Localization of Specific Relaxin-Binding Cells in the Ovary and Testis of Pigs¹

^c Department of Molecular and Integrative Physiology ^d and College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

	ABSTRACT

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It is not known whether relaxin has physiological roles in the gonads in mammalian species. Limited evidence indicates that relaxin may act locally to regulate ovarian function in pigs. The possibility of a role for relaxin in testicular function in pigs has not been investigated. A major initial step toward the establishment of direct effects of relaxin on the ovary and/or testis is to demonstrate that relaxin binds with specificity to the gonads. Accordingly, the first objective of this study was to employ an immunohistochemical localization technique to determine whether relaxin-binding cells are present in the ovaries and/or testis of pigs. Once they were found to be present, the second objective was to determine whether relaxin-binding sites noticeably change either within the ovary at different stages of the estrous cycle and pregnancy, or within the testis at sexual maturity.

Ovaries were collected from four stages of the estrous cycle (midfollicular, late follicular, early luteal, and midluteal) and three stages of the pregnancy (Day 40, Day 80, and Day 110). Two gilts were used for each of the stages of the estrous cycle and pregnancy. Testes were collected from a 5-mo-old immature boar and a 36-mo-old mature boar. Tissues were cut into cubes (3–4 cm³), frozen in liquid nitrogen, and cryosectioned (8 µm). Specific cell types that bind relaxin were identified by sequential application of a biotinylated relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver for signal amplification. In the ovary, specific relaxin-binding sites were localized in both the theca and granulosa cells of developing follicles, luteal cells, and blood vessels. In the testis, specific relaxin-binding sites were localized in the Leydig cells. There were no apparent differences in relaxin-binding distribution within the ovary at different stages of the estrous cycle and pregnancy in gilts, or within the testis at sexual maturity in boars.

We conclude that the specific relaxin-binding cells within the ovary and testis of the pig may contain relaxin receptors. Therefore, relaxin may have effects in the ovary and testis of pigs.

	INTRODUCTION

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Relaxin is produced by the ovaries during both the estrous cycle and pregnancy in pigs. During the approximately 21-day estrous cycle, relaxin is produced by the theca interna layer of developing preovulatory follicles and by the corpora lutea formed after ovulation [1–3]. Three lines of evidence indicate that a portion of the relaxin that is released by the theca interna during the follicular phase of the cycle may diffuse into adjacent cells to bring about local effects on remodeling and growth within the pig ovary. First, the levels of relaxin in porcine follicular fluid were found to increase with development of the follicles [3]. Second, when granulosa cells obtained from developing pig follicles were incubated in culture media, porcine relaxin enhanced FSH-stimulated plasminogen activator activity [3]. Plasminogen activator is thought to be involved in disruption of follicular wall connective tissue during follicular growth and ovulation [3, 4]. Third, when granulosa cells or theca cells obtained from developing follicles of nonpregnant pigs were incubated in vitro in serum-free media, porcine relaxin not only stimulated DNA synthesis but also increased cell proliferation in a dose-dependent manner [3, 5, 6].

In the pregnant pig, relaxin accumulates in dense membrane-bound cytoplasmic granules within luteal cells during most of the 114-day pregnancy [7, 8]. Relaxin bioactivity levels within the ovary [7] and relaxin immunoactivity within the peripheral serum [9, 10] increase steadily from about Day 20 of pregnancy until about Day 110. Relaxin bioactivity within the ovary declines rapidly [7], and serum relaxin surges [9, 10] during the 2–3 days before birth when degranulation of luteal cells occurs. Whereas there is presently no evidence in the pregnant pig, one in vitro study with cow luteal cells provided limited evidence that relaxin may have autocrine effects on luteal function in species that produce relaxin within the corpus luteum. Porcine relaxin stimulated progesterone secretion in a dose- and time-dependent manner in primary cultures of cells obtained from bovine corpora lutea during late pregnancy [11].

