Ovarian factors inhibit and fetal factors stimulate the secretion of rat placental lactogen

Removal of fetuses at day 14 of gestation (Ftx14) in the pregnant rat leads to a marked suppression of serum levels of rat placental lactogen (rPL-II). One might attribute this to compromised placental growth in the absence of a fetus. However, if ovariectomy and fetectomy (Ftx14 Ovx14) are carried out at the same time, a great increase in serum rPL-II levels is seen. This occurs despite a significant decrease in placental weight. When Ftx14 was performed on day 14 and Ovx was delayed 1, 2, or 3 days, the expected large increase in serum rPL-II was progressively attenuated compared to that seen when Ftx and Ovx were carried out simultaneously. Daily administration of 17 beta-estradiol (4 micrograms/rat X day) to Ftx14 Ovx14 pregnant rats resulted in a significant suppression of rPL-II and elevation of rPRL levels, a reversal of what is seen for these hormones in untreated Ftx14 Ovx14 animals. To test whether 17 beta-estradiol was acting through rat PRL (rPRL), serum levels of rPRL were elevated in Ftx13 Ovx13 animals with pimozide (0.6 mg/kg), a dopamine receptor blocker. There was no effect of this treatment on rPL-II levels. In late pregnancy (day 17) serum rPL-II levels remained high after removal of half the fetuses (1/2 Ftx), compared to the rapid fall in pregnant rats in which all fetuses were removed (Ftx17). Serum levels were also elevated in 1/2 Ftx animals compared to those in which half of the fetuses and placentas were removed by hemi-hysterectomy, suggesting that the increase in rPL-II levels in 1/2 Ftx animals was due to a stimulatory effect of the remaining fetuses on all of the placentas. These results indicate that the presence of the fetus is necessary for the normally observed increase in rPL-II levels in late pregnancy. In conclusion, fetal stimulators and ovarian inhibitors influence rPL-II secretion.     

Ovarian and fetal control of rat placental lactogen and prolactin secretion at midpregnancy

The role of the fetus and ovarian steroids in stimulating rat placental lactogen (rPL) secretion and in regulating nocturnal PRL surges was investigated. On day 10, pregnant rats were ovariectomized (O) and/or fetectomized (F). Those rats injected with steroids received 0.1 microgram estradiol benzoate (E) or 4 mg progesterone (P) in oil sc, or both (PE). Blood samples were taken by cardiac puncture at 0500 h, on days 10 through 14. OPE rats showed slightly depressed serum rPL levels on days 11 and 12 and normal growth of the conceptuses. Ovariectomized rats given P alone also showed significant growth of the conceptus, although less than the OPE rats, demonstrating the necessity for estrogen during pregnancy. PRL surges were no longer present in these groups on day 11, similar to controls. Ovariectomized rats had very low rPL levels, and the nocturnal PRL surges continued for 2 extra days. Fetectomized and OF animals also had very low rPL levels and one extra PRL surge. P counteracted the detrimental effects of fetectomy on rPL secretion; yet placental uterine growth was not greater than in F animals. Thus, increased placental mass was not always correlated with increased rPL secretion. The results show that P is necessary to maintain rPL secretion in the pregnant rat, although estradiol and P in combination is most effective. The fetus also is essential to stimulate both placental growth and rPL secretion. The inverse correlation between rPL and PRL levels on days 11 and 12 suggests that rPL normally terminates the PRL surges at midpregnancy.     

