Serum Of Pregnant Rats Research Articles

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.

Serum concentrations of testosterone throughout pregnancy in rats.

Testosterone concentrations in serum of rats bled throughout pregnancy and post partum were measured using Celite microcolumn chromatography and a radioimmunoassay for testosterone. Mean serum levels of testosterone ranged from about 170 to 340 pmol/l during the first 10 days of pregnancy. Significant increases in concentrations of testosterone in serum of pregnant rats were found on days 12, 15 and 18 of gestation. The highest testosterone concentrations occurred on days 18 and 20 of pregnancy when mean levels were 3228 and 3685 pmol/l respectively. Testosterone levels declined before parturition on day 22 (mean = 1449 pmol/l and declined further after parturition (mean = 315 pmol/l). In order to determine whether serum testosterone concentrations varied during the day in the pregnant rat, samples were collected at 6-h intervals on days 6–7 and 14–15 of gestation. Diurnal variations in serum testosterone concentrations were not evident during early or late pregnancy, unlike the rhythmic changes in serum prolactin levels found at these times during early pregnancy. The possible sources of the increased titres of serum testosterone during the second part of gestation in rats are discussed.

Effects of the uterus and mid-pregnancy conceptus on luteal lifespan of the rat.

Hysterectomy on or after Day 10 resulted in a quick return of pseudopregnant rats to oestrus but the interval to oestrus in pregnant rats was significantly longer (P less than 0.01), indicating that the gravid uterine horn interfered with uterine-induced luteolysis by Day 10. When pseudopregnant rats were injected with extracts of conceptuses or pregnant rat serum (PRS), samples collected on Days 10 or 12 of pregnancy delayed the return to oestrus (P less than 0.01 for conceptus extracts, P less than 0.05 for PRS) and serum progesterone concentrations on Day 13 of pseudopregnancy were higher (P less than 0.01) than in controls. Bioassays utilizing mammary development or inhibition of oestrus in virgin cyclic rats as endpoints failed to detect lactogenic activity in Day 10 conceptus extracts or PRS, and radioreceptor assays using bovine prolactin (NIH-B5) as a standard showed that Day 10 extracts had only about 2--3% of the lactogenic activity found in Day 12 extracts. Injections of dilutions of Day 12 extracts with 2 or 4 times the lactogenic activity as Day 10 extracts did not extend pseudopregnancy. These results suggest that by Day 10 the conceptus produces a factor capable of interfering with luteolysis and that this factor is not placental lactogen.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum.

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

The effect of pinealectomy on serum progesterone and oestradiol levels during the last stage of pregnancy in rats.

Pinealectomy leads to significantly higher levels of progesterone (on days 19 and 20 of gestation) and oestradiol (on days 21 and 22) in the serum of pregnant rats. These results indicate that during the last phase of pregnancy the pineal gland may be modifying the levels of gonadal hormones, although not affecting the sequence of the physiological events.

Studies on the irreversible nature of prolactin binding to receptors.

Studies on hormone-receptor interactions generally assume that the formation of a hormone-receptor complex is a reversible process. This assumption has been examined directly in three experiments using liver membrane receptor preparations from pregnant rats and ovine PRL (oPRL). In Exp 1, Receptors were preincubated with a range of concentrations of oPRL at 23 C for periods up to 60 min, washed thereafter to remove free oPRL, and subsequently incubated with [125I]iodo-oPRL (23 C) to determine specific binding. Preincubation of receptors (0.25 mg membrane protein) with oPRL (5 ng) for periods as brief as 10 min reduced subsequent binding of [125I]iodo-oPRL to receptor, suggesting incomplete dissociation of oPRL even after 30 h. In Exp 2 after preincubation for 30 min with oPRL and subsequent incubation with [125I]iodo-oPRL for 19 h, membranes were washed, and the dissociation (23 or 37 C) of [125I]iodo-oPRL from the hormone-receptor complex in the presence or absence of 1000 ng oPRL was studied. After 48 h, only 35-50% of the [125I]iodo-oPRL dissociated from the hormone-receptor complex even in the presence or excess oPRL, indicating a heterogeneity of binding sites (i.e. 50-65% irreversible; 35-50% reversible). When pregnant rat serum was used in place of oPRL or when rabbit mammary glands were used instead of rat livers to prepare receptor preparations, results were similar to those described above, except for the nearly complete dissociation (90%) obtained at 37 C using rabbit mammary gland receptors. In Exp 3 after incubation (10 min, 2 h, or 15 h) of rat liver receptors with [125I]iodo-oPRL plus various amounts of oPRL, the hormone-receptor complex could be completely dissociated with 5 M MgCl2, restoring binding affinity and capacity of receptor to their original values. Labeled oPRL dissociated by MgCl2 treatment from such a complex is capable of binding to fresh receptor. These data strongly suggest that the PRL-receptor interaction, particularly the rat liver receptor interaction with PRL under usual in vitro conditions, is not reversible to a significant degree. This is not due to hormone or receptor damage but to a significant number of binding sites (50-65%) in the receptor preparation which are not reversible except under extreme conditions.

