Chromium-51 ethylenediaminetetraacetate for estimation of globerular filtration rate.

Renal clearances of inulin and chromium-51 ethylenediaminetetraacetate were compared in sheep. In 41 clearance periods from five animals the mean clearance ratio of the chelate to inulin was 0.95 +/- 0.03 (S.D.). Part of the difference between the two sets of results may have been due to binding of the metal chelate by plasma protein since 1.5 to 2 percent of chromium-51 ethylenediaminetetraacetate in plasma could not be removed by dialysis.

Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum.

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.

Search for a progesterone-binding protein in secretory granules of the ovine corpus luteum.

The hypothesis that electron-dense granules present in the corpus luteum contain progesterone in a protein-bound form was examined using differential and sucrose gradient centrifugation. Ovine luteal tissue was fractionated by differential centrifugation at 1,000 X g (P1 pellet), 10,000 X g (P2), and 82,000 X g (P3; supernatant S3). Samples of P2, P3, and S3 were further fractionated o 20-40% (P2 and P3) or 5-25% (S3) sucrose gradients and examined for progesterone-binding activity by measuring the progesterone content and/or the specific binding of [3H]progesterone of sucrose gradient samples. In addition, saturation binding assays were performed with steroid-free samples of P2 and S3. Saturable binding of progesterone was not found in P2, the fraction containing electron-dense granules. In S3, two progesterone-binding proteins with sedimentation rates of 3.2S and 8.6S and an affinity of 7.1 X 10(5) M-1 for progesterone were detected. The sedimentation behavior of these proteins was distinct from that of ovine plasma transcortin, a 4S protein. The view that a binding protein is released into the interstitial fluid during the exocytosis of granules was examined by measuring the progesterone-binding activity of protein released by slices of corpus luteum in vitro. No binding activity was found. The results of this investigation do not support the hypothesis that putative progesterone-secreting granules observed in luteal tissue contain a binding protein

Oxytocin, oxytocin-associated neurophysin, and prostaglandin F2 alpha concentrations in the utero-ovarian vein of pregnant and nonpregnant sheep.

Oxytocin, oxytocin-associated neurophysin (neurophysin), prostaglandin F2 alpha (PGF2 alpha), and progesterone concentrations were measured in the utero-ovarian vein (UOV) of sheep during the estrous cycle and early pregnancy. On days 13-16 of the cycle, large pulses of PGF2 alpha, oxytocin, and neurophysin were measured in samples collected at hourly intervals from the UOV draining a corpus luteum (UOV/CL). Most of the PGF2 alpha pulses (96.5%) coincided with a pulse of both oxytocin and neurophysin, whereas only 55.6% of oxytocin pulses coincided with a pulse of PGF2 alpha. Therefore, during luteolysis in sheep, uterine PGF2 alpha release is closely associated with ovarian oxytocin release, and oxytocin release is unlikely to be dependent upon a uterine PGF2 alpha stimulus. During frequent sampling, coincident oxytocin pulses were measured in 1) both UOVs when a CL was present in both ovaries and 2) the jugular vein, carotid artery, and UOV/CL, with a significantly higher oxytocin pulse concentration occurring in jugular venous compared with carotid arterial plasma. Pituitary and luteal release of oxytocin may, therefore, occur simultaneously and be controlled by a circulating factor in sheep. Compared to days 13-16 of the cycle, significantly (P less than 0.001) fewer pulses of PGF2 alpha, which were significantly (P less than 0.001) smaller in amplitude, were measured in UOV samples collected frequently during early pregnancy. The frequency of oxytocin pulses observed in the UOV/CL of pregnant sheep was not significantly (P greater than 0.1) different from that observed in cyclic ewes, although most (86.4%) oxytocin pulses occurred in the absence of a PGF2 alpha pulse. In contrast, when a pulse of PGF2 alpha was observed in the UOV/CL of pregnant ewes, it usually coincided with a pulse of oxytocin. The suppression of uterine PGF2 alpha release during early pregnancy is not considered to result from a lack of stimulation by oxytocin.

Purification and characterization of a fetal somatomedin from the sheep: similarity to insulin-like growth factor II.