Differential action and secretion of rat placental lactogens

The abilities of the two different mol wt forms of rat placental lactogen (rPL) to maintain luteal steroidogenesis in pregnant rats in the absence of PRL were determined. The two forms of rPL present in day 12 pregnant rat serum were separated by gel chromatography, lyophilized, and reconstituted in a volume of saline equal to the original volume of serum. Rats were injected with 0.4 mg ergocryptine (ECO) on day 6 of pregnancy to suppress PRL and were then treated with a preparation of the large mol wt (LMW) hormone, the small mol wt (SMW) hormone, or both molecules (2 ml/day). Control animals received saline. Twice daily injections of the amount of LMW hormone contained in 1 ml day 12 pregnant rat serum reversed the abortifacient effect of ECO. In contrast, administration of the SMW hormone did not maintain either pregnancy or progesterone levels. Administration of both mol wt forms of rPL was also capable of maintaining luteal function. Sera obtained from day 18 pregnant rats containing only the SMW hormone had no luteotropic activity when administered, yet treatment with sera of day 12 pregnant rats sustained progesterone synthesis and fetal survival after ECO treatment. rPL were measured in the peripheral circulation and in the uterine vein throughout pregnancy by radioreceptor assay (RRA) using particulate membranes from either rabbit mammary gland or rat ovaries. Two peaks of activity were observed in the peripheral circulation by the two RRAs: one between days 11-14 and another between days 17-21, with a decline in activity between days 14-16. In contrast, levels of rPL in the uterine vein remained elevated throughout pregnancy. Concentrations of the LMW placental luteotropin were 5-10 times higher by ovarian RRA (O-RRA) than by mammary gland RRA (MG-RRA) between days 11-13, but concentrations of the SMW placental lactogen were found by the two RRAs to be similar in the later stages of pregnancy. Gel filtration of day 12 pregnant rat serum revealed two peaks of PRL-like activity. The O-RRA detected 19 times more LMW placental luteotropin than did the MG-RRA in the first peak of activity, yet measured equivalent amounts of the SMW placental lactogen in the second peak. Similar to results found in the peripheral circulation, levels of LMW placental luetotropin in the uterine vein measured by MG-RRA were significantly lower than those determined by O-RRA until day 14 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat chorionic gonadotropin: augmentation of bioactivity in the absence of the pituitary

Significant gonadotropin-like bioactivity, as measured by rat interstitial cell testosterone assay, is present in the serum and placenta of pregnant rats. The patterns of activity for this putative rat chorionic gonadotropin (rCG) in serum and placenta are distinct from those observed for rat placental lactogen (rPL) radioreceptor activity. Placental and serum rCG (and rPL) activities are substantially increased, as are placental weights, following removal of the maternal pituitary on day 12 of pregnancy. These findings strengthen the evidence for the existence of a rat CG and suggest a novel role for the maternal pituitary in regulating placental function during normal gestation.     

Circulating maternal immunoreactive inhibin levels during pregnancy in the rat: effects of oophorectomy, hypophysectomy, hemihysterectomy, delayed implantation and pseudopregnancy.

This study examines the source of inhibin in the maternal circulation of pregnant rats by measuring serum immunoactive inhibin levels following a range of experimental procedures. Ovariectomy at days 7, 13 or 19 of gestation, with maintenance of pregnancy by supplementation with progesterone and oestradiol dipropionate, led to a profound fall of serum inhibin levels in comparison with controls, demonstrating that the ovary is a major source of circulating inhibin. This conclusion was supported by the inhibition of the late rise (days 16-22) in serum inhibin in pregnant rats which were hypophysectomized on day 15 and maintained with oestrogen and progesterone supplementation. These data support the view that the rise in serum inhibin from days 16 to 22 is due to re-activation of follicular development in preparation for the post-partum oestrus. Reduction of fetal numbers by hemihysterectomy on days 7, 13 or 19 did not alter serum inhibin levels. Induction of delayed implantation by ovariectomy on day 3 and progesterone supplementation together with initiation of reimplantation by the addition of oestradiol dipropionate on day 7 or 11 did not significantly alter inhibin levels. The induction of pseudopregnancy by mating to vasectomized rats did not result in the maintenance of stable serum inhibin levels until oestrous cycles recommenced. Taken together, the studies have identified the ovary as the predominant source of circulating maternal inhibin levels throughout pregnancy in the rat.     

