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Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

Ovine placental lactogen (oPL) is structurally similar to PRL, is a product of the chorionic epithelium, and has been implicated in playing a supportive role in fetal growth. This study examined the concentration and cellular location of oPL mRNA at five stages of pregnancy (days 60, 90, 105, 120, and 135) in 21 cross-bred ewes, and results were compared to maternal and fetal serum oPL concentrations, cotyledonary DNA and actin mRNA concentrations, and total fetal weight. The concentration of oPL mRNA in fetal cotyledonary tissue increased (Por = 0.05) from day 60 (15.4 pg/micrograms total cellular RNA) to day 120 (73.7 pg/micrograms total cellular RNA) of gestation and then plateaued, whereas no significant changes occurred in the concentration of actin mRNA over the gestational ages examined. The concentration of DNA in cotyledonary tissue (micrograms per mg wet tissue) increased (Por = 0.05) from days 60 through 120 and remained constant through day 135, such that when oPL mRNA was expressed on a picogram per microgram DNA basis, no stage of gestation effect (Por = 0.10) was observed. The maternal serum oPL concentration increased (Por = 0.05) from day 60 (7.1 ng/ml) to day 105 (417.7 ng/ml), followed by a large but nonsignificant (Por = 0.10) increase in maternal serum oPL occurring on day 135 (902.0 ng/ml). Fetal serum oPL concentrations increased (Por = 0.05) from day 60 (11.0 ng/ml) to day 90 (29.0 ng/ml) and then remained relatively constant. Maternal serum oPL (r = 0.68; Por = 0.01) and cotyledonary oPL mRNA levels (r = 0.61; Por = 0.05) were correlated with total fetal weight when adjusted for fetal number and gestational age, and together accounted for 80.6% (r2 value) of the variation found in total fetal weight. The correlation between fetal serum oPL concentrations and total fetal weight was nonsignificant (Por = 0.10). Examination of placentome cross-sections by immunocytochemistry and in situ hybridization at the five gestational ages indicated that the chorionic binucleate cell was the sole source of oPL. These data provide evidence that, like maternal serum concentrations of oPL, oPL mRNA expression by chorionic binucleate cells increases until late gestation, whereas fetal serum concentrations of oPL plateau during midgestation.(ABSTRACT TRUNCATED AT 400 WORDS)

This study examined the influence of ovarian steroids on the uterotropic actions of relaxin (RLX) in ovariectomized prepubertal gilts. Ovariectomized gilts received (im) corn oil (CO), estradiol benzoate (EB), or EB and progesterone (P) for 0-16 days. Steroid administration was patterned to approximate the plasma concentrations of endogenous ovarian steroids observed during 1) the follicular phase (EB), 2) luteal phase (EB+P), and 3) early pregnancy (EB+P+EB). Half of each group also received PBS or 0.5 mg RLX every 6 h for 54 h, coinciding with the final 2 days of the experimental period. After hysterectomy, uterine tissues were analyzed for water, dry matter, protein, DNA, glycosaminoglycans (GAGs), and collagen contents. Administration of EB or P increased uterine weight 5- to 6-fold, but no differences were observed between EB+P- and EB+P+EB-treated gilts. Cotreatment with RLX enhanced steroid-induced uterine growth 40-70%, and RLX stimulated growth in CO- and EB+CO control gilts 2- to 3-fold. The water content of uterine tissues was greater in EB-, EB+P-, and EB+P+EB-treated gilts than in their respective controls, and this response was augmented by RLX in all treatment groups. Administration of steroids stimulated a 4- to 5-fold increase in uterine dry weight compared to that in controls, with responses not differing between EB+P- and EB+P+EB-treated gilts. In all groups, RLX increased uterine dry weight. Protein and DNA contents of uterine tissue increased with steroid treatment, but neither variable differed between EB+P- and EB+P+EB-treated gilts. Administration of RLX, alone or in combination with steroids, increased protein and DNA contents of uterine tissues. The tissue content of GAGs increased in response to steroids, and