A high efficiency procedure for the purification to homogeneity of an ovine fetal somatomedin is described. Fetal sheep serum was used as the source material, and activity was followed throughout purification by an insulin-like growth factor (IGF) II RRA. IGF-II-like activity was initially enriched through binding at acid pH to a column of SP-Sephadex C-25 and elution with a neutral pH high-salt buffer. Further chromatography on SP-Sephadex resulted in a preparation containing less than 0.1% of the original serum protein content, but retaining much of the IGF-II-like activity. This enriched fetal IGF preparation was then purified to homogeneity using reverse phase HPLC. However, chromatography on an HPLC gel filtration column was found to be essential to ensure the stability of the purified peptide during storage, although this procedure did not result in any apparent increase in purification. The final yield of purified ovine fetal IGF was 80 micrograms from 400 ml fetal sheep serum. Only one polypeptide chain was detected during amino-terminal sequencing of the fetal somatomedin, and the preparation gave a single band on both gel electrophoresis and isoelectric focusing, indicating that the peptide was essentially pure. The sequence (eight amino acid residues) was identical to the equivalent sequence in IGF-II from human, rat, and bovine sources. In additional, amino acid analysis of the ovine fetal IGF showed close similarity to the amino acid content of IGF-II from other species. The mol wt of the purified peptide, estimated by HPLC gel filtration, was approximately 7000, close to that of previously purified somatomedins, and the isoelectric point, obtained by chromatofocusing, was around pH 6.8. Thus, the purified ovine fetal somatomedin appears to be similar to IGF-II from other species, and may be the ovine homolog of human IGF-II.

Fetal sheep serum contains a high molecular weight insulin-like growth factor (IGF) binding protein that is acid stable and specific for IGF-II.

We have used radiolabeled ovine insulin-like growth factor II (oIGF-II) and human IGF-I (hIGF-I) to investigate the nature of the IGF binding proteins in fetal sheep serum. Incubation of fetal sheep serum with [125I]oIGF-II, followed by chromatography on Fractogel TSK HW55(S), revealed the presence of two major binding protein species, a lower molecular weight binding protein (apparent mol wt approximately 60,000) and a much higher molecular weight binding protein (apparent mol wt approximately 500,000). Only the lower molecular weight binding protein complex was seen in serum from adult nonpregnant sheep. The lower molecular weight binding protein in fetal and adult sheep serum bound both [125I]oIGF-II and [125I]hIGF-I, both of which could be displaced by unlabeled oIGF-II or hIGF-I. However, the very high molecular weight binding protein bound only [125I]oIGF-II, and this could only be displaced by unlabeled oIGF-II. The very high molecular weight binding protein appears to bind approximately 40% of the endogenous oIGF-II. We have purified the very high molecular weight binding protein from fetal sheep serum using anion exchange, Concanavalin A Sepharose, and hydrophobic interaction chromatography. The purified binding protein preparation did not contain any lower molecular weight binding protein and did not bind [125I]hIGF-I. In addition, this binding protein was stable at pH 3.2 for 1 h. Thus, fetal sheep serum contains a very high molecular weight IGF binding glycoprotein that is acid stable and specific for IGF-II.

Control of oxytocin secretion by ovine corpora lutea: effects of arachidonic acid, phospholipases, and prostaglandins.