Correlation of luteinizing hormone surges with estrogen nuclear and progestin cytosol receptors in the hypothalamus and pituitary gland. I. Estradiol dose response effects

The present studies were designed to answer three questions: (1) how will a progressive increase in serum estradiol (E2) in ovariectomized (OVX) rats affect progesterone (P4)-induced luteinizing hormone (LH) surge concentrations? (2) Can steroid-induced LH surges be correlated with estrogen nuclear receptor (E2Rn) and progestin cytosol receptor (PRc) levels in brain regions known to regulate LH secretion, and (3) do differences in pituitary responsiveness to LHRH in E2- or E2P4-treated OVX rats parallel changes in E2Rn and PRc concentrations in this gland? 1 week after ovariectomy of adult cyclic rats (day 0), Silastic E2 capsules were placed subcutaneously at 09.00 h and produced serum E2 levels of 6-8 (low), 12-19 (medium) and 27-37 (high) pg/ml, respectively. 2 days later (day 2), some rats also received Silastic P4 capsules subcutaneously which elevated serum P4 concentrations to 10-12 ng/ml. In rats with low serum E2, P4 treatment induced peak serum LH levels of 913 ng/ml. When serum E2 was increased to the medium or relatively high physiologic range, P4 treatment resulted in LH surge levels of 4,686 and 5,030 ng/ml. OVX controls and E2-treated OVX rats were sacrificed at 10.00 h on day 2 and E2Rn and PRc were measured concurrently in the preoptic area (POA), mediobasal hypothalamus (MBH), corticomedial amygdala (CMA) and pituitary gland (PIT). Raising serum E2 from OVX levels to the low range significantly increased both E2Rn and PRc in MBH and PIT, but not in the POA or CMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of circulating levels of IGF-I in pregnant rats: changes in nitrogen balance correspond with changes in serum IGF-I concentrations

Serum IGF-I concentrations in rats decrease significantly in late pregnancy. To determine if the reduction in serum IGF-I concentrations is attributable to circulating GH or maternal nutritional status, we investigated the effect of treatment with recombinant human GH (rhGH: 100 microgram/rat per day) on IGF-I concentrations during late pregnancy, and evaluated the relationship between maternal nitrogen balance and IGF-I concentrations. Serum IGF-I concentrations and maternal nitrogen balance ((nitrogen intake)-(nitrogen content in faeces and urine)-(nitrogen content in fetus and placenta)) were measured by RIA and the Dumas method. In non-pregnant rats treated with rhGH for 3 days, serum IGF-I concentrations (835.4+/-59.5 ng/ml; P<0.01) were significantly greater than in those animals treated with saline (319.6+/- 95.6 ng/ml). In the pregnant rats, however, there was no significant difference in serum IGF-I between those treated with rhGH (151. 1+/-43.0 ng/ml) and those treated with saline (142.0+/- 39.9 ng/ml) from day 17 to 19 of pregnancy. Maternal nitrogen balance in the pregnant rats increased significantly from day 4 to day 10 of pregnancy (169.5+/-57.4 and 196.1+/- 33.4 mg/day, respectively; P<0. 05) compared with non-pregnant controls (31.9+/-19.9 mg/day) and decreased markedly from day 12 of pregnancy (79.8+/-60.1 mg/day; P<0. 05) onwards, to 14.9+/-47.8 mg/day on day 20 of pregnancy (P<0.01), significantly different from the value on day 10 of pregnancy. The mean difference in maternal nitrogen balance between pregnant and non-pregnant rats was positively correlated (r=0.87, P<0.01) with the mean difference in maternal IGF-I concentrations, using linear regression analysis. These results support the conclusion that the circulating concentration of IGF-I in pregnant rats is associated with the change in nitrogen balance, but not with circulating GH.     

Pituitary regulation of human growth hormone binding sites in rat liver membranes.

We have studied the binding of 125I-human growth hormone (hGH) to crude 100,000 X g membrane preparations from rat liver, and have studied factors which might regulate the capacity and affinity of hGH binding sites. Membrane preparations have livers of pregnant rats bound between 8% and 18% of the 125I-hGH initially added, and 70%-80% of that bound was displaced by 1 mug of unlabeled hGH. Humans prolactin (hPrl) displaced 125 I-hGH in a manner parallel to hGH itself but with about one-third the potency. Ovine, porcine, and rat Prl, and rat and bovine GH were much less effective. Scatchard analysis of specific hGH binding by a variety of different rat liver membrane preparations revealed a single order of binding site in each case with a binding affinity of 0.93-1.62 X 10(-9) M-1. Membranes from pregnant rats had twice the binding capacity of membranes from nonpregnant female rats, and about six times the capacity of sites present in preparations from normal adult male rats and hypophysectomized (Hx) male or female rats. Female or male rats with extremely high circulating GH an Prl levels, due to the presence of transplantable GH/Prl secreting pituitary tumors showed a significantly greater binding capacity than did the pregnant rats. Estradiol (E2) treatment (25 mug/day for 10-12 days) of normal male rats led to an increase in specific hGH binding. Treatment of hypophysectomized male rats with bovine GH (100 or 500 mug/day) +/- E2 (25 mug/day) for 5-10 days stimulated both body weight gain and the incorporation of sulfate by cartilage from the treated rats, but no significant increase was observed in the characteristics of 125I-hGH binding. These results indicate that high levels of E2, GH, and/or Prl play an important role in the regulation of hGH binding sites in rat liver membranes. The restoration of binding sites in liver from hypophysectomized rats, however, apparently requires additional factors which are as yet unidentified. The role of the hGH binding sites in the physiologic actions of GH also remains to be determined.     