HORMONAL EFFECTS ON RAT MAMMARY GLAND IN VITRO

The mammogenic and lactogenic effect of various hormones on rat mammary gland has been studied in vitro using an organ culture technique. The effects have been assessed mainly by light microscopy. The study has been confined mainly to the effects and interactions of the lactogenic protein hormones, corticosteroids and progesterone. It establishes the importance of the presence of insulin and serum and of the type of serum for the hormonal effects in vitro. These effects have been studied mainly on the explants obtained from 13 days pregnant rats. In addition explants from non-pregnant rats and at various stages of pregnancy and lactation were studied.

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.

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.

Conversion of Progesterone to Testosterone in the Rat Leydig Cell

SummaryThe bioassay for LH-like hormones, based on the amount of testosterone produced in vitro by collagenase-dispersed rat Leydig cells in response to the hormone to be tested, was used to see whether a chorionic gonadotrophin (CG)-like hormone could be detected in the serum of pregnant rats. The curve of the serum “CG” concentration (i.e. in vitro testosterone produced) during the course of pregnancy was different from that of serum LH, determined by RIA, but was very similar to that of serum progesterone. This similarity proved to be due entirely to the conversion of progesterone to testosterone by the Leydig cells, since known amounts of progesterone (10-200 ng) dissolved in hypophysectomized rats'serum were converted quantitatively to testosterone when added to the cells under the same conditions used to test the pregnancy serum. The bioassay may thus be used as a specific measure of LH-like hormones only under conditions in which it is certain that steroids which can be converted to testosterone ar...

Epoxide-metabolizing enzyme activities and cytochrome P-450 content of rat ovaries during pregnancy

Glutathione S -transferase activity was determined in 176,000 g supernatant fraction of ovaries, adrenals, and liver and in the serum of virgin, pregnant, lactating, and post-lactating female rats. A 90% increase in glutathione S -transferase specific activity was observed in the ovaries of pregnant rats whereas activity in the other tissues was not affected by pregnancy. The increase in ovarian glutathione S -transferase activity was not a function of the estrous cycle of the rat. The activity reached a maximum in midpregnancy and then remained constant until late pregnancy. The specific content of cytochromes P-450 and b 5 in microsomes prepared from rat ovaries was induced 3.7- to 4.7-fold during mid- and late pregnancy. No concomitant changes occurred in mitochondrial cytochrome P-450 concentrations. Microsomal aryl hydrocarbon hydroxylase and epoxide hydrase activities of ovaries from pregnant rats were not affected.

The effect of a placental mammotrophin and cortisol on the ultrastructure of rat mammary gland in organ culture.

Mammary gland explants obtained from 13 days pregnant rats were cultured for 3, 6 and 9 days in the presence of serum of either virgin rats or 13 days pregnant rats. Insulin was added alone or in combination with cortisol. The way these different forms of treatment affected the ultrastructure was studied. With virgin rat serum plus insulin alone the mammary gland regressed to a lower state of development comparable to that of the virgin rat in vivo. When virgin rat serum was replaced by 13 days pregnant rat serum--which contains rat chorionic mammotrophin--the initial development was maintained, but synthesis of secretory products ceased. When cortisol was added to the virgin rat serum plus insulin, cortisol maintained the differentiated state originally present. Even some secretory activity was induced, but at a minimal level. With cortisol added to the pregnant rat serum plus insulin, the explants were stimulated to synthetize the extrude secretion products.

