Determination of the source of immunoreactive relaxin in the cat

We have recently reported the secretory profile of relaxin throughout gestation in the cat. Because the appearance of relaxin begins at about Day 20 (Day O = ovulation) and because implantation begins shortly before this at Days 13-14, we hypothesized that relaxin was of feto-placental origin. To test this hypothesis, we used 4 experimental groups: 1) Control (laparotomy-only at Day 23 or 42, n = 4); 2) Early Ovariectomy (Ovx, bilateral ovariectomy between Days 23 and 26, n = 4); 3) Late Ovx (bilateral ovariectomy between Days 40 and 44, n = 4); 4) Tissue Removal (removal of feto-placental units, uterus, and one ovary on Days 16, 21, 28 and 35, n = 1 per day). Pregnancies were maintained in both Ovx groups by progesterone administration. Relaxin secretory patterns in Ovx groups were similar to the Control data. Relaxin was detectable in plasma beginning at about Day 20, with maximum concentrations reached by Day 30. Relaxin concentrations were highest (immunoactivity per mg tissue) in homogenates of placental tissues as compared to luteal, fetal, or uterine tissues. Altogether, these data indicate that the feto-placental unit is the source of relaxin in the cat.     

Laparoscopy in the ewe: A photographic record of the ovarian activity of ewes experiencing normal or abnormal oestrous cycles

Daily observation of ovarian activity in cycling ewes by laparoscopy shows that visual estimation of the age of corpora lutea must be primarily related to changes in their colour rather than to changes in either their shape or size. The shape and size of corpora lutea varies widely depending upon their depth of origin within the cortex of the ovary.A 36-day oestrous cycle in one ewe was found to be associated with the maintenance of a mature corpus luteum rather than with a silent ovulation. By contrast, a 6-day oestrous cycle in another ewe was associated with the premature regression of the corpus luteum.     

Relaxin activity in the pregnant cat

Plasma relaxin activity was measured by radioimmunoassay (RIA) in the domestic cat utilizing two different antisera developed against highly purified porcine relaxin. One was the 5858 antiserum from our laboratory and the other was the R6 antiserum of Dr. Bernard Steinetz. Relaxin activity could not be detected during the estrous cycle or during pseudopregnancy. Relaxin immunoactivity during early gestation was not detected by either antiserum. Plasma relaxin immunoactivity was first detected by both antisera on about Day 25 of gestation. Relaxin concentrations then increased rapidly, with a plateau reached between Days 30 and 35 that was maintained until 10-15 days prepartum. Relaxin concentrations then declined gradually until parturition. No prepartum increase was observed. Relaxin concentrations were undetectable within 24 h of delivery. Although amounts of immunoactivity measured with the R6 antiserum were consistently higher than measurements with the 5858 antiserum, the patterns of secretions observed were similar for both antisera.     

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

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.     

Uterine response to ovariectomy during the proliferative and luteal phases in the marsupial, Trichosurus vulpecula.

The uterine luteal phase in T. vulpecula is not dependent upon the secretions of the CL throughout its duration. Ablation of the CL or ovariectomy after Day 7 of the 26-day oestrous cycle does not result in the termination of the uterine secretory phase. The dependence of the luteal phase on the secretions of the CL is demonstrated by ablation of the CL or ovariectomy on Days 2, 4, 8, 12 and 24 of the oestrous cycle. Ablation of the CL before Day 8 resulted in the inhibition of the impending luteal phase, and the commencement of a follicular phase resulting in oestrus 8 to 9 days later. Removal of the CL or ovariectomy on Days 8 or 12 does not completely inhibit the uterine luteal phase since sufficient precursor of uterine milk is stored in the uterine basal glandular epithelium, thus enabling the endometrium to maintain the secretion of uterine milk.     

Rabbit endometrial relaxin: immunohistochemical localization during preimplantation, pregnancy, and lactation

Relaxin was localized in rabbit endometrium (but not ovary) on Days 4-30 of pregnancy and Days 2-5 of lactation. The hormone was not observed on Days 2 and 3 post coitus. Relaxin was found in endometrial glands throughout the length of the uterus on Days 4-9 post coitus. Later, on Days 11-23, relaxin was localized in both uterine endometrial gland cells and luminal epithelial cells. At this time, staining was observed only in the endometrium directly associated with implantation sites. Areas between implantation sites were devoid of staining. On Days 25-30 of pregnancy, relaxin was found mainly in uterine luminal epithelial cells. Few glands were observed with relaxin. During the first week of lactation, the staining profile was the same as that observed on Days 25-30. Relaxin was not found in the endometrium of pseudopregnant rabbits (Days 1, 4, 8, 12, and 16). The early appearance of uterine relaxin at the time the blastocyst migrates into the uterine cavity coupled with the hormone's later confinement to implantation sites suggests that the blastocyst initiates and the conceptus maintains uterine relaxin.     