The involvement of arachidonic acid and arachidonic acid metabolites in the control of oxytocin secretion by ovine corpus luteum was investigated, using slices of luteal tissue incubated in vitro. Oxytocin was secreted at steady rates by luteal slices, during 60-min incubations (315.0 +/- 45.3 pg/mg.h). The secretion of oxytocin was stimulated by arachidonic acid, phospholipase A2 (PLA2), and phospholipase C (PLC) in a dose-dependent manner. The highest doses of arachidonic acid, PLA2, and PLC used stimulated oxytocin secretion by 145.8 +/- 23.0% (P less than 0.01; n = 6), 331.5 +/- 42.4% (P less than 0.02; n = 4), and 955.5 +/- 278.6% (P less than 0.01; n = 4), respectively. Oxytocin secretion by luteal slices was not affected by either prostaglandin F2 alpha (PGF2 alpha) or PGE2 over a concentration range from 3-3000 nM. Furthermore, inhibitors of the cyclo-oxygenase pathway of arachidonic acid metabolism did not consistently affect arachidonic acid and PLA2-stimulated oxytocin secretion. Nordihydroguaiaretic acid, which inhibits 5-lipoxygenase, however, totally abolished arachidonic acid- and reduced PLA2-stimulated oxytocin secretion. The presence of CoCl2 in the incubation medium also significantly reduced basal and PLA2- and PLC-stimulated oxytocin secretion [P less than 0.05 (n = 5), P less than 0.05 (n = 5), and P less than 0.01 (n = 6), respectively]. We have shown that oxytocin secretion from slices of ovine corpus luteum incubated in vitro is stimulated by exogenous and endogenously released arachidonic acid. The data show that PGF2 alpha and PGE2 do not have a role in luteal oxytocin secretion in vitro and PG formation does not appear to be involved in the stimulation of oxytocin secretion elicited by arachidonic acid or PLA2. Arachidonic acid may have its effect via the lipoxygenase pathway.

Release of E prostaglandins into the cerebrospinal fluid and its inhibition by melatonin after cervical stimulation in the rabbit.

The release of the E type of prostaglandin (PGE) into the cerebrospinal fluid of mature female rabbits was studied using an indwelling catheter in the cisterna magna. The cervix of the rabbit was stimulated 5 min after the central injection of either vehicle or melatonin solution. PGE was measured by RIA, and in unstimulated animals, the level of PGE remained steady at about 4 ng/ml. After stimulation of the cervix of control rabbits, PGE levels rose by 31 ng/ml 40--70 min after the initiation of stimulation in those animals which subsequently ovulated. PGE levels rose by only 15 ng/ml 70--115 min after stimulation in those rabbits which did not ovulate. In the melatonin-treated rabbits, PGE levels remained close to those in unstimulated controls, and ovulation did not occur. Thus, stimulation of the cervix of the rabbit produces a rise in PGE levels in cerebrospinal fluid. This endogenous PGE surge may, in turn, cause the release of LHRH and LH and induce ovulation if levels exceed a critical threshold. A weaker stimulus leads to only a smaller surge of PGE, insufficient to cause ovulation. Melatonin blocked the stimulation-induced rise in cerebrospinal fluid PGE levels and ovulation, and appears to exert its antigonadotropic activity in the rabbit by the inhibition of central PGE synthesis.

Prostaglandin E2 administered to fetal sheep increases the plasma concentration of adrenocorticotropin (ACTH) and the proportion of ACTH in low molecular weight forms.

This study investigated the effects of repeated short term (2-h) intrafetal infusions of prostaglandin E2 (PGE2) on ACTH and cortisol release in fetal sheep during late gestation (119-144 days). We compared the effects of administration of PGE2 (2 micrograms/min) into the fetal carotid artery or jugular vein. PGE2 infusion significantly (P < 0.001) increased fetal plasma immunoreactive (ir-) ACTH and cortisol concentrations regardless of the vessel used for administration. Saline infusion did not alter the concentrations of ir-ACTH or cortisol for the duration of the experiment. To compare the responses of fetal ir-ACTH and cortisol to repeated intracarotid infusions of PGE2, the hormone data were grouped into five gestational age ranges (119-125, 126-130, 131-135, 136-140, and 141-145 days). Fetal ir-ACTH was stimulated by PGE2 infusion at all gestational ages studied; the greatest response was achieved at the earliest gestational age range, 119-125 days. PGE2 infusion preferentially stimulated the release of low mol wt ACTH [ACTH-(1-39); 60 min from the start of infusion] at all gestational ages (P < 0.01), but basal low mol wt ACTH did not increase with gestational age until after 140 days. Cortisol concentrations were increased within 30 min of infusion at all gestational ages studied. These results suggest that PGE2 may play a role in maintaining elevated ir-ACTH concentrations in the face of high levels of cortisol in fetal sheep before parturition.

Chronic administration of low doses of adrenocorticotropin to hypophysectomized fetal sheep leads to normal term labor.