Although it remains to be established that relaxin is produced in the boar, there is limited evidence that a relaxin-like peptide may be produced in boar testis. Lobb et al. [12], employing reverse transcription and polymerase chain reaction (RT-PCR), reported that a relaxin-like peptide gene is expressed in boar testis. There is presently no evidence that relaxin has effects within the boar testis.

A major step toward the establishment of direct effects of relaxin on the ovary and/or testis is to demonstrate that relaxin binds with specificity to the gonads. Recent studies from our laboratory identified specific relaxin-binding cells in several target tissues in the pregnant pig by employing an in vitro immunohistochemical localization technique [13]. The first objective of this study was to employ that technique to determine whether relaxin-binding cells are present in the ovaries and/or testis of pigs. Once they were found to be present, the second objective was to determine whether relaxin-binding sites noticeably change either within the ovary at different stages of the estrous cycle and pregnancy in gilts, or within the testis at sexual maturity in boars.

	MATERIALS AND METHODS

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Preparation and Characterization of Biotinylated Relaxin

Porcine relaxin was isolated as described by Sherwood and O'Byrne [14] and biotinylated as previously described [15]. In brief, porcine relaxin was dissolved in 0.2 M N-methylmorpholine-HCl buffer (pH 7.5) at a final concentration of 2 µmol/ml. To supply the biotinylating reagent in excess, 10 molar equivalents of biotinyl--aminocaproic acid-N-hydroxysuccinimide ester (Sigma, St. Louis, MO) in dimethylformamide at a concentration of 100 µmol/ml were added to the relaxin. The reaction mixture was stirred at room temperature for 4 h, and the reaction was stopped by the addition of acetic acid until a 1 M acetic acid solution was obtained. The contents of the reaction mixture were separated from the biotinyl--aminohexanoyl-relaxin (biotinylated relaxin) by ultrafiltration using an Amicon model 402 stirred ultrafiltration apparatus with a Diaflo Ultrafilter type YM1 membrane (molecular weight cut-off 1000; Amicon, Beverly, MA). The N-methylmorpholine HCl buffer and acetic acid were replaced with PBS (0.01 M NaH₂PO₄ and 0.15 M NaCl, pH 7.4) in the ultrafiltration unit. The biotinylated relaxin was stored at a final concentration of 9 nmol/ml at -70°C.

The mean number of biotin molecules per biotinylated relaxin molecule as determined by spectrophotometric 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assay (Pierce Chemical Co., Rockford, IL) was 3.5. We previously used the mouse interpubic ligament bioassay [16] to demonstrate that biotinylated porcine relaxin retained essentially full biological activity [15]. With that bioassay, comparable dose-response values were obtained with doses of 0.25, 0.5, and 1 µg of both biotinylated porcine relaxin and unmodified porcine relaxin. Accordingly, as a quality control measure in the present study, the bioactivity of 1 µg of the biotinylated relaxin was compared to that of 1 µg of unmodified porcine relaxin in the mouse interpubic ligament bioassay [16]. Biotinylated relaxin elicited a strong biological response that did not differ when analyzed by means of t-test from that of unmodified porcine relaxin. The mouse interpubic ligament lengths (mean ± SE) after the injection of vehicle control, 1 µg of unmodified porcine relaxin, and 1 µg of biotinylated porcine relaxin were 0.7 ± 0.07, 2.4 ± 0.17, and 2.2 ± 0.18 mm, respectively (n = 20/group).