Release of Mammotrophic Activity by the Rat Placenta

Release of mammotrophic activity into the culture medium by placenta fragments in vitro could be demonstrated with the aid of an organ culture of mammary explants. Maintenance of the placenta fragments was optimal with less than 8 fragments per ml medium. Release of activity was already high after one hour of culture, but continued for at least four days if the medium was favourable. To obtain this latter effect the presence of salts and glucose in the medium was sufficient, amino acids, serum and insulin being of no or minor importance. The activity observed can be explained partly by a slow release of hormone(s) already present in the fragments. Activity was released by freezing and thawing the placenta fragments. If placenta fragments were kept at + 4°C or + 20°C release of activity continued for several days. Moreover, the release at + 37°C was not inhibited by cycloheximide. On the other hand intact fragments released more activity than frozen fragments, even after one day of culture, while the release continued at a high level for several days in intact fragments and stopped after one day in frozen fragments. The placenta fragments had an active metabolism with glucose consumption and lactate production. When virgin rat serum was incubated with placenta fragments in vitro, its mammotrophic activity became equal to that of pregnant rat serum. Pregnant rat serum seemed to inhibit the release of mammotrophic activity from placenta fragments in some experiments, but the assay method does not permit a definite conclusion. The medium activated by incubation with placenta fragments showed synergism with cortisol as regards secretion, and with progesterone as regards proliferation. An effect of cortisol, progesterone, oestradiol, oestrone or oestriol on the release of mammotrophic activity from the placenta fragments could not be demonstrated. Rat placental giant cells seem to be involved in the production of rat chorionic mammotrophin (rCM). Activity appeared in the maternal serum at day 8-9 of pregnancy when mainly the trophoblastic giant cells adjacent to the foetal membranes are in contact with the maternal circulation. Those placenta fragments which on organ culture released activity into the culture medium contained giant cells. Undifferentiated giant cells and/or basal zone cells also may be capable of producing rCM. The giant cells could be maintained viable in vitro. They incorporated thymidine, uridine and leucine. The giant cells did not stain with acidophilic staining for pituitary hormones. Electron microscopy suggested that if the giant cells do in fact produce rCM, the Golgi apparatus may not be involved in its secretion. Secretion granules could not be demonstrated in the giant cells. Sephadex G-100 gel chromatography fractions were obtained from pregnant rat serum, placenta extract and placenta culture medium. The fractions were tested for the presence of mammotrophic activity. In 13 and 19 days pregnant rat serum the fraction with molecular weight of approximately 35 000 showed high activity. In placenta extracts obtained from 13 and 19 days pregnant rats activity was present in the fraction with molecular weight of approximately 18 000, but other fractions with lower and higher molecular weight also possessed high mammotrophic activity. Media collected from a placenta culture similarly contained multiple fractions with activity.

The importance of insulin, cortisol, progesterone and prolactin for the mammotrophic effect of pregnant rat serum.

ABSTRACT In order to explain the increased mammotrophic activity of pregnant rat serum, some hormones known to have an in vitro effect on the mammary gland were added to non-pregnant rat serum and their effect compared with that of pregnant rat serum. The addition of insulin to the culture medium increased the mammotrophic effect of pregnant rat serum, but a difference between non-pregnant and pregnant rat serum could be demonstrated either in the presence or absence of a high concentration of insulin. While pregnant rat serum had a mammogenic (i. e. growth-promoting) effect, cortisol had no such effect and antagonized the mitotic activity induced by the pregnant serum. Pregnant rat serum was active at such low concentrations, that the progesterone content of pregnant rat serum could not explain the mammogenic activity of the latter. Moreover, the combination of progesterone with non-pregnant rat serum did not produce other changes produced by pregnant rat serum alone such as cytoplasmic opalescence and the localized development of vacuolization and secretion. Prolactin with non-pregnant rat serum produced a mammotrophic effect which was in all respects similar to that of pregnant rat serum. An effect could be demonstrated at 0.08 μg prolactin/ml medium. However, again pregnant rat serum was active at such low concentrations that the prolactin content of pregnant rat serum could not explain the mammotrophic effect of pregnant rat serum. Moreover, the effect of pregnant rat serum could not be blocked with rabbit anti-rat prolactin serum, but the anti-serum blocked the effect of rat prolactin added to non-pregnant rat serum. The results exclude the possibility that insulin, cortisol, progesterone or prolactin can account for the mammotrophic activity in pregnant rat serum. They provide indirect evidence for the importance of rat chorionic mammotrophin.

