of pups born x 100; 5) percentage of pups surviving 48 h postpartum&; number of pups alive after 48 h/number of pups born alive x 100.

Preparation of Cervical Tissue for Light Microscopy

Uterine cervices were fixed by immersion in 10% buffered formalin for 24 h, dehydrated in graded concentrations of alcohol, embedded in paraffin, and sectioned at 5 µm. Serial sections of whole cervices were taken along a cervical canal and stained with either Picrosirius-hematoxylin or Sirius Red in alkaline solution.

Measurement of Eosinophil Invasion

Cervical sections were stained for 60 min in 0.5% Sirius Red (Direct Red 80; Aldrich, Milwaukee, WI) dissolved in alkaline solution (NaOH, pH 10.5) and then counterstained with Harris' hematoxylin (Biopur, Rosario, Argentina) for 10 min. This method permits the identification of eosinophils in tissue sections: their specific granules are deeply stained in red, which strongly contrasts against a pale background [7, 27]. Because eosinophilic infiltration does not occur uniformly throughout the cervical stroma, the quantification in each section was performed in the whole cervical stroma. For each cervical specimen, three sections were evaluated, and at least 40 fields were counted in each section [14, 28]. Means for each rat were calculated and used for statistical analysis.

Measurement of Organization of Collagen Fiber Bundles

Cervical sections were stained for 30 min in a 0.1% Sirius Red solution in saturated picric acid, washed rapidly in tap water, counterstained with Harris' hematoxylin for 15 min, dehydrated, and mounted in synthetic resin. Picrosirius-hematoxylin-stained sections were evaluated by polarization microscopy. This method is specific for orientated collagen molecules, in the sense that only these structures present a bright birefringence [22]. Collagen fibers normally form thick bundles of densely packed and regularly arranged fibers, and they appear as brightly birefringent structures throughout the whole microscopic field. When collagen fibers are not dense and/or not regularly arranged, they are weakly birefringent. In order to quantify the influence of hormone treatment on collagen fiber organization, measurement of the intensity of birefringence was performed in specimens from the different experimental groups. Polarization was obtained with two Polaroid filters, one located below the condenser and the other, above the objective lens. Light intensity was measured with a Reichert (Reichert Jung, Nossloch, Germany) model 6 A E 9/65 microspectrophotometer at a 585-nm wavelength. A low-power (x2.6) Zeiss aplanatic objective (Carl Zeiss, Inc., Thornwood, NJ) was used in these measurements. The light intensity of the whole cervical stroma (more than 40 fields) from one section of each specimen was measured. Optical conditions were regulated so that the transmittance light intensity of each cervix was expressed as the percentage area of birefringence. This technique has been used previously to measure the extent of change in collagen fiber organization [6, 14, 22].

Statistics

The point-counting procedure [28] was used to obtain the data used for morphometric analysis of the number of eosinophils invading the cervical tissue. Eosinophils were counted using a glass disc with a squared grid inserted in a focusing eyepiece and a x100 immersion objective [29]. The fraction of points occurring within the structure of eosinophils (stained in red) was determined and then compared to the total number of points lying within the cervical stroma. The volume fraction was calculated by applying the formula given by Weibel [30]: Vv = Pi/P, where Vv is the estimated volume fraction of the object; Pi is the number of incident points over the eosinophils; and P is the total number of incident points over the volume unit.

In order to investigate the differences in eosinophilic infiltration of the cervical stroma among experimental groups, values were subjected to the Kruskal-Wallis one-way ANOVA [31]. To assign probabilities of the difference between two groups, the Mann-Whitney U test was used [31].

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of Hormone Treatments on Birth Parameters

Table 1 contains data clearly indicating that administering relaxin restored normal birth parameters. Treatment with estrogen alone (group OE) resulted in a significant increase in duration of straining, a decrease in the percentage of pups born alive, and a decrease in pup survival rate. In contrast, when rats were treated with relaxin (group OR or OER), birth parameters did not differ from those in intact controls (group C).