Effect of adrenalectomy at different pregnancy stages on maternal and fetal serum corticosteroid binding globulin and corticosterone in the rat

The response of pregnant rat corticosteroid binding globulin to maternal adrenalectomy was studied as a function of the stage of pregnancy. Non-pregnant or pregnant rats were deprived of their adrenal glands during 4 days. In non-pregnant animals, adrenalectomy led to undetectable corticosterone levels and to the doubling of corticosteroid binding globulin. In pregnant rats adrenalectomized at 12 days and studied at 16 days, the serum corticosterone was likewise undetectable and the corticosteroid binding globulin was doubled as compared with pregnant rats of the corresponding age. In contrast, adrenalectomy from day 14 to 18 or from day 16 to 20 did not deplete the maternal serum corticosterone and the corticosteroid binding globulin remained unchanged. Under these conditions neither fetal corticosteroid binding globulin nor fetal corticosterone were modified. However, when the pregnant rats adrenalectomized from day 16 to 20 also received an injection of 30 mg of metyrapone on days 19 and 20 in order to inhibit fetal adrenal secretion, the maternal response was again a depletion of serum corticosterone together with an increase in corticosteroid binding globulin. Under these conditions, the fetus also reacted by a fall of corticosterone and a rise of corticosteroid binding globulin. Our results suggest that the maternal response of corticosteroid binding globulin to adrenalectomy depends on the pregnancy stage inasmuch as it may be influenced by a supply of corticosterone from the fetus during late pregnancy. Moreover, they show that in this late period, fetal corticosteroid binding globulin is regulated independently.     

Relative sensitivity of the corpus luteum of different days of pregnancy to LH-deprivation in the rat and hamster.

Deprivation of endogenous LH by LH antiserum (LH A/S) in 6-day pregnant rats did not affect the luteal or serum progesterone within 24 h. LH A/S treatment on day 7 or 8 of pregnancy, however, caused a 70 and 92% reduction in luteal progesterone, respectively, within 24 h. Serum levels of progesterone showed a similar reduction. In the case of pregnant hamster, unlike the rat, there was a significant decrease in progesterone in the serum, luteal and non-luteal compartments whether the A/S was administered on day 4, 5 or 6. There was more than a 10-fold increase in the luteal cholesterol esters within 24 h whether the A/S was given on day 6, 7 or 8 of pregnancy in the rat. Rat corpora lutea of days 6 and 8 of pregnancy reacted in a like manner to LH-deprivation, showing an increased utilization of [U-14C]glucose to form 14CO2 in vitro. In the rat, LH (25 mug NIH-S19) administration in vivo either on day 6 or day 8 of pregnancy, caused within 2 h an increase in serum and non-luteal progesterone, but luteal progesterone was unchanged. On the other hand, LH administration to hamsters on day 8 of pregnancy caused an increase in progesterone levels in serum, luteal and non-luteal tissue. Incubation of corpora lutea isolated from untreated 6- and 8-day pregnant rats with LH brought about an increase in progesterone secretion into the medium in both cases. The results show that, even though LH-deprivation does not apparently affect progesterone concentration in the corpus luteum of 6-day pregnant rats, it does affect other metabolic parameters such as glucose utilization and cholesterol turnover, suggesting that the corpus luteum of early pregnancy exhibits a continuous dependency on LH for the maintainence of metabolic functions.     