Animals

The animal experimentation described in this study was approved by the University of Illinois Laboratory Animal Care Advisory Committee. Fourteen cycling cross-bred gilts (Camborough-15 X Pig Improvement Co.; 8 mo of age; ~120 kg) were obtained from the Swine Research Center at the University of Illinois Urbana-Champaign (UIUC). Animals were monitored for estrous behavior with vasectomized mature boars. The day when gilts were receptive for mating (estrus) was designated Day 0 of the estrous cycle. After the second estrus (Day 0), eight animals were assigned to one of four stages of the estrous cycle (2 gilts/stage): early luteal (Day 4), midluteal (Day 9), midfollicular (Day 18), and late follicular (Day 20). Six cycling animals were mated at estrus (Day 0) and assigned to one of three stages of pregnancy (2 gilts/stage): Day 40, Day 80, and Day 110. Gilts were housed in individual confinement crates, fed a diet of corn and soybean (12% protein) once daily, and allowed free access to water.

Tissue Collection and Processing for Immunohistochemistry

On each day of the four stages of the estrous cycle and the three stages of pregnancy, gilts were electrostunned and killed by exsanguination at the University of Illinois Meat Science Laboratory. Testes from a 5-mo-old immature boar (~100 kg) and a 36-mo-old mature boar (~290 kg) were obtained, immediately after their removal, from the Swine Research Center at UIUC. Ovaries and testes were cut into cubes (3–4 cm³) and individually placed in peel-A-way plastic embedding molds (Polysciences Inc, Warrington, PA). The tissues were frozen with Tissue-Tek OCT compound (Miles Scientific, Elkhart, IN) in liquid nitrogen and stored at -70°C until sectioning. Frozen sections (8 µm) were cut on an HR Mark II cryostat (Slee Medical Equipment Limited, London, England) at -20°C and thaw-mounted on microscope slides coated with 0.2% poly-L-lysine (M_r 300 000).

Immunohistochemical Localization of Biotinylated Relaxin

The tissue slides were brought to room temperature, and subsequent immunohistochemical procedures were performed at room temperature. Tissue slides were incubated for 30 min in 50 mM glycine in PBS (pH 7.4) and then incubated for 3 h with blocking buffer 1 (1% BSA fraction V, 0.2% fish gelatin [Amersham, Arlington Heights, IL], 5% normal pig serum, and 2 mM NaN₃ in PBS). Tissue slides were incubated for 3 h in incubation buffer 1 (1% BSA fraction V, 0.2% fish gelatin, 1% normal pig serum, and 2 mM NaN₃ in PBS) in four different ways. The first treatment incubated each tissue with biotinylated relaxin probe (4 µg/ml) in order to localize relaxin receptors. The second treatment incubated each tissue with unmodified porcine relaxin (4 µg/ml). Since the unmodified relaxin does not contain biotin, this treatment was used as a negative control. The third treatment incubated each tissue with biotinylated relaxin plus a 2000-fold excess of porcine insulin (ILETIN II; Eli Lilly, Indianapolis, IN) or human insulin (Humulin R; Eli Lilly) in order to determine hormonal specificity of binding of the biotinylated relaxin probe. The fourth treatment incubated each tissue with biotinylated relaxin plus a 2000-fold excess of porcine relaxin [14] in order to determine whether there are finite numbers of relaxin receptors in the tissue. After incubation, tissue slides were rinsed for 2 h with ten changes of wash buffer (1% BSA fraction V, 0.2% fish gelatin, and 2 mM NaN₃ in PBS). The tissues were then postfixed for 10 min in 2% glutaraldehyde in PBS, rinsed briefly with double-distilled water, and incubated for 30 min in 50 mM glycine. The tissues were then incubated for 4 h in blocking buffer 2 (1% BSA fraction V, 0.2% fish gelatin, 5% normal goat serum, and 2 mM NaN₃ in PBS), and for 2 h in 800 µl of antibiotin immunoglobulin G conjugated to 1 nm colloidal gold (Auroprobe One anti-biotin; Amersham) diluted 1:25 with incubation buffer 2 (1% BSA fraction V, 0.2% fish gelatin, 1% normal goat serum, and 2 mM NaN₃ in PBS). The tissues were rinsed for 2 h with ten changes of wash buffer and were postfixed in 2% glutaraldehyde for 10 min. All slides were rinsed with copious amounts of double-distilled water for 30 min before silver intensification of the gold particles. Silver intensification was performed by incubating sections in IntenSE M silver solution (Amersham) for 5 min at room temperature. The slides were rinsed with copious amounts of double-distilled water for 10 min, and the silver intensification step was repeated. The tissue sections were dehydrated in an ascending series of ethanol, cleared in Clear-Rite 3 (Richard Allen, Richland, MI), and coverslipped using mounting medium (Richard Allen).