Measurement of Eosinophilic Invasion

Consistent with previous studies [7, 14] in which pregnant intact rats were killed during delivery, pregnant sham-ovariectomized rats (group C) that were killed at 1200 h on Day 23 (immediately before the time of expected parturition) showed high eosinophilic infiltration in the cervical stroma (see Figs. 1A and 2A). When pregnant rats were ovariectomized at 0600 h on Day 22 and given no subsequent hormone replacement therapy (group O), cervical eosinophilic infiltration was extremely low (Fig. 2A). When ovariectomized rats were treated with estrogen alone (group OE), a massive eosinophilic infiltration was found (Figs. 1C and 2A). Levels of eosinophils in cervices from group OE rats were as great as those in group C rats (Fig. 2A). Treatment with relaxin did not increase cervical eosinophil levels. In ovariectomized rats treated with relaxin alone (group OR), the eosinophilic infiltration did not exceed that found in group O (Figs. 1E and 2A). Moreover, when ovariectomized rats were treated with estrogen plus relaxin (group OER; Fig. 1G), eosinophilic infiltration in the cervical stroma did not differ from that of group OE (Fig. 2A).

View larger version (136K): [in this window] [in a new window]   FIG. 1. Photomicrographs of sections across the uterine cervices of rats in different experimental conditions. At left, sections were stained by Sirius Red in alkaline solution and counterstained with hematoxylin for the detection of eosinophils. At right, sections from the same rats were studied by the Picrosirius-polarization method, which detects the degree of orientation of collagen fibers in tissue sections. All brightly birefringent structures, which shine against a dark background, are collagenous. The cervical stroma of an intact pregnant control (group C) shows a massive eosinophilic infiltration (A) and a low level of organization of collagen (low birefringence) (B). In ovariectomized estrogen-treated rats (group OE), eosinophilic invasion occurred (C), but the collagen framework showed a highly oriented arrangement (high birefringence) (D). In contrast to group OE animals, the ovariectomized relaxin-treated animals (group OR) show a lack of eosinophil invasion (E) and low birefringence (F). In ovariectomized estrogen- and relaxin-treated rats (group OER), cervical eosinophilic infiltration (G) and collagen degradation (H) appeared similar to those in the pregnant intact controls (group C). x800 (left); x450 (right).

View larger version (35K): [in this window] [in a new window]   FIG. 2. Mean (+SE) eosinophilic infiltration (A) and area of birefringence (B). Means with different letters differ significantly (p < 0.01). n = 4 or 5.

Measurement of Organization of Collagen Fiber Bundles

In pregnant intact rats (group C), low birefringence was observed in the cervical stroma (Figs. 1B and 2B), correlating with the widespread reduction in density and orientation of collagen fiber bundles that occurs during late pregnancy. When pregnant rats were ovariectomized at 0600 h on Day 22 and given no subsequent hormone therapy (group O), collagen birefringence was high (Fig. 2B), which is indicative of a dense and highly organized collagen framework. When ovariectomized rats were treated with estrogen alone (group OE), birefringence was high. There was no indication that either collagen density or orientation in the cervical stroma was lower than in group O (Figs. 1D and 2B). In ovariectomized rats treated with relaxin alone (group OR) or estrogen plus relaxin (group OER), collagen birefringence was low and not different from that in the sham-ovariectomized (group C) controls (Figs. 1, F and H, and 2B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 
The current study indicates that eosinophilic infiltration and remodeling of the collagen within the rat cervix at term are under control of different hormones. Estrogen stimulates eosinophilic invasion but not remodeling of collagen. Relaxin promotes remodeling of the collagen but not eosinophilic invasion.

The present study not only confirms our previous finding that estrogen promotes infiltration of eosinophils into the cervix in pregnant rats [14] but also extends our understanding by demonstrating that eosinophilic infiltration is dependent upon the continued presence of elevated blood estrogen levels during late pregnancy. In rats that were ovariectomized on Day 22 and not given hormone replacement therapy with estrogen (groups O and OR), cervical eosinophil levels were low by Day 23. The present study is consistent with previous observations that treatment with estrogen alone does not reduce the density and organization of collagen fibers [20], and that it results in prolonged delivery with reduced fetal survival [26].