coadministration of RLX did not alter this response. Although the uterine tissue concentration of collagen was reduced in steroid- and RLX-treated gilts, the collagen content of the uterus was not affected by the various treatments. The results of this study indicate that RLX is a potent stimulator of uterine growth under a variety of steroidal environments. RLX- or steroid-induced uterine growth was manifest by increased water, dry matter, protein, and DNA and GAG contents, but the uterine content of collagen was not affected. The overall growth-promoting effects of EB and the stimulation of DNA accretion by RLX were not observed when gilts were cotreated with P.(ABSTRACT TRUNCATED AT 400 WORDS)

The endometrium of the pig secretes retinol-binding protein (RBP) under the influence of progesterone (P4). The objective of this study was to determine how conceptus-derived estrogen might modulate this production of RBP around days 11-13 of pregnancy when conceptuses elongate from spheres to long thread-like forms. Concentrations of retinol and RBP were low (35 +/- 7 ng/ml) in uterine flushings obtained on days 10-12 of the estrous cycle or from pregnant gilts in which conceptuses had not elongated. Concentrations of retinol and RBP increased 7- to 8-fold (P less than 0.01) in flushings where filamentous conceptuses were present. Size exclusion and ion exchange chromatography demonstrated that virtually all retinol assayed in uterine flushings was associated with RBP. Northern blot analysis with a cDNA representing uterine RBP revealed a single endometrial mRNA 1.1 kilobases in length. Expression of RBP mRNA in uterine endometrium was measured in ovariectomized prepubertal gilts after the administration of steroids according to the following regimens: I, corn oil (days 0-16; n = 10); II, estradiol benzoate (EB; days 13-14; n = 11); III, EB (days 1-2; n = 12); IV, EB (days 1-2) plus P4 (days 3-16; n = 12); and V, EB (days 1-2) plus P4 (days 3-16) plus EB (days 13-14; n = 12). EB (200 micrograms) and P4 (100 mg) were administered twice daily. Treatment IV was designed to stimulate the estrous cycle, and treatment V simulated early pregnancy. All gilts were hysterectomized on day 16, and total uterine mRNA (3 micrograms) was analyzed by Northern blotting. No RBP mRNA was detected in groups I, II, or III. In group IV, 5 of 12 gilts had detectable RBP mRNA, as measured by densitometric scanning (OD = 0.35 +/- 0.14). RNA isolated from all gilts in group V (12 of 12) gave a strong hybridization signal (OD = 1.58 +/- 0.22) for RBP. Finally, RBP mRNA was examined in the uterine endometrium of mature gilts on day 13 of the estrous cycle (n = 4), day 13 of pseudopregnancy (2.5 mg EB given on days 11-12; n = 4), or day 13 of pregnancy after conceptuses had elongated (n = 4). RBP mRNA was present in all groups, but was enhanced approximately 12-fold (P less than 0.01) in pregnant and pseudopregnant gilts compared to that in control gilts.(ABSTRACT TRUNCATED AT 400 WORDS)

The corpus luteum is the primary source of circulating relaxin (RLX) in female pigs. However, a preliminary experiment in our laboratory identified RLX in the uterine endometrium of a dav 16 nreanant ailt that had been ovariectomized on day 8 of pregnancy, Hnd the pregnancy maintained via progesterone replacement therapy. Therefore, our objective was to examine the uterus as a potential extraovarian source of RLX during the estrous cycle and early pregnancy in pigs. Reproductive tissues were collected from pregnant (n 2 3/day) and nonpregnant (n 2 S/day) gilts on days 10 (n = 6), 12 (n = 6), 14 (n = 6), 16 (n = 8), 18 (n = 6), and 20 (n = 6) of the estrous cycle and pregnancy and on day 42 (n = 2) of gestation. To verify that the RLX identified in uterine tissues was not of ovarian origin, three additional pregnant gilts were ovariectomized on day 8, and the pregnancy was maintained by progesterone replacement therapy until day 16, when the reproductive tissues were collected for immunohistochemistry. The uterine tissues were scored for specific RLX immunostaining and analyzed for the effects day and pregnancy by analysis of variance. Within the uterine endometrium, the luminal epithelium