Statistics

Bioassay data were analyzed by ANOVA, and significant differences among groups were determined by t-test.

	RESULTS

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Immunohistochemistry results from sections of ovaries obtained from the midfollicular phase of the estrous cycle are shown in Figure 1. Relaxin binding was observed in the granulosa cells, theca cells, cells associated with blood vessels, and smooth muscle cells (Fig. 1, A–C). No signal was detected in ovarian sections incubated with unmodified porcine relaxin (Fig. 1D). Binding of the biotinylated relaxin to the ovaries was hormone-specific and saturable. Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (Fig. 1E) but not in the presence of a 2000-fold excess of porcine relaxin (Fig. 1F).

View larger version (170K): [in this window] [in a new window]   FIG. 1. Localization of relaxin-binding sites in the ovary of cycling pigs at midfollicular stage (Day 18) of the estrous cycle. Relaxin binding was localized in ovaries incubated with biotinylated relaxin (A–C). B and C are higher magnifications of the middle left and upper middle, respectively, of A. Relaxin was not localized in ovaries incubated with unmodified porcine relaxin (D). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (E) but not in the presence of a 2000-fold excess of porcine relaxin (F). f, Follicles at different stages of development; ca, corpus albicans; bv, blood vessel; sm, smooth muscle; t, theca cells; g, granulosa cells. Bar in A = 2333 µm; A and D–F are the same magnification. Bar in B = 583 µm; B and C are the same magnification.

Results from sections of ovaries obtained from the midluteal stage of the estrous cycle are shown in Figure 2. The most prominent labeling was observed in the luteal cells (Fig. 2A). Relaxin binding was also observed in theca cells and to a lesser degree in granulosa cells of follicles at different stages of development (Fig. 2, A–C). It should also be noted that relaxin binding was associated with blood vessels (Fig. 2A). No signal was detected in ovarian sections incubated with unmodified porcine relaxin (Fig. 2D). Binding of the biotinylated relaxin to the ovaries was hormone-specific and saturable. Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (Fig. 2E) but not in the presence of a 2000-fold excess of porcine relaxin (Fig. 2F).

View larger version (154K): [in this window] [in a new window]   FIG. 2. Localization of relaxin-binding sites in the ovary of cycling pigs at midluteal stage (Day 9) of the estrous cycle. Relaxin binding was localized in ovaries incubated with biotinylated relaxin (A). B and C are a higher magnification of upper middle and lower right areas, respectively, of A. Relaxin binding was not localized in ovaries incubated with unmodified porcine relaxin (D). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (E) but not in the presence of a 2000-fold excess of porcine relaxin (F). cl, Corpus luteum; f, follicles at different stages of development; g, granulosa cells; t, theca cells; bv, blood vessel. Bar in A = 2333 µm; A and D–F are the same magnification. Bar in B and C = 583 µm.

Relaxin binding was also associated with luteal cells, both theca and granulosa cells of developing follicles, and blood vessels in ovaries obtained from pregnant gilts on Day 80 (Fig. 3). There were no apparent differences in relaxin binding distribution in pig ovaries obtained at different stages of either the estrous cycle or pregnancy.