Immunocytochemical localization of relaxin in corpora lutea of sows throughout the estrous cycle

Porcine relaxin has been sought by localization in the corpus luteum of sows on Days 3, 7, 9, 11, 12, 15, 18, 19, and 21 of the estrous cycle, using the avidin-biotin immunoperoxidase method and an antiserum to purified porcine relaxin. Simultaneous localization of relaxin in corpora lutea from sows on Days 108 and 113 of pregnancy was used to compare the intensity of immunostaining with that of corpora lutea of cyclic animals. However, the antiserum dilution necessary for optimal localization differed considerably in these two states (1:10,000 in pregnancy and 1:750 in the cycle), suggesting that lower levels of antigen are present in the luteal cells of the cycle. Relaxin immunostaining was undetectable on Day 3 of the cycle but became evident by Days 7 and 9. At Day 11 staining intensity increased and persisted through Day 15. On Day 18 some stain was still evident, but by Days 19, 20, and 21 there was complete absence of immunostain. Relaxin immunostaining appeared to be located throughout the cytoplasm of the luteal cell, as clear areas in the nuclear region were often observed. The results suggest that relaxin is produced in low amounts by the luteal cells of the cyclic sow and that the levels fluctuate with stage of the cycle. Lack of evidence from radioimmunoassay for a surge of relaxin secretion into the systemic circulation prior to luteolysis in the pig estrous cycle suggests that the relaxin localized in the luteal cells of the cycle may have an intraovarian function.     

Rabbit relaxin: the influence of pregnancy and ovariectomy during pregnancy on the plasma profile.

A porcine relaxin radioimmunoassay was developed to evaluate the profile of immunoreactive relaxin in rabbit plasma. Relaxin was nondetectable in pseudopregnant (Days 1, 4, 5-8, 12, and 16), nonpregnant, and male rabbits. However, in pregnant rabbits, relaxin was detected during the peri-implantation period (Days 4-9). Peak concentrations were reached on Day 15 and were maintained until parturition (Day 32). Relaxin concentrations abruptly decreased on Day 1 postpartum to low but detectable concentrations that were unchanged during the first week postpartum. In contrast, progesterone concentrations peaked earlier (Day 13), decreased after Day 25, and were not detectable on Day 1 postpartum. The effect of ovariectomy on the profile of plasma relaxin was evaluated. Four pregnant rabbits were ovariectomized (Day 13) and treated with medroxyprogesterone acetate to maintain pregnancy. As in normal pregnant rabbits, relaxin was observed initially during the peri-implantation period (Days 4-9) and increased to peak concentrations by Day 16. These concentrations were maintained until parturition and abruptly decreased on Day 1 postpartum to low yet detectable concentrations during the first week postpartum. The concentrations of relaxin in the plasma of ovariectomized medroxyprogesterone-treated rabbits were not different from those in three sham controls. These results indicate that the ovary is not a significant source of relaxin in pregnant rabbits. The unique observation of the presence of relaxin during the peri-implantation period suggests that this hormone has a role in preparing the rabbit uterus for implantation. The continued presence of relaxin during the first week postpartum may represent residual hormone, or it may suggest a physiological role during the early postpartum period.     

The effect of the stage of the oestrous cycle and exogenous sex steroids on the transport of starch grains through the genital tract of the ewe, a preliminary study

The starch grain test was studied in two ewes to determine the effect of the stage of the oestrous cycle on their transport through the Fallopian tubes. There was very little difference when the test was performed at oestrus or day 10 of the oestrous cycle. Bilateral ovariectomy had very little effect on the transport of starch grains. When progesterone in oil was administered by intramuscular injection to a bilaterally ovariectomised ewe and an intact ewe there was a marked increase in the number of grains which were recovered. Oestradiol benzoate in oil delayed the recovery of grains and reduced their numbers.     

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.     

Plasma relaxin immunoactivity in the pig at parturition and during nuzzling and suckling.