The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy.

Pregnant rats, hypophysectomized-hysterectomized on day 12 (day 1=insemination), secreted progesterone (P) at about 50% of the day 12 level for 3-5 days. Daily treatment with estradiol 100 microng (E100) but not with E25 or E50, from day 12 to day 20, of such rats, restored P secretion until day 16 to that of intact pregnant rats; on day 17 a drastic permanent fall occurred. E100 had no effect after ovariectomy, and did not change the metabolic clearance rate of P. This E100 effect was absent in decidual tissue (DT)-bearing, or hysterectomized pseudopregnant (PSP) rats, and in pregnant ones before day 11. When pregnant rats were hypophysectomized-hysterectomized on day 10, and were treated with rat placental luteotrophin (rPL) (in the form of day 12 pregnant rat serum: "PRS-12") on days 10 and 11, however, E100 increased P secretion above that found with either PRS-12 or E100 alone. DT-bearing PSP rats, similarly operated on on day 12 and treated with PRS-12 on day 12, responded in the same way to E100. In these rats also the E100 effect lasted for four days. In the day 12 hypophysectomized-hysterectomized pregnant rat, the E100 effect could not be prolonged by single treatments with PRS-12 on either days 13, 14 or 16, but when PRS-12 was given daily from day 12 to 19, P was secreted until day 20 only slightly below the day 12 level; this treatment plus E100 raised P secretion, prolonged it to day 18, and led to a marked fall by day 20 similar to that of the intact pregnant rat at term. The marked increase in P secretion between days 12 and 15 of normal pregnancy may thus be a response to intraluteal estrogen; the pattern of P secretion from day 12 to term may reflect the effects of both estrogen and rPL. rPL probably induces this effect by generating luteal estrogen and LH receptors. The placenta may also secrete an LH-like hormone (rCG?) which, through the LH receptors, could stimulate intraluteal estrogen production.

MAMMOTROPHIC ACTIVITY IN RAT SERUM DURING THE OESTROUS CYCLE, PREGNANCY AND LACTATION

The mammotrophic activity of various serum samples was assessed on the mammary gland of pregnant rats, in organ culture. Insulin was added to the medium. Serum samples from virgin rats showed little activity, and variation in mammotrophic activity during the oestrous cycle was slight. During pregnancy a significant increase in proliferative activity was not seen during the first week, but around day 8-9 mammotrophic activity increased sharply. The activity showed maxima around days 12 and 19. A decrease in the activity between these maxima was not consistently observed. Mammotrophic activity was still present in sera collected during parturition, and 30 min after parturition. It had disappeared completely within 24 h. The mammotrophic factor detectable by organ culture in the serum of pregnant rats could be rat chorionic mammotrophin. Activity in the serum on day 0 and 1 of lactation was comparable to that of virgin rats, but some activity appeared on day 2. High actvities were foound frequently around day 4 to 6 of lactation. On other days the activity showed fluctuations without a definite pattern. Litter size was of minor importance but the combination of a larger litter size and fasting-overnight seemed to suppress the presence of mammotrophic activity in the serum. Nursing was important: after weaning the activity has disappeared, while renewed nursing after weaning resulted in the appearance of high levels of activity. The mitotic activity obtained with lactating rat serum in the culture was suppressed by addition of rabbit anti-rat prolactin serum to the medium. This suggests that the main mammotrophic factor detectable by organ culture in the serum of lactating rats, is prolactin.

Characterization of rat chorionic mammotropin.

A placental lactogen or chorionic mammotropin (rCM) has been identified in the serum of pregnant rats by radioreceptor assay (RRA). Two peaks of activity were found, the first between days 11-13 and the second between days 17-21 of pregnancy. Gel filtration of day 12 serum or placental extracts revealed two peaks of lactogenic activity, the first eluting ahead of and the second appearing immediately after 125I-hPRL (human prolactin). On the other hand, when serum or placental extracts from day 17-21 pregnant rats were fractionated in the same manner, only a single peak of lactogenic activity was eluted after 125I-hPROL. The placental concentration of rCM increased from 11 mug/g wet weight at day 12 to 74 mug/g at days 14-15, after which the concentration declined to 29 mug/g on day 21. Electrophoresis of a fraction from G-100 column of a day 12 0r day 20 placental extract or serum with a V-e/V-o ratio of 2.08 revealed a single peak of lactogenic activity by RRA with an Rf equal to 0.43. Large MW rCM (V-e/V-o ratio of 1.35) from serum had an Rf equal to 0.52 whereas the large MW species from placental extracts had a mobility similar to that of small MW fraction. The half-time disappearance rate of serum rCM on day 12 is 19.5 min compared to 1.2 min on days 17-21 of pregnancy.