View larger version (155K): [in this window] [in a new window]   FIG. 3. Localization of relaxin-binding sites in the ovary of pregnant pigs on Day 80. Relaxin binding was localized in ovaries incubated with biotinylated relaxin (A) (B is a higher magnification of middle right area of A) but not in ovaries incubated with unmodified porcine relaxin (C). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of porcine insulin (D) but not in the presence of a 2000-fold excess of porcine relaxin (E). cl, Corpus luteum; f, follicles at different stages of development; g, granulosa cells; t, theca cells; bv, blood vessel; lv, lymphatic vessel. Bar in A = 2333 µm; A and C–E are the same magnification. Bar in B = 583 µm.

Immunohistochemistry results from sections of testis obtained from an immature boar are shown in Figure 4. Prominent labeling was observed exclusively in interstitial cells (Fig. 4A). No signal was detected in sections incubated with unmodified porcine relaxin (Fig. 4B). Binding of the biotinylated relaxin to the testes was hormone-specific and saturable. Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (Fig. 4C), but not in the presence of a 2000-fold excess of porcine relaxin (Fig. 4D). In the pig, the interstitial space is almost completely filled with closely packed Leydig cells [17]. The relaxin binding within the interstitium was associated with Leydig cells, as shown most clearly in Figure 4E. Relaxin binding was also associated with the Leydig cells in the testis obtained from a mature boar (Fig. 5).

View larger version (156K): [in this window] [in a new window]   FIG. 4. Localization of relaxin-binding sites in the testis of an immature boar (5 mo old). Relaxin binding was localized in testis incubated with biotinylated relaxin (A, E) but not in testis incubated with unmodified porcine relaxin (B). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (C) but not in the presence of a 2000-fold excess of porcine relaxin (D). st, Seminiferous tubule; i, interstitial Leydig cells. Bar in A = 442 µm; A–D are the same magnification. Bar in E = 110 µm.

View larger version (104K): [in this window] [in a new window]   FIG. 5. Localization of relaxin-binding sites in the testis of a mature boar (36 mo old). Relaxin binding was localized in testis incubated with biotinylated relaxin (A) but not in testis incubated with unmodified porcine relaxin (B). Tissue sections incubated with biotinylated relaxin showed binding in the presence of a 2000-fold excess of human insulin (C) but not in the presence of a 2000-fold excess of porcine relaxin (D). st, Seminiferous tubule; i, interstitial Leydig cells. Bar in A = 442 µm; all panels are the same magnification.

	DISCUSSION

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This study demonstrates that specific relaxin-binding sites are localized in both the theca and granulosa cells of developing follicles, luteal cells, and cells associated with blood vessels within the pig ovary. It also demonstrates that relaxin binds with specificity to Leydig cells within the boar testis.

The relaxin-binding sites identified in this study may not be relaxin receptors. It is possible that relaxin binds to one or more insulin-like growth factor binding proteins (IGFBP) in the ovaries and/or testis. There are six high-affinity IGFBPs and four additional proteins that were recently postulated to be low-affinity IGFBPs [18]. Whereas it is known that insulin does not bind to any of the six high-affinity IGFBPs, it is possible that relaxin binds to one or more IGFBPs. There is a second possibility. In the boar testis, the relaxin-binding sites may serve as receptors for a testicular hormone other than relaxin. In 1993, Adham and coworkers [19] reported the cloning of a novel insulin-like peptide whose gene is expressed exclusively in the Leydig cells within the testis in pigs and other species including humans. This peptide, which was originally designated Ley I-L [19], has since been designated relaxin-like factor [20]. Whereas relaxin-like factor (RLF) has only about 30% amino acid sequence homology with relaxin, it contains a five-amino acid putative receptor-binding motif (Arg-x-x-x-Arg) near the middle of the B-chain that is required for relaxin bioactivity. There is limited evidence that RLF shows weak cross-reactivity with mouse uterine and brain relaxin receptors but not with insulin receptors [21]. Thus the relaxin-binding sites within the Leydig cells may normally serve as receptors for locally produced RLF.