One sow bled at 30--60-min intervals for 48 h at 5 and 4 days before parturition had mean +/- s.e.m. relaxin levels of 5.0 +/- 0.48 ng/ml and 5.5 +/- 0.44 ng/ml for each 24-h period respectively. This sow and another were bled at frequent intervals during parturition; both showed considerable fluctuations in their relaxin levels but no consistent peaks in relation to each birth. Mean levels during parturition were 10.7 +/- 0.46 ng/ml and 13.4 +/- 0.81 ng/ml respectively, both significantly higher than the levels at 4 and 5 days before birth. Relaxin levels in two lactating sows rose acutely during nursing, showing a 3-fold rise in one animal and an 8-fold rise in the other. Results from a third sow during an extended period of nuzzling and sucking by the piglets showed multiple peaks of relaxin immunoactivity associated with each nuzzling/sucking stimulus.     

Immunohistochemical co-localization of placental lactogen II and relaxin in the golden hamster (Mesocricetus auratus)

Two hormones with lactogenic activity are produced by the hamster placenta during the second half of pregnancy. One of these hormones, hamster placental lactogen II (haPL-II), has been well characterized; however, its cellular source is not known. In the present study, haPL-II was localized in placental tissues using a specific antibody and the avidin-biotin-peroxidase immunohistochemical technique. Because relaxin has been localized in the hamster placenta, it was of interest to determine if haPL-II and relaxin are localized in the same cells. haPL-II immunoactivity was observed in primary and secondary giant trophoblast cells of the placenta on Days 12, 14, and 15 of pregnancy. On Day 15 positive staining was also observed in large cells located within mesometrial arteries and in eosinophilic bodies associated with degenerating sheathed arteries of the decidua basalis. haPL-II-positive staining was not observed in placentae from Days 8 or 10 of pregnancy. On Day 14, haPL-II was colocalized with relaxin in 75% of the giant trophoblast cells observed. Therefore, it is probable that these hormones are synthesized and secreted by the same cell.     

Purification and sequence determination of canine relaxin

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C₁₈ column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.     

Progesterone, oestradiol-17 beta and LH during the oestrous cycle of muskoxen (Ovibos moschatus)

Progesterone, oestradiol-17 beta and LH were measured in plasma from 6 non-pregnant, captive, female muskoxen during the 1984 and 1985 breeding seasons. Jugular blood samples were taken on an alternating 3/4-day schedule in 1984 and daily or at 4-h intervals over oestrus, via indwelling jugular cannulae, for 6 weeks in 1985. Oestrous cycle length was 19.6 +/- 0.96 (s.d.) days (n = 19) and did not vary between the first and subsequent cycles of the season. Progesterone was lowest at oestrus (less than or equal to 0.1 ng/ml), began to rise on Days 4-5, peaked on Days 10-12 (mean = 2.6 ng/ml) and returned to baseline 2-5 days before the next oestrus. A small rise in progesterone before the first cycle of the breeding season was observed on 7 of 12 occasions. Oestradiol-17 beta was significantly higher (P less than 0.001) 1-4 days before, or coincident with, oestrus. The average duration of the LH peak was 24.6 h (n = 7) and coincided with observations of behavioural oestrus. In one animal behavioural oestrus and an LH peak preceded a small progesterone rise at the beginning of the breeding season. The temporal relationship of these three hormones during the muskox oestrous cycle is very similar to that seen in domestic ruminants.     

Plasma prolactin in relation to changes in oestrous cyclicity following olfactory bulbectomy in post-pubertal pigs

Prolactin was determined by radioimmunoassay in the peripheral plasma of five post-pubertal Large White gilts before and after olfactory bulbectomy. In the oestrous cycle before bulbectomy and in cycles after bulbectomy, the highest concentrations of prolactin were found during the period from the late luteal phase to oestrus. Throughout the prolonged summer anoestrus which occurred after bulbectomy, the amounts of prolactin were similar to base-line quantities found during the oestrous cycle between Days 3 and 12. These results differ from those found for the seasonally breeding European wild sow where high prolactin levels are associated with a summer anoestrus.     

Uterine blood flow of cows during the oestrous cycle and early pregnancy: effect of the conceptus on the uterine blood supply.