Parturition in the sheep is preceded by an increase in the plasma concentration of fetal ACTH and an increase in the plasma cortisol concentration. The role and importance of the increase in fetal ACTH in stimulating fetal glucocorticoid synthesis and the subsequent onset of labor require closer examination, as it has been demonstrated that the fetal adrenal becomes more responsive to ACTH in late gestation. This study sets out to determine whether the increase in plasma ACTH in the late gestation fetal sheep is essential for maturation of the fetal adrenal gland and normal delivery. Fetal sheep were either hypophysectomized (HX) and cannulated or cannulated only (intact) at 125 days gestation. Immediately after surgery, HX fetuses were infused with a constant dose of ACTH-(1-24) (ACTH/HX; 100 ng/h.kg, i.v.) or saline (SAL/HX) until uterine electromyography indicated the onset of labor or 161 days gestation was reached (term = 147 +/- 2.6 days). The mean gestational age at labor of the ACTH/HX group was 147 +/- 2.9 days, whereas none of the animals in the SAL/HX entered labor, and they were killed at 161 days gestation. The concentration of ACTH in both ACTH/HX and SAL/HX fetal plasma was less than 2.2 pg/ml throughout the study. The concentration of cortisol in ACTH/HX fetuses mimicked that in intact fetuses in late gestation, reaching 80 ng/ml at term. The concentration of cortisol in SAL/HX fetuses remained less than 5 ng/ml. This study supports the hypothesis that the ovine fetal adrenal becomes increasingly responsive to ACTH in late gestation and indicates that ACTH may only be permissive in the activation of adrenal function. In intact fetal sheep there may be endogenous inhibition of the fetal adrenal, requiring relatively high plasma concentrations of ACTH [100-250 pg/ml ACTH-(1-39)] in late gestation.

Diurnal rhythms in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation.

We have measured plasma melatonin (MT) concentrations in the pregnant ewe and fetal sheep during 24-h periods between 114 and 142 days gestation. There was a clear diurnal rhythm in the plasma MT concentrations in both the ewe and fetus from 114 days gestation. Blood samples were also collected from the pregnant ewe and fetus during the day every 2-3 days from 112 days gestation to term. There was no gestational age trend in maternal or fetal day time plasma MT concentrations during late pregnancy. To establish whether there was transplacental transfer of MT, pregnant ewes were injected with [3H]MT, and total radioactivity (disintegrations per min) was measured in maternal and fetal arterial plasma and in amniotic fluid collected before and for 1 h after the [3H]MT injection. Two minutes after [3H]MT injection, radioactivity was detected in both maternal and fetal sheep plasma. Extraction of fetal plasma with chloroform indicated that [3H]MT accounted for 48.0 +/- 7.2 (SE) % of total radioactivity at 2 min after the injection. In one pregnant ewe infused with unlabeled MT (0.3 microgram/ml saline.min for 20 min) maternal and fetal plasma MT concentrations increased within 6 min after the start of the MT infusion. We conclude that there is a diurnal rhythm in the plasma concentrations of MT in the fetal lamb and pregnant ewe between 114 and 142 days gestation, and that MT crosses the ovine placenta from the maternal to the fetal circulation. Therefore, the MT present in the fetal sheep circulation may be solely of maternal origin or it may be derived from both fetal and maternal sources.

Continuous intrafetal infusion of prostaglandin E2 prematurely activates the hypothalamo-pituitary-adrenal axis and induces parturition in sheep.