Whereas there are reasons for caution in the interpretation of findings in this study, there are also reasons to think that the relaxin-binding sites associated with porcine gonad cells are, at least in part, functional relaxin receptors. First, in all previously examined tissues in which cells that bind relaxin with specificity were identified in pigs and rats with the method used in this study, relaxin has been demonstrated to bring about a biological response [13, 15, 22–24]. Second, in this study, specific relaxin binding was found in porcine theca and granulosa cells—two ovarian cell types in which porcine relaxin demonstrated a biological response in vitro [5, 6]. Third, other investigators employed concentrations of biotinylated hormone probe similar to the concentration used in the present study to identify putative receptors for growth hormone and motilin in cultures of human lymphoid cells and rabbit gastric smooth muscle cells, respectively [25, 26]. Accordingly, the remainder of this discussion is grounded on the premise that the relaxin-binding sites identified in the present study are, at least in part, relaxin receptors.

The demonstration that relaxin binds with specificity to both the theca and granulosa cells of developing follicles supports previous reports that porcine relaxin may contribute to theca and granulosa cell growth during follicular development [3–6]. Likewise, the finding that relaxin binds with specificity to luteal cells is supportive of a previous report that relaxin promotes progesterone secretion in bovine luteal cells [11].

The physiological significance of relaxin binding in the boar testis is entirely speculative. Whereas relaxin has been reported to be produced in the human prostate gland [27], there is less evidence that relaxin is produced in boars. Circulating relaxin levels were reported to be low (about 0.3–0.4 ng/ml) in mature boars and somewhat higher (about 1 ng/ml) in the seminal plasma of mature boars [28, 29]. The source of the relaxin in boars is not established. An immunohistochemical localization study that used anti-porcine relaxin serum provided limited evidence that the secretory epithelial cells of the seminal vesicles of intact boars contain relaxin immunoactivity [30]. Lobb et al. [12] employed RT-PCR to find that the relaxin gene is expressed in the boar testis, whereas no consistent relaxin signal was obtained from the sex accessory organs. If small amounts of relaxin are produced within the boar testis, it may influence testicular function through actions on the Leydig cells. The prominent labeling of relaxin to Leydig cells in this study is consistent with the possibility that relaxin plays a role in the regulation of steroidogenesis. Supraphysiologic levels of porcine relaxin reportedly inhibited testosterone production when added to nuclei-free tissue homogenate [31] obtained from adult macaque (Macaca fascicularis) testes. The present study provides no evidence that relaxin binds to germ cells within the testis. This finding is inconsistent with limited evidence that endogenous relaxin within the seminal plasma of humans and pigs contributes to normal motility of the spermatozoa [32–34]. In view of the limited number of studies and the inconsistencies among existing findings, it remains to be established that relaxin has a physiologic role(s) in the male in any species.

In summary, we report that specific relaxin-binding sites are localized in both the theca and granulosa cells of developing follicles, luteal cells, and blood vessels within the pig ovary. Relaxin-binding sites are also localized in the Leydig cells within the pig testis. We conclude that these cells may contain relaxin receptors. Therefore, relaxin may have effects in the ovary and testis of pigs.

	ACKNOWLEDGMENTS

 
The authors thank the employees of the University of Illinois Swine Research Center for their assistance with maintenance of animals, Mr. R.T. Gladin for his assistance with preparation of the photographs, and the College of Medicine Document Management Center for assistance with the preparation of the manuscript.

	FOOTNOTES

 
¹ This work was supported by USDA Grant AG 93-37203-9562 (to O.D.S.).

² Correspondence: O.D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, IL 61801-3704. FAX: (217) 333-1133; od-sherw{at}uiuc.edu

Accepted: March 31, 1998.