of pregnant pigs contained more RLX than that of nonpregnant pigs (P 5 0.001). However, there was no difference in either the glandular epithelium or stromal tissues (P 2 0.10). Increased RLX in the luminal epithelium of pregnant pigs was detected on both days 18 (P 5 0.05) and 20 (P 5 0.001) of gestation compared to that in nonpregnant pigs on similar days of the estrous cycle. An effect of day was also observed for RLX immunostaining in the luminal epithelium (P 5 0.005), but not in glandular or stromal tissues (P 2 0.10). In both pregnant and nonpregnant pigs, luminal epithelial RLX immunostaining was faint on days 10-12. Thereafter, RLX immunostaining increased on day 14 to reach peak levels on day 16. In nonpregnant pigs, RLX immunostaining dropped to low levels on day 18 and became faint to absent by day 20 of the estrous cycle. In pregnant pigs, RLX immunostaining remained elevated, but appeared to decline slightly by day 20 and became undetectable by day 42 of gestation. RLX immunolocalization in the glandular epithelium was detected on all days in pregnant and nonpregnant pigs, whereas stromal-specific immunostaining was not observed. In addition, RLX was immunolocalized in both the luminal and glandular epithelium of uteri from pregnant gilts 8 days after ovariectomy. Northern blot analysis identified the approximately l-kilobase RLX messenger RNA in RNA isolated from luteal and endometrial tissues. Reverse transcription of endometrial messenger RNA and subsequent polymerase chain amplification with porcine RLX gene-specific primers identified RLX gene expression in both pregnant and nonpregnant endometrial samples between days 10 and 20. We conclude that the porcine uterine endometrium is an additional source of RLX production during early pregnancy and the estrous cycle. Local uterine production may allow RLX to exert its uterotropic actions during the period of pregnancy establishment. (Endocrinology 135: 2517-2525, 1994)

Secretion of a tissue inhibitor of metalloproteinases (TIMP-1) is initiated by ovine preovulatory follicles after the gonadotropin surge. In addition, TIMP-1 is a major secretory product of ovine corpora lutea. We have isolated an approximately full-length cDNA clone for TIMP-1 from an ovine luteal cDNA library. The 887-basepair cDNA obtained was 95%, 86%, and 77% identical to the reported nucleotide sequences of bovine, human, and mouse TIMP-1 cDNAs, respectively. Total cellular RNA was isolated from preovulatory follicles collected before (presurge; n = 5) or 12-14 h after (postsurge; n = 4) a LHRH-induced gonadotropin surge (36 h after prostaglandin F2 alpha-induced luteolysis); from luteal tissue collected on days 3, 7, 10, 13, and 16 postestrus (n = 5, 5, 4, 5, and 5, respectively); and from purified populations of small (n = 4) and large (n = 3) luteal cells. Concentrations of TIMP-1 mRNA (picograms per micrograms tissue DNA) were increased in preovulatory follicles after exposure to a gonadotropin surge (Por = 0.01). TIMP-1 mRNA was localized primarily to the granulosa layer of postsurge follicles by in situ hybridization. Concentrations of TIMP-1 mRNA in luteal tissue did not differ throughout the luteal phase (P = 0.07). However, TIMP-1 mRNA was localized predominantly to specific cells located in the connective tissue surrounding and within day 3 corpora lutea. In situ hybridization of day 10 corpora lutea localized TIMP-1 mRNA predominantly to specific cells that were randomly dispersed throughout the luteal tissue. TIMP-1 mRNA was expressed by purified populations of both small and large luteal cells collected from day 10 corpora lutea. Concentrations of TIMP-1 mRNA (picograms per micrograms DNA) were greater in the large luteal cell populations (Por = 0.0001). We conclude that 1) expression of TIMP-1 mRNA by the granulosa layer of ovine preovulatory follicles increased after the gonadotropin surge, whereas TIMP-1 mRNA concentrations during the luteal phase remained constant; 2) during the luteal phase, TIMP-1 mRNA was localized to specific cells surrounding (day 3) or located within (day 10) the corpus luteum; and 3) expression of TIMP-1 mRNA was greatest in large luteal cells.