This study has investigated the effect of continuous intrafetal infusion of PGE2 or saline on hormone concentrations and the length of gestation in sheep. Fetal and maternal vascular catheters were surgically implanted at 112.3 +/- 1.3 days (n = 10), and the infusions were started at 121 +/- 1.2 days of gestation (term = 147). Fetuses were infused with either PGE2 (n = 5; 2 micrograms/min for 48 h and then increased to 4 micrograms/min for the remainder of the experiment) or the vehicle solution (n = 5; sterile isotonic saline) via the fetal carotid artery. In the PGE2-infused group, fetal and maternal plasma PGE2 concentrations increased (P < 0.001) after the change to the higher dose rate (4 micrograms/min) and remained elevated, fetal plasma immunoreactive ACTH (ir-ACTH) concentrations dramatically increased after the start of the infusion being maximal at 11 h before decreasing to match concentrations exhibited by the saline-infused group. Fetal plasma cortisol concentrations increased after the start of the PGE2 infusion (P = 0.05) and increased further after the change to the higher dose rate of 4 micrograms/min (P < 0.001). Concentrations of PGE2, ir-ACTH, and cortisol in the saline-infused group did not change until labor. Plasma concentrations of PGE2 (P < 0.001) and ir-ACTH (P < 0.005) increased on the day of labor in both treatment groups, and fetal cortisol concentrations increased (P < 0.001) in both groups in the last few days before labor. The proportion of low molecular weight ir-ACTH in the plasma of PGE2-infused fetuses was significantly higher than that of saline-infused fetuses (P < 0.001) during the first 15 days of infusion. In the saline-infused group, the proportion of low molecular weight ir-ACTH increased in the last few days before labour (P = 0.001), whereas no change was seen in PGE2-infused fetuses at this time. Maternal plasma progesterone concentrations decreased in both groups in the last few days before labor (P < 0.001). Fetuses infused with PGE2 delivered at 138.4 +/- 2.1 days, whereas control fetuses infused with saline delivered at 148.2 +/- 0.5 days (P < 0.001). The spontaneous increase in PGE2 preceding normal labor may thus play an important role in activation and maturation of the hypothalamic-pituitary-adrenal axis in fetal sheep.

Hypophysectomy of the fetal lamb leads to a fall in the plasma concentration of insulin-like growth factor I (IGF-I), but not IGF-II.

The role of the pituitary gland in the regulation of the plasma concentrations of insulin-like growth factors (IGFs) in the late gestation sheep fetus has been examined. Singleton sheep fetuses were either hypophysectomized or sham-operated between days 110-120 of gestation. Blood samples were then collected via carotid cannulae at least three times weekly for the remainder of gestation. In some hypophysectomized fetuses T4 was administered (100 g/day) to overcome the hypothyroidism caused by hypophysectomy. Blood samples were also obtained from lambs during the perinatal period, neonatal lambs within 1-10 days after birth, and pregnant and nonpregnant adult ewes. All plasma samples were subjected to Sephadex G-50 gel filtration under acidic conditions (pH 2.3) to eliminate IGF-binding protein activity. The fractions containing the free IGF peptides were collected and assayed for IGF-I by heterologous RIA, and IGF-II by a homologous RRA. Plasma concentrations of IGF-I and IGF-II did not change with advancing gestational age in any fetal group and were not affected by the prolonged gestation that results from hypophysectomy. The mean plasma IGF-I and IGF-II concentrations in the sham fetuses were 112 +/- 8 and 1340 +/- 112 ng/ml, respectively. Hypophysectomy without thyroid hormone replacement resulted in a significant decrease in plasma IGF-I concentrations to 50 +/- 5 ng/ml, whereas IGF-II concentrations were not affected (1096 +/- 124 ng/ml). IGF-I concentrations in the hypophysectomized fetuses that received T4 were significantly increased (67 +/- 6.0 ng/ml) compared to those in the hypophysectomized fetuses that did not receive T4. The IGF-II concentrations in the hypophysectomized fetuses that received T4 were similar to those in the sham-operated fetuses (1120 +/- 112 ng/ml). At term IGF-I concentrations were increased (180 +/- 21 ng/ml) and IGF-II concentrations were decreased (264 +/- 25 ng/ml) compared to fetal values. Plasma IGF-I concentrations in the prepubertal lamb were similar to the fetal values. Pregnancy in the adult ewe was associated with a significant increase in IGF-II, but had no effect on IGF-I plasma concentrations. These data show that circulating IGF-I concentrations in the fetal lamb are under some pituitary and thyroid control, whereas IGF-II concentrations are independently of pituitary or thyroid status. We confirm, using a homologous assay, that fetal IGF-II concentrations are high and then decrease at term. These data also support the concept that a pregnancy-related factor may regulate plasma IGF-II concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor receptor in fetal lamb liver: characterization and developmental changes.