Received: December 8, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Denning-Kendall PA, Guldenaar SEF, Wathes DC. Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig. J Reprod Fertil 1989; 85:261–271.[Abstract]
2. Bagnell CA, Zhang Q, Ohleth K, Connor ML, Downey BR, Tsang BK, Ainsworth L. Developmental expression of the relaxin gene in the porcine corpus luteum. J Mol Endocrinol 1993; 10:87–97.[Abstract]
3. Bagnell CA, Zhang Q, Downey B, Ainsworth L. Sources and biological actions of relaxin in pigs. J Reprod Fertil Suppl 1993; 48:127–138.[Medline]
4. Bagnell CA, Domondon MC, Bryant-Greenwood GD. Plasminogen activator (PA) activity in rat and pig follicles during follicular development. In: Ryan RJ, Tofts DO (eds.), Proceedings of the 5th Ovarian Workshops. Champaign, IL: Ovarian Workshops; 1984: 233–238.
5. Zhang QI, Bagnell CA. Relaxin stimulation of porcine granulosa cell deoxyribonucleic acid synthesis in vitro: interactions with insulin and insulin-like growth factor I. Endocrinology 1993; 132:1643–1650.[Abstract]
6. Zhang Q, Bagnell CA. Trophic action of relaxin on porcine theca cells: interactions with insulin and insulin-like growth factor-I in vitro. Endocr J 1994; 2:349–355.
7. Anderson LL, Ford JJ, Melampy RM, Cox DF. Relaxin in porcine corpora lutea during pregnancy and after hysterectomy. Am J Physiol 1973; 225:1215–1219.
8. Fields PA, Fields MJ. Ultrastructural localization of relaxin in the corpus luteum of the nonpregnant, pseudopregnant, and pregnant pig. Biol Reprod 1985; 32:1169–1179.[Abstract]
9. Sherwood OD, Chang CC, BeVier GW, Dziuk PJ. Radioimmunoassay of plasma relaxin levels throughout pregnancy and at parturition in the pig. Endocrinology 1975; 97:834–837.[Abstract]
10. Anderson LL, Adair V, Stromer MH, McDonald WG. Relaxin production and release after hysterectomy in the pig. Endocrinology 1983; 113:677–686.[Abstract]
11. Musah AI, Schwabe C, Anderson LL. Relaxin, oxytocin, and prostaglandin effects on progesterone secretion from bovine luteal cells during different stages of gestation. Proc Soc Exp Biol Med 1990; 195:255–260.[Abstract]
12. Lobb DK, Yeo JE, Raeside JI, Porter DG. Identification of relaxin gene expression in tissues of the boar. In: MacLennan AH, Tregear GW, Bryant-Greenwood GD (eds.), Progress in Relaxin Research. Singapore: Global Publications Services; 1995: 579–587.
13. Min G, Sherwood OD. Identification of specific relaxin-binding cells in the cervix, mammary glands, nipples, small intestine, and skin of pregnant pigs. Biol Reprod 1996; 55:1243–1252.[Abstract]
14. Sherwood OD, O'Byrne EM. Purification and characterization of porcine relaxin. Arch Biochem Biophys 1974; 160:185–196.[CrossRef][Medline]
15. Kuenzi MJ, Sherwood OD. Immunohistochemical localization of specific relaxin-binding cells in the cervix, mammary glands and nipples of pregnant rats. Endocrinology 1995; 136:1367–1373.[Abstract]
16. Steinetz BG, Beach VL, Kroc RL, Stasilli NR, Nussbaum RE, Nemith PJ, Dun RK. Bioassay of relaxin using a reference standard: a simple and reliable method utilizing direct measurement of interpubic ligament formation in mice. Endocrinology 1960; 67:102–115.
17. Setchell BP. The Mammalian Testis. Ithaca, NY: Cornell University Press; 1978: 1–29.