Effect of ACTH and histamine stress on serum corticosterone and adrenal cyclic AMP levels in immature rats.

Adrenal cAMP and plasma corticosterone levels were determined in pre-weanling rats subjected to treatment with either ACTH (50 mU/rat) or histamine dihydrochloride (0.2 mg/g body wt). ACTH injection elevated both serum corticosterone and adrenal cAMP levels on all days tested. However, the ACTH-induced elevation of adrenal cAMP and serum corticosterone both diminished steadily from day 2 to day 8 and then increased from day 8 to day 16. Histamine injection resulted in elevated serum corticosterone levels in a pattern similar to that of the corticosterone response to ACTH. However, histamine injection did not result in any significant increase in adrenal cAMP from day 2 to day 10. From day 12 to day 16 the adrenal cAMP concentration rose steadily in parallel with ther serum corticosterone levels. These results indicate: (1) that a functional, ACTH-sensitive adenyl cyclase system is present in the adrenal gland of the immature rat, (2) that the responsiveness of this system diminishes during the first postnatal week before returning to its previous 2-day-old capacity by day 16, and (3) that during the first few days after birth, histamine stress results in elevated serum corticosterone levels without elevating adrenal cAMP levels.     

Differential regulation of estrogen receptor alpha and beta mRNAs in the rat uterus during pregnancy and labor: possible involvement of estrogen receptors in oxytocin receptor regulation

Oxytocin receptor (OTR) mRNA levels in the uterus dramatically increase in the near term human and rat. Estrogen is believed to be a potent stimulator of OTR mRNA expression. However, estrogen does not stimulate rat OTR mRNA expression on day 18 of pregnancy or in progesterone-treated rats. Thus, the regulation of uterine responsiveness to estrogen in the near term rat appears to be an important mediator of estrogen action. To determine the effect of altering uterine responsiveness to estrogen on OTR induction, uterine ERalpha and ER beta mRNA levels were examined by competitive RT-PCR in pregnant and parturient rats, progesterone-treated ovariectomized (OVX) virgin rats and OVX pregnant rats. In pregnant and parturient rats, OTR mRNA levels were highest at 2200-2230 h on day 21 of pregnancy (P21pm) and during labor when compared with other groups. ERalpha mRNA levels significantly increased during labor compared with days 15-21 of pregnancy. Compared with control animals, ERalpha mRNA levels decreased significantly in OVX virgin rats implanted with tubes containing progesterone for one week; 24 h following the removal of the progesterone tubes, ERalpha mRNA levels were found to be similar to control levels. Estrogen treatment following OVX on day 18 of pregnancy caused increased OTR mRNA levels, whereas ovariectomy alone increased ERbeta mRNA but not ERalpha mRNA. Results from the present study suggest that ERalpha and ERbeta mRNA expressions are differentially regulated in the rat uterus. Moreover, during spontaneous labor our findings appear to suggest that ERalpha plays a more prominent role than ERbeta in mediating estrogen action in the induction of uterine OTR mRNA before labor.     

Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of interleukin 6

The purpose of this study was to determine the role of interleukin (IL) 6 in mediating the increase in arterial pressure (AP) in response to chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. AP was higher in RUPP rats (138+/-1 mm Hg) than in normal pregnant (NP) rats (104+/-1 mm Hg). Serum IL-6 levels in the RUPP rats were 104.5+/-28.6 pg/mL as compared with 36.6+/-7.4 pg/mL in NP rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma IL-6 on renal function and AP in pregnant rats, we infused IL-6 for 5 days (2.5 ng/day) in NP rats starting at day 14 of gestation. Five days later, serum IL-6 levels were 55.5+/-6.5 pg/mL in the control NP rats and 157.0+/-36.1 pg/mL in the IL-6-treated NP rats. AP was higher in the IL-6-treated NP rats (115+/-3 mm Hg) as compared with NP controls (101+/-1 mm Hg) at day 19 of gestation. Renal plasma flow and GFR were lower in the IL-6-treated NP rats than in the NP group. IL-6 increased plasma renin activity but did not affect endothelin in IL-6-treated NP rats. In contrast to the NP rats, IL-6 had no effect on AP or renal hemodynamics in virgin rats. In summary, these data indicate that plasma IL-6 is elevated in response to chronic reductions in uterine perfusion in pregnant rats and that a comparable elevation in plasma IL-6 increases AP and reduces renal function in pregnant rats.     