The trophoblast of the sheep conceptus secretes a low mol wt, acidic polypeptide called ovine trophoblast protein-1 (oTP-1) from days 13-21 of pregnancy. This protein has been proposed to have an antiluteolytic role during pregnancy, since infusion of oTP-1 into the uterine lumen of nonpregnant ewes lengthened their estrous cycles. In the present study, mRNA from day 16 conceptuses was translated using a cell-free wheat germ lysate, and oTP-1 was immunoprecipitated from the translation mixture by means of a specific rabbit antiserum. The predominant protein synthesized under these conditions was oTP-1, which had a mol wt (21,000) higher than that of oTP-1 secreted by intact conceptuses into culture medium (mol wt, 17,000). In a separate experiment, in vitro secretion of oTP-1 from conceptuses on days 13, 17, and 21 of pregnancy was examined. Conceptuses were cultured for 24 h in the presence of [3H]leucine, and oTP-1 was immunoprecipitated from the culture medium. Incorporation of [3H]leucine into secreted macromolecules (mol wt, greater than 3500) increased 12-fold from days 13 to 21, while release of immunoprecipitable 3H-labeled oTP-1 increased 27-fold from days 13 to 17 and declined by over 50% from days 17 to 21. When results were expressed per microgram DNA, secretion of total macromolecules declined from days 13 to 17 and then remained constant until day 21. The pattern of oTP-1 secretion held whether results were expressed per conceptus or per unit DNA. The interestrous intervals for the donor ewes were shorter for ewes which had their conceptuses removed on day 13 than for ewes flushed on day 17 or 21. Thus, the period of oTP-1 synthesis corresponds closely to the time at which the conceptus acts to extend luteal lifespan.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) are newly discovered proteins produced by embryonic tissues for a limited period in early gestation. They appear to act as agents that prevent regression of the corpus luteum during early pregnancy in the ewe and cow. Ovine TP-1 [mol wt (Mr), 17,000] consists of three or four isoelectric variants (pI 5.4-5.7), whereas bTP-1, which cross-reacts with antiserum to oTP-1, is found as two predominant Mr classes (Mr, 22,000 and 24,000), each with several isoelectric variants (in the pI range 6.3-6.8). Cell-free translation of ovine conceptus mRNA yields pre-oTP-1 also consists of three or four isoelectric variants, assumed to have arisen by translation of multiple mRNA species. Ovine TP-1 is not glycosylated. When bovine conceptus mRNA is translated, a group of four or five isoforms of pre-bTP-1 are generated, each with a Mr of 19,000. In the presence of microsomes the Mr shifts upward to about 21,500. Bovine conceptuses cultured in presence of either [3H]glucosamine or [3H]mannose incorporate label into both size classes of bTP-1 (Mr, 22,000 and 24,000). Culture in presence of [35S]methionine and tunicamycin gave rise to a nonglycosylated form of bTP-1 with an apparent Mr of 18,000. Treatment of [35S]methionine-labeled bTP-1 with either endoglycosidase-H or peptide:N-glycosidase F yielded products with Mr of 17,000 and 16,000, respectively. bTP-1, although functionally and structurally related to oTP-1, appears to be a glycoprotein carrying at least two Asn-linked oligosaccharides. The two Mr classes of bTP-1 arise as a result of differences in either the number or structure of the carbohydrate chains. Like oTP-1, bTP-1 is probably translated from multiple mRNA species.