18. Kim H-S, Nagalla SR, Oh Y, Wilson E, Roberts CT Jr, Rosenfeld RG. Identification of a family of low affinity insulin-like growth factor binding proteins (IGFBPs): characterization of connective tissue growth factor as a member of the IGFBP superfamily. Proc Natl Acad Sci USA 1997; 94:12981–12986.[Abstract/Free Full Text]
19. Adham IM, Burkhardt E, Kenahmed M, Engel W. Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem 1993; 268:26668–26672.[Abstract/Free Full Text]
20. Ivell R, Balvers M, Domagalski R, Ungefroren H, Hunt N, Schulze W. Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis. Mol Hum Reprod 1997; 3:459–466.[Abstract/Free Full Text]
21. Büllesbach EE, Schwabe C. A novel Leydig cell cDNA-derived protein is a relaxin-like factor. J Biol Chem 1995; 270:16011–16015.[Abstract/Free Full Text]
22. Min G, Hartzog MG, Jennings RL, Winn RJ, Sherwood OD. Evidence that endogenous relaxin promotes growth of the vagina and uterus during pregnancy in gilts. Endocrinology 1997; 138:560–565.[Abstract/Free Full Text]
23. Zhao S, Kuenzi MJ, Sherwood OD. Monoclonal antibodies specific for rat relaxin. IX. Evidence that endogenous relaxin promotes growth of the vagina during the second half of pregnancy in rats. Endocrinology 1996; 137:425–430.[Abstract]
24. Min G. Identification of relaxin target tissues and target cells in the pig. Urbana, IL: University of Illinois-Urbana; 1998: 105–140. Ph.D. Dissertation.
25. Bentham J, Aplin R, Norman MR. Histochemical detection of binding sites for human growth hormone using biotinylated ligand. J Histochem Cytochem 1994; 42:103–107.[Abstract]
26. Sakai T, Satoh M, Hayashi H, Fujikura K, Sano I, Koyama H, Tatemoto K, Itoh Z. Biotinyl C-terminal-extended motilin as a biologically active receptor probe. Peptides 1994; 15:257–262.[CrossRef][Medline]
27. Ivell R, Hunt N, Khan-Dawood F, Dawood MY. Expression of the human relaxin gene in the corpus luteum of the menstrual cycle and in the prostate. Mol Cell Endocrinol 1989; 66:251–255.[CrossRef][Medline]
28. Juang HH, Musah AI, Schwabe C, Anderson LL. Immunoactive relaxin in boar seminal plasma and its correlation with sperm motility. Anim Reprod Sci 1990; 22:47–53.
29. Juang HH, Musah AI, Schwabe C, Ford JJ, Anderson LL. Relaxin in peripheral plasma of boars during development, copulation, after administration of hCG and after castration. J Reprod Fertil 1996; 107:1–6.[Abstract]
30. Kohsaka T, Takahara H, Sasada H, Kawarasaki T, Bamba K, Masaki J, Tagami S. Evidence for immunoreactive relaxin in boar seminal vesicles using combined light and electron microscope immunocytochemistry. J Reprod Fertil 1992; 95:397–408.[Abstract]
31. Kwan TK, Poh CH, Perumal R, Gower DB. Pregnenolone metabolism in testicular homogenates of macaques (Macaca fascicularis): some effects of relaxin and freezing. Biochem Mol Biol Int 1994; 34:661–670.[Medline]
32. Sarosi P, Schoenfeld C, Berman J, Basch R, Randolph G, Amelar R, Dubin L, Steinetz BG, Weiss G. Effect of anti-relaxin antiserum on sperm motility in vitro. Endocrinology 1983; 112:1860–1861.[Abstract]
33. Juang HH, Musah AI, Schwabe C, Anderson LL. Effect of relaxin and antirelaxin serum on porcine sperm motility. Anim Reprod Sci 1989; 20:21–29.
34. Sokol RZ, Okuda H, Johnston PD, Swerdloff RS. Videomicrographic analysis of the effects of antihuman relaxin antibody on human sperm motility. Fertil Steril 1988; 49:729–731.[Medline]