Blood flow to each uterine horn of cows during the oestrous cycle and early pregnancy was determined daily by use of electromagnetic blood flow probes placed around both middle uterine arteries. The pattern of blood flow to uteri of pregnant and non-pregnant cows was similar until Day 14 after mating or oestrus. Between Days 14 and 18 of pregnancy blood flow to the uterine horn containing the conceptus increased (P less than 0.01) 2- to 3-fold, whereas blood flow to the non-gravid uterine horn in these cows remained constant. No corresponding increase in blood flow to the uterine horn ipsilateral to the ovary bearing the CL was observed in non-pregnant cows during this 4-day period. By Day 19 of pregnancy, blood flow to the gravid uterine horn had returned to a level similar to that observed on Day 13. Blood flow to both uterine horns of pregnant cows remained constant from Days 19 to 25 and then increased to the gravid horn (P less than 0.01) markedly until Day 30 whereas blood flow to the non-gravid horn remained low. Uterine blood flow during the oestrous cycle of non-pregnant cows was positively correlated (P less than 0.01) with systemic concentrations of oestradiol and the ratio of oestradiol (pg/ml) to progesterone (ng/ml). There was no association between oestradiol concentrations and blood flow to the gravid uterine horn. These data indicate local control of uterine blood flow by the bovine conceptus which may function to create optimal conditions for the continuation of pregnancy.     

The effect of relaxin on in vivo uterine electromyographic activity in the conscious ovariectomized ewe is oestrogen-dependent

Electromyographic (EMG) activity of the uterus was recorded in vivo in 4 conscious ovariectomized ewes treated with oestrogen (one injection of 100 micrograms of oestradiol benzoate). Doses of 500 micrograms of purified porcine relaxin were injected intravenously 12, 16, 20, 24, 28 and 32 hrs after oestrogen treatment. Relaxin had an inhibitory effect on uterine activity between 16 and 32 hrs after oestrogen treatment. Because the period during which relaxin had a biological effect corresponded to the moment when oestrogen-induced uterine activity was maximal, it is suggested that the ability of relaxin to inhibit uterine activity in the ewe is oestrogen-dependent.     

Secretion of gonadotrophins change during the luteal phase of the bovine oestrous cycle in the absence of corresponding changes in progesterone or 17β-oestradiol

The hypothesis in the present study was that changes in circulating luteinizing hormone (LH) and follicle stimulating hormone (FSH) would occur during the luteal phase of the oestrous cycle (Days 4–19; Day 0, day of behaviourial oestrus) that were not related to corresponding changes in concentrations of progesterone and 17β-oestradiol. The stage of the oestrous cycle of cows (n = 18) was synchronised to obtain cows that were on alternate days of the cycle. Blood samples were collected every other day at 15 min intervals for 12 h from all cows: Days 4, 6, 8, 10, 12, 14, 16, 18 (n = 9) and Days 5, 7, 9, 11, 13, 15, 17, 19 (n = 9). Concentrations of LH, FSH, 17β-oestradiol and progesterone were determined in these samples. Data were compared across days to determine when significant changes occurred in concentrations or patterns of secretion of the gonadotrophins and ovarian steroid hormones during the oestrous cycle. There were significant changes in mean concentrations of FSH in circulation between Days 6 and 12. The most striking changes in secretion of gonadotrophins that could not be explained by changes in gonadal steroids were the fluctuations in amplitude of LH pulses between Days 7 and 12. Amplitude of LH pulses increased between Days 7 and 11 and subsequently decreased between Days 11 and 12 of the oestrous cycle. Some changes in gonadotrophin secretion that occurred in the present study can be explained by fluctuations in concentrations of progesterone and 17β-oestradiol in circulation. Other changes cannot be explained by fluctuations in circulating concentrations of these steroids. We accept our hypothesis because the concomitant changes in mean concentration of FSH between Days 6 and 11 and amplitude of LH pulses between Days 7 and 12 of the bovine oestrous cycle cannot be explained by changes in circulating concentrations of progesterone and 17β-oestradiol.     

Effects of oxytocin and an oxytocin antagonist on testosterone secretion during the oestrous cycle of the goat (Capra hircus)

Oxytocin at a dose of 100 i.u. injected subcutaneously (s.c.) daily to goats between Days 3 and 6 of the oestrous cycle caused a significant increase in testosterone secretion compared with saline-treated animals. An oxytocin antagonist (0.2 micrograms/kg) injected intra-arterially between Days 12 and 18 of the oestrous cycle or simultaneously with oxytocin between Days 3 and 6 blocked the increased release of testosterone and occurrence of oestrus. It is suggested that oxytocin-induced oestrus may occur via testosterone secretion.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).