Termination of prolactin surges with development of placental lactogen secretion in the pregnant rat

It has been hypothesized that it is rat placental lactogen (rPL) which causes PRL surges to terminate at mid-pregnancy. Using a hormonally induced model of delayed implantation, the temporal relationship between the secretion of rPL and the nocturnal PRL surges was followed. Implantation was prevented by removing the ovaries, the source of estrogen, on day 3 of pregnancy. Blastocysts were maintained free-floating in the uterine lumen by sc injection of 4 mg progesterone in oil daily for 0, 5, 7, or 9 days. Implantation was induced and the subsequent pregnancy was maintained with 1 microgram estrone plus 4 mg progesterone daily. Nocturnal PRL surges (0500 h) were followed for 10 days after the first estrone injection. Control animals last exhibited PRL surges on day 10. Animals with 5, 7, or 9 days of implantation delay had their last PRL surge on days 15, 17, and 18, respectively. Levels of rPL in control animals, as measured by Nb2 lymphoma cell bioassay, were low on day 6, slightly higher on day 10, and significantly elevated on day 12. Delaying implantation delayed the increase in rPL secretion in the experimental groups. This proportionately prolonged the number of days the PRL surges were present. These data suggest that the termination of the nocturnal PRL surges requires the secretion of rPL by the developing conceptus.     

The effect of hypophysectomy on rat chorionic somatomammotropin as measured by prolactin and growth hormone radioreceptor assays: possible significance in maintenance of somatomedin generation.

We have previously reported that serum somatomedin concentrations are maintained in pregnant rats after hypophysectomy. Because rat chorionic somatomammotropin (rCS) might replace pituitary GH during pregnancy, the concentration of rCS was measured at intervals after hypophysectomy on day of pregnancy. By day 16 of pregnancy, the serum rCS of hypophysectomized (hypox) rats was actually higher than that of normal pregnant rats when measured by a lactogenic radioreceptor assay. Increased levels of lactogenic radioreceptor activity (L-RRA) were maintained in the serum of hypox rats throughout the remainder of pregnancy. The GH radioreceptor activity (GH-RRA) of serum of hypox pregnant rats was also greater than that of normal rats during the last days of pregnancy, but the activity was only about 1/20th that of the L-RRA with the assays employed. There was no significant difference between placental L-RRA and GH-RRA of normal and hypox pregnant rats. The difference in concentration could not be attributed to differences in the number of fetuses. We conclude that the high levels of rCS were sufficient to maintain serum somatomedin concentration in hypox pregnant rats. This effect of rCS could have been due to binding by GH or PRL receptors of the maternal liver.     

Comparison of in vivo effects of insulin-like growth factors I and II and of growth hormone in hypophysectomized rats

Pure human IGF I (43 and 103 micrograms/day) and IGF II (131 micrograms/day) were infused into hypophysectomized rats during 6 days by means of sc implanted minipumps. Their effects on several growth indices were compared with those of various doses of sc infused human growth hormone. Growth hormone infusion produced a dose-dependent rise of endogenous rat IGF from 39 (without growth hormone) to 86 microU equivalents/ml (with 400 mU hGH/day) as determined by a competitive protein binding assay with a human IGF standard. In rats receiving the two doses of IGF I, total serum IGF levels rose to 83 and 99 microU equivalents/ml, respectively, in those receiving the IGF II dose the total serum IGF level rose to 146 microU equivalents/ml. These increases corresponded to steady state levels of 168 and 286 ng/ml of immunoreactive insulin-like growth factor (IR-IGF) I and 320 ng/ml of IR-IGF II. IGF I, but not IGF II led to an increase in body weight similar to that induced by the low doses of hGH (12.5 and 25 mU, respectively). The rise of endogenous rat IGF as well as the infused human IGF I and II caused a widening of the tibial epiphysis and an increase of the [3H]thymidine incorporation into costal cartilage. With respect to these two indices IGF II was clearly less potent that IGF I. When expressed in microU equivalents of the protein binding assay, endogenous rat IGF induced by hGH appeared to be relatively more effective than infused human IGF I or II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a homologous radioimmunoassay for rat relaxin.