The present study also extends our understanding of the hormonal regulation of eosinophil infiltration by providing evidence that relaxin does not increase the level of eosinophil infiltration in the cervix beyond that promoted by estrogen alone. Thus, relaxin does not appear to play a role in cervical eosinophil infiltration during the second half of rat pregnancy. The widespread disruption of collagen fibers and the absence of eosinophilic invasion found in pregnant ovariectomized rats treated only with relaxin (group OR) indicates that relaxin's actions on the rat cervix are not mediated by the promotion of eosinophil invasion. The absence of eosinophils in rats treated with relaxin alone could be attributable to either the lack of estrogen stimulation [14] or relaxin-induced eosinophilic degranulation. Since remodeling of collagen occurred in ovariectomized rats treated with estrogen plus relaxin (group OER) in the presence of eosinophils, it appears that degranulation of eosinophils it is not the correct explanation.

Relaxin's effects on the cervix in pregnant rats are estrogen-dependent [32]. It seems likely that the ability of relaxin alone in this study to reduce collagen organization is due to the fact that the rats were ovariectomized near the expected time of delivery (approximately 30 h before delivery), when the animals were essentially primed with the endogenous estrogen that rises in the peripheral blood during late pregnancy [32].

The results of this study are consistent with earlier findings that relaxin is required for rapid and safe delivery in the rat [26, 33–37]. Neither induction of early delivery nor abortion occurred after ovariectomy. There was no need for progesterone replacement because the ovariectomy was performed about 32 h before delivery. This is the time relative to delivery when functional luteolysis occurs, and serum levels of progesterone fall precipitously [25]. Previous work showed that rats ovariectomized 30 h before parturition and treated with either estrogen plus relaxin or relaxin alone restored pup survival and all parturition parameters to values not different from those of intact controls [26]. In the present study, we also found no differences in pup survival and delivery trauma in either ovariectomized rats treated with relaxin alone or estrogen plus relaxin.

Perhaps the process of cervical dilation should be visualized as an epiphenomenon, in which several factors act to bring about its success. Polymorphonuclear leukocyte infiltration may be related to an immunological process [38] that offers some protection from infections that could be provoked during the opening of cervical canal at parturition. A link between the immune system and induction of labor is supported by evidence of an association of preterm labor with infection [38]. Another possible role for the polymorphonuclear leukocyte invasion of the cervix in late pregnancy may be the formation of intercommunicating canals through the cervical extracellular matrix. These channels would permit a rapid diffusion of hormones or other collagenase-activating factors that would act on pre-existing collagenases (such as collagen-bound collagenases) at term, since it has been shown that the migration of polymorphonuclear leukocytes increases the permeability of certain tissues [39, 40].

In conclusion, this investigation does not support the hypothesis that relaxin brings about its effects on rat cervical collagen remodeling through eosinophilic invasion and degranulation. Eosinophilic infiltration and collagen remodeling in the rat cervix at term are under control of different hormones; it appears that estrogen stimulates eosinophilic invasion and relaxin promotes collagen remodeling.

	ACKNOWLEDGMENTS

 
We are very grateful to Professor G.S. Montes (School of Medicine, Sao Paulo, Brazil) for helpful discussion, to Dr. Elia Caldini for expertise and assistance in photomicrography, and to Mr. Juan C. Villarreal for indispensable assistance in animal care.

	FOOTNOTES

 
¹ This study was supported by grants from the Argentine National Research Council (CONICET, PID 3752/92&BID 802/OCAR PCIT442) and the Universidad Nacional del Litoral (CAI+D 468/89). E.H.L. is a Career Investigator of the CONICET.

² Correspondence: Enrique H. Luque, Department of Human Physiology, Faculty of Biochemistry and Biological Sciences, Casilla de Correo 530, Santa Fe, Argentina. FAX: 54 42 550944; eluque{at}fbcb.unl.edu.ar

Accepted: May 11, 1998.