Trypsin inhibition in serum and urine of neonatal and lactating rats and in rat colostrum and milk

1. 1. Trypsin inhibiting capacity (TIC) and total protein were measured in serum, urine, colostrum and milk of lactating rats and in serum and urine of neonatal, juvenile, adult and pregnant rats. A spectrophotometric method was used with benzoyl- dl-arginine- pnitroanilide (BAPNA) as a substrate. Casein agarose gel electrophoresis was used for electrophoretic localization of tryptic inhibition. 2. 2. TIC is very low in rat colostrum but increases within one day to a constant level, while the protein concentration is very high in colostrum and decreases within one day. 3. 3. Total protein concentration in serum from lactating rats is lower than in adult rats while TIC shows adult levels. TIC and protein in serum increase in parallel during the neonatal period. 4. 4. The electrophoretic properties are identical for TIC in milk and serum. Low TIC was registered in urine of late pregnant and lactating rats. 5. 5. The results are compared with previous findings on neonatal piglets. It seems that a correlation exists between TIC in colostrum/milk and the ability of the intestinal epithelium of the neonate to absorb colostral proteins into blood. 6. 6. The results indicate that the trypsin inhibitors of rat milk are transported from serum to milk via the mammary gland of the lactating rat. No passage of inhibitors into the urine of the neonatal rat was found.

Estradiol binding to rat corpora lutea during pregnancy.

The corpus luteum of the pregnant rat bound estradiol in a manner similar to that observed in classical target tissues. A luteal cell cytosol component exhibited specific, high affinity and low capacity binding for estradiol with an equilibrium dissociation constant of 0.22 nM. The cytosol es-tradiol binding component appeared to have charac-teristics of an intracellular receptor distinct from the estradiol binding protein found in serum of pregnant rats. In the presence of \3H]estradiol a cytosol component, but not the serum protein, bound to DNA; in the presence of diethylstilbestrol, the binding of \3H]estradiol to cytosol, but not to serum, was inhibited. The content of estradiol receptor complex in luteal cell nuclei changed during preg-nancy as determined by nuclear exchange assay. Estradiol receptor complex in luteal cell nuclei increased between days 3–12, reached a maximum in the second half of pregnancy and decreased on day 22 of pregnancy. Similar changes in the amount of \3H]estradiol specific...

Liaison du sulfate d'oestrone par les proteines seriques du rat au cours de son developpement

The binding of estrone sulfate by rat serum has been studied comparatively, with an equilibrium dialysis technique, in the embryo and the pregnant rat at the 19th day of pregnancy, as well as during post-natal development. The sera of the embryo and the new-born rat almost bind no estrone sulfate; in contrast, this binding is high in the 21-days-old and in the adult animal. A significant reduction of the affinity index is shown in the pregnant rat serum. The binding of testosterone sulfate is 3–4 times lower than that of estrone sulfate. There is no fixation of estradiol glucuronide in any of the tested sera. A positive correlation has been found between the albumin level of the different sera and their affinity index for the estrone sulfate. The albumin purified from embryo sera and the adult serum albumin have similar high affinity indexes for estrone sulfate. The binding curves for the estrogen conjugate with these two purified proteins and the whole sera of normal and pregnant adult rats are not appreciably different. In all cases, two kinds of binding sites are demonstrated: relatively strong binding sites, with an association constant of about 1.5·10 5 M −1 at 25°C and numerous low affinity binding sites, with an association constant of about 3·10 3 M −1 at 25 °C. There are probably two strong binding sites per serum albumin molecule ( n 1 ∼ 2). From these results, it is concluded that albumin is the main protein that binds estrone sulfate in the rat serum. The absence of binding in the embryo is due to the low levels of this protein and not to a qualitatively different behavior of the foetal serum albumin. The lack of binding of estrone sulfate in the embryo is in contrast with the high affinity of the embryo sera for estrone and estradiol-17β.