Highly purified rat relaxin has been radioiodinated to specific activities of approximately 100 micro Ci/microgram with the Bolton and Hunter reagent [N-succinimidyl 3-(4-hydroxy-5-[125I]iodophenyl) propionate]. A rabbit antirat relaxin serum, applicable in a final dilution of 1:100,000, was developed in a rabbit using unconjugated highly purified rat relaxin. A specific and precise double antibody RIA for rat relaxin sufficiently sensitive to routinely measure from 32--2000 pg rat relaxin was developed. Using this RIA, relaxin immunoactivity levels in extracts of pregnant rat ovaries were found to rise from 0.8 microgram/geq ovarian fresh tissue on day 8 of pregnancy to 723 microgram/geq ovarian fresh tissue on day 20 of pregnancy and then to drop precipitously to 6 microgram/geq ovarian fresh tissue on day 1 of lactation. Consistent with the occurrence and relative levels of relaxin in the ovarian extracts, levels of relaxin in pregnant rat serum were less than 2 ng/ml on day 10 of pregnancy, approximately 150 ng/ml on days 20 and 22 of pregnancy, and 12 ng/ml on day 2 of lactation.     

Regulation of a thyroid hormone-dependent protein by growth hormone: the induction of hepatic alpha 2U globulin and its messenger ribonucleic acid in adult male hypothyroid rats

Studies in the adult male hypothyroid rat, a known GH-deficient animal, have shown hepatic alpha 2U-globulin mRNA to be dependent on thyroid hormones. To study the effects of GH on alpha 2U-globulin synthesis in the absence of thyroid hormones, adult male rats were rendered hypothyroid before hormone treatment. The relative effects of bovine GH or T3 were studied by RIA of alpha 2U-globulin in hepatic cytosol in rats 6 weeks after thyroid ablation. alpha 2U-Globulin levels in vehicle-treated controls were 1.3 +/- 0.7 micrograms (+/- SD) alpha 2U-globulin/mg protein. After 2 days, GH (200 micrograms/100 g X day) resulted in an increase to 5.7 +/- 1.0 micrograms alpha 2U-globulin/mg (P less than or equal to 0.05), and T3 50 micrograms/100 g X day) resulted in an increase to 11.5 +/- 3.6 micrograms/mg (P less than or equal to 0.01). After 7 days, GH resulted in an increase to 12.4 +/- 4.6 micrograms/mg (P less than or equal to 0.01), and T3 resulted in an increase to 28.7 +/- 8.7 micrograms/mg (P less than or equal to 0.01). After 4 months of thyroid ablation, baseline hepatic alpha 2U-globulin levels fell to 4.8 ng alpha 2U-globulin/mg protein. Hepatic alpha 2U-globulin was determined 4 and 8 h after the injection of GH (200 micrograms/100 g). In these animals with markedly diminished hepatic alpha 2U-globulin levels, significant (P less than or equal to 0.01) increases occurred 4 h (25.4 ng/mg) and 8 h (57.2 ng/mg) after GH injection. The effects of treatment with bovine GH (200 micrograms/100 g X day) for 3 days on hepatic alpha 2U-globulin synthesis in liver slices and alpha 2U-globulin poly (A)+ RNA levels were measured in rats 10 weeks after thyroid ablation. GH significantly (P less than 0.05) increased alpha 2U-globulin synthesis as a percentage of total protein synthesis (from 0.01% to 0.035%) and alpha 2U-globulin mRNA as a percentage of total mRNA (from 0.03% to 0.24%). The results show that GH rapidly and specifically stimulates hepatic alpha 2U-globulin and its mRNA activity in thyroid hormone-deficient rats.     

Regulation of FSH beta messenger ribonucleic acid levels in the rat by endogenous inhibin.