Received: February 24, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Liggins GC, Forster CS, Grieves SA, Schwartz AL. Control of parturition in man. Biol Reprod 1977; 16:39–56.[Abstract]
2. Calder AA. Control of parturition: scientific and clinical aspects. In: Garfield RE (ed.), Uterine Contractility. Norwell, MA: Serono Symposia; 1990: 283–293.
3. Sherwood OD, Downing SJ, Lao Guico-Lamm M, Hwang J-J, O'Day-Bowman MB, Fields PA. The physiological effects of relaxin during pregnancy; studies in rats and pigs. In: Milligen SR (ed.), Oxford Reviews of Reproductive Biology, vol. 15. Oxford: Oxford Press; 1993: 143–189.
4. Kitamura K, Ito A, Mori Y, Hirakawa S. Changes in uterine cervical collagenase with specific reference to cervical ripening. Biochem Med 1979; 22:332–337.[CrossRef][Medline]
5. Rajabi MR, Dean DD, Beydoun SN, Woessner JF. Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition. Am J Obstet Gynecol 1988; 159:971–976.[Medline]
6. Junqueira LCU, Zugaib M, Montes GS, Toledo OMS, Krisztán RM, Shigihara KM. Morphologic and histochemical evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical dilation. Am J Obstet Gynecol 1980; 138:273–281.[Medline]
7. Luque EH, Montes GS. Progesterone promotes a massive infiltration of the rat uterine cervix by the eosinophilic polymorphonuclear leukocytes. Anat Rec 1989; 223:257–265.[CrossRef][Medline]
8. Osmers R, Rath W, Adelmann-Grill BC, Fittkow C, Kuloczik M, Szeverényi M, Tschesche H, Kuhn W. Origin of cervical collagenase during parturition. Am J Obstet Gynecol 1992; 166:1455–1460.[Medline]
9. Fosang AJ, Handley CJ, Santer V, Lowther DA, Thorburn GD. Pregnancy-related changes in the connective tissue of the ovine cervix. Biol Reprod 1984; 30:1223–1235.[Abstract]
10. Hegele-Hartung C, Chwalisz K, Beier HM, Elger W. Ripening of the uterine cervix of the guinea-pig after treatment with the progesterone antagonist onapristone (ZK 98,299): an electron microscopic study. Hum Reprod 1989; 4:369–377.[Abstract/Free Full Text]
11. Duchesne M-J, Badia E. Immunohistochemical localization of the eosinophil major basic protein in the uterus horn and cervix of the rat at term and after parturition. Cell Tissue Res 1992; 270:79–86.[CrossRef][Medline]
12. Owiny JR, Gilbert RO, Wahl CH, Nathanielsz PW. Leukocytic invasion of the ovine cervix at parturition. J Soc Gynecol Invest 1995; 2:593–596.[CrossRef][Medline]
13. Luque EH, Bassani MM, Ramos JG, Maffini M, Canal A, Kass L, Caldini EG, Ferreira JMC, Muñoz de Toro MM, Montes GS. Leukocyte infiltration and collagenolysis in cervical tissue from intrapartum sheep. J Vet Med A 1997; 44:501–510.
14. Luque EH, Ramos JG, Rodriguez HA, Muñoz de Toro MM. Dissociation in the control of cervical eosinophilic infiltration and collagenolysis at the end of pregnancy or after pseudopregnancy in ovariectomized steroid-treated rats. Biol Reprod 1996; 55:1206–1212.[Abstract]
15. Lee YH, Howe RS, Sha S-J, Teuscher C, Sheehan DM, Lyttle R. Estrogen regulation of an eosinophil chemotactic factor in the immature rat uterus. Endocrinology 1989; 125:3022–3028.[Abstract]
16. Fields PA, Lee AB, Haab LM, Hwang J-J, Sherwood OD. Evidence for a dual source of relaxin in the pregnant rat: immunolocalization in the corpora lutea and endometrium. Endocrinology 1992; 130:2985–2990.[Abstract]
17. Sherwood OD, Crnekovic VE, Gordon WL, Rutherford JE. Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. Endocrinology 1980; 107:691–698.[Abstract]
18. Hwang J-J, Shanks RD, Sherwood OD. Monoclonal antibodies specific for rat relaxin. IV. Passive immunization with monoclonal antibodies during the antepartum period reduces cervical growth and extensibility, disrupts birth, and reduces pup survival in intact rats. Endocrinology 1989; 125:260–266.[Abstract]