This study investigated the role of endogenous inhibin in regulating FSH beta mRNA levels subsequent to the gonadotropin surge in the immature, estradiol (E2)-treated female rat. Rats which undergo FSH surges on day 29 have low to undetectable levels of FSH beta mRNA at 0900 h on day 30, whereas those treated simultaneously with E2 and progesterone (P) implants to block these surges have considerably higher levels of FSH beta mRNA. In view of the profound inhibitory effect of inhibin on FSH beta mRNA, we examined the possibility that increased inhibin secretion is responsible for the decline in FSH beta mRNA levels on the morning after the FSH surge by immunoneutralization of endogenous inhibin. Twenty-eight day-old rats which received E2 and blank (B1) or P implants were injected iv with 0.4 ml of a potent anti-rat inhibin serum (anti I alpha, prepared in sheep against rat inhibin alpha (1-26)-Tyr27 coupled to human alpha-globulins) or normal sheep serum at 1700 to 1830 h on day 29 and were killed at 0900 h on day 30. Animals which received the inhibin antiserum showed significantly (P less than 0.001) elevated serum FSH levels (22.9 +/- 1.9 ng/ml [E2 + B1] and 17.1 +/- 0.6 ng/ml [E2 + P]) compared to those which received normal serum (4.4 +/- 0.1 [E2 + B1] and 4.2 +/- 0.1 [E2 + P]). Serum LH was undetectable (less than 0.6 ng/ml) in all groups. Free glycoprotein alpha-subunit was also increased (P less than 0.001) by antiserum to inhibin in E2 + B1-treated rats but was significantly suppressed by P after injection of either normal serum or anti I alpha. Total pituitary RNA was extracted and hybridized to cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit by Northern blot analysis; RNA levels were normalized with beta-actin or cyclophilin probes. As expected, in rats which received normal serum, FSH beta mRNA levels were about 4-fold higher after treatment with E2 + P implants than after treatment with E2 + B1 implants. However, injection with anti-inhibin serum resulted in a striking elevation of FSH beta mRNA levels: 13-fold in animals treated with E2 + B1 implants and 5-fold in animals treated with E2 + P implants. There were no significant differences in levels of LH beta or alpha-subunit mRNAs between rats which received anti-inhibin or normal serum although there was a 30-40% decrease in alpha mRNA after P treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actions of pregnant mare serum gonadotropin in the immature female rat: correlative changes in blood steroids, gonadotropins, and cytoplasmic estradiol receptors of the anterior pituitary and hypothalamus.

Several blood steroids, serum gonadotropins and cytosol estradiol receptors of the anterior pituitary and hypothalamus were quantified in immature female rats which were induced to ovulate with pregnant mare's serum gonadotropin (PMSG). Studies revealed that serum levels of progesterone, 17-hydroxyprogesterone, testosterone, androstenedione and estradiol were initially elevated at 6 PM (day 30) after administration of 8 IU of PMSG at 10 AM day 30. Serum levels of estradiol and testosterone rose progressively from day 30 through the AM of day 32. A further increase in serum concentrations of progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, and dehydroepiandrosterone occurred on the PM of day 32 whereas serum estradiol levels declined. Serum levels of all steroids declined on the day of estrus (33) and only progesterone levels were further elevated on day 34 (diestrus). Dihydrotestosterone concentrations were minimally altered by PMSG treatment. Saline administration resulted in no significant alterations in levels of any steroid quantified from day 29 to 34 in control animals. A progressive decline in cytosol estradiol receptor content of the anterior pituitary and hypothalamus was documented following PMSG treatment of intact female rats; there was no depletion of receptors following PMSG administration to ovariectomized immature rats. Maximal depletion of cytosol estradiol receptors occurred on day 32 with replenishment of cytosol estradiol receptor levels on estrus (day 33). The preovulatory gonadotropin surge was found to occur on the PM of day 32 after maximal receptor depletion. The cycle of depletion and replenishment of receptors was repeated during a second spontaneous estrous cycle four days later which coincided with a rise and fall in serum estradiol levels. It is suggested that the depletion of cytosol estradiol receptors of the anterior pituitary/hypothalamic unit may be causally related to the preovulatory gonadotropin surge resulting from PMSG administration to immature female rats. In addition, changes in blood steroids and gonadotropins after PMSG treatment are similar to those reported for proestrus-estrus-diestrus I of the normal adult estrous cycle. These findings further demonstrate the validity of the PMSG-primed immature female rat preparation as a model for the estrous cycle of the adult rat.