19. Lee AB, Hwang J-J, Haab LM, Fields PA, Sherwood OD. Monoclonal antibodies specific for rat relaxin. VI. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts histological changes associated with cervical softening at parturition in rats. Endocrinology 1992; 130:2386–2391.[Abstract]
20. Cheah SH, Ng KH, Johgalingam VT, Ragavan M. The effects of oestradiol and relaxin on extensibility and collagen organisation of the pregnant rat cervix. J Endocrinol 1995; 146:331–337.[Abstract]
21. Junqueira LCU, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979; 11:447–455.[CrossRef][Medline]
22. Montes GS. Structural biology of the fibres of the collagenous and elastic systems. Cell Biol Int 1996; 20:15–27.[CrossRef][Medline]
23. Sherwood OD, O'Byrne EM. Purification and characterization of porcine relaxin. Arch Biochem Biophys 1974; 160:185–190.[CrossRef][Medline]
24. Downing SJ, Sherwood OD. The physiological role of relaxin in the pregnant rat. IV. The influence of relaxin on cervical collagen and glycosaminoglycans. Endocrinology 1986; 118:471–479.[Abstract]
25. Sherwood OD, Downing SJ, Golos TG, Gordon WL, Tarbell MK. Influence of light-dark cycle on antepartum serum relaxin and progesterone immunoactivity levels and on birth in the rat. Endocrinology 1983; 113:997–1003.[Abstract]
26. Cheah SH, Sherwood OD. Effect of preparturient 17ß-estradiol and relaxin on parturition and pup survival in the rat. Endocrinology 1988; 122:1958–1963.[Abstract]
27. Bogomoletz W. Advantages de la coloration par le rouge Sirius de l'amyloide et des éosinophiles. Arch Anat Cytol Pathol 1980; 28:252–253.[Medline]
28. Gundersen HJG, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A, West MJ. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988; 96:379–394.[Medline]
29. Anderson JM. Histometry. In: Bancroft JD, Stevens A (eds.), Theory and Practice of Histological Technique, 2nd ed. London: Churchill Livingston; 1982: 428–457.
30. Weibel ER. Stereological principles for morphometry in electron microscopic cytology. Int Rev Cytol 1969; 26:235–302.[Medline]
31. Siegel S. Nonparametric statistics for the behavioral sciences. New York: McGraw-Hill; 1956.
32. Sherwood OD. Relaxin. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, vol I, 2nd ed. New York: Raven Press Ltd; 1994: 861–1009.
33. Steinetz BG, O'Byrne EM. Speculations on the probable role of relaxin in cervical dilation and parturition in rats. Semin Reprod Endocrinol 1983; 1:335–342.
34. Downing SJ, Sherwood OD. The physiological role of relaxin in the pregnant rat I. The influence of relaxin on parturition. Endocrinology 1985; 116:1200–1205.[Abstract]
35. Lao Guico-Lamm M, Sherwood OD. Monoclonal antibodies specific for rat relaxin. II. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts birth in intact rats. Endocrinology 1988; 123:2479–2485.[Abstract]
36. Hwang J-J, Sherwood OD. Monoclonal antibodies specific for rat relaxin. III. Passive immunization with monoclonal antibodies throughout the second half of pregnancy reduces cervical growth and extensibility in intact rats. Endocrinology 1988; 123:2486–2490.[Abstract]
37. Burger LL, Sherwood OD. Evidence that cellular proliferation contributes to relaxin-induced growth of both the vagina and the cervix in the pregnant rat. Endocrinology 1995; 136:4820–4826.[Abstract]
38. Kelly RW. Pregnancy maintenance and parturition: the role of prostaglandin in manipulating the immune and inflammatory response. Endocr Rev 1994; 15:684–706.[CrossRef][Medline]
39. Hibbs MS, Mainardi CL, Kang AH. Type-specific collagen degradation by eosinophils. Biochem J 1982; 207:621–624.[Medline]
40. Milks LC, Conyers GP, Cramer EB. The effect of neutrophil migration on epithelial permeability. J Cell Biol 1986; 103:2729–2738.[Abstract/Free Full Text]