Multiplication-stimulating activity (MSA), an insulin-like growth factor (IGF) (rat IGF II), binds to extracts of many tissues from fetal lambs. We now report the presence of high concentrations of a glycoprotein receptor with a high affinity for MSA-II in microsomes prepared from fetal lamb liver. The binding of radiolabeled MSA-II is inhibited by IGF but not by insulin, human, and ovine GH, ovine PRL, ovine placental lactogen, and mouse epidermal growth factor. The relative potencies of human IGF II, human IGF I, and MSA-II in competing with [125I]MSA-II for binding to this receptor are 100:17:3.5 by weight. Binding is pH, time, and temperature dependent. Gel permeation chromatography of the Triton X-100 soluble receptor indicates a hydrodynamic radius of 6.8 nm. Specific binding increases from mid- to late gestation and is associated with changes in both the affinity and concentration of receptors. Receptor concentration increases from 7.95 +/- 3.94 pmol/mg (mean +/- SE) at 78 days gestation to 15.8 +/- 4.3 pmol/mg at 134-140 days (P less than 0.05), whereas receptor affinity decreases from 1.14 +/- 0.34 X 10(9) liter/mol to 0.63 +/- 0.14 X 10(9) liter/mol over this period (P less than 0.05). The presence of very high concentrations of an IGF receptor in fetal lamb liver suggest that this organ may be a major target for IGF action in fetal life. The increase with advancing gestational age of the concentration of IGF receptors which have preferential specificity for IGF II may function to increase the responsiveness of the fetal lamb liver to IGF II stimulation and so compensate for the decline in plasma concentrations of this growth factor which occur near birth.

Effect of hypophysectomy with and without thyroxine replacement on growth and circulating concentrations of insulin-like growth factors I and II in the fetal lamb.

The role of the pituitary gland in the regulation of skeletal growth and plasma insulin-like growth factor (IGF)-I and IGF-II concentrations in the late gestation sheep fetus has been studied. Singleton fetuses were either hypophysectomized (n = 14) or sham operated (n = 8) between days 110 and 125. Fetal and maternal blood samples were collected three times weekly through the remainder of gestation. In some hypophysectomized fetuses (HXT4 group, n = 4), T4 was administered (100 micrograms L-T4/day) to overcome the hypothyroidism caused by hypophysectomy. The other hypophysectomized fetuses received no replacement therapy (HXNR group, n = 10). Six HXNR fetuses were allowed to remain in utero post term and were killed at day 163 of gestation. All other animals were killed at day 147. All values are group means +/- SE. Hypophysectomized fetuses had significantly shorter limbs and long bones and delayed osseous maturation at term compared to sham controls. Plasma free T4 concentrations in HXT4 fetuses were not significantly different from those measured in sham fetuses (P greater than 0.05). Bone maturation at term was normal in HXT4 fetuses although there was no improvement in limb or bone elongation. Retention of hypophysectomized fetuses in utero until 16 days past term yielded fetuses which were heavier than controls but whose limb and bone lengths were no greater than hypophysectomized fetuses killed at term. Osseous maturation was appropriate for term in five of the six postterm hypophysectomized fetuses. The plasma IGF-I and IGF-II concentrations were not significantly affected by hypophysectomy, hypophysectomy with T4 replacement, gestational age, or prolonged gestation. The plasma IGF-I concentrations in the sham, HXNR, and HXT4 fetuses were 35.4 +/- 6.6, 28.2 +/- 3.0, and 34.4 +/- 1.7 ngeq human (h)IGF-I/ml, and the IGF-II concentrations were 656.3 +/- 59.2, 635.3 +/- 56.3, and 645.5 +/- 71.9 ngeq hIGF-II/ml, respectively, and remained within these ranges throughout the experiment. Fetal IGF-I concentrations were significantly lower than mean maternal IGF-I concentrations (88.0 +/- 6.8 ngeq hIGF-I/ml, P less than 0.05), and fetal IGF-II concentrations were significantly higher than mean maternal IGF-II concentrations (362.4 +/- 24.0 ngeq hIGF-II/ml, P less than 0.05). We conclude that in the late gestation fetal sheep, elongation of the appendicular skeleton is under some direct pituitary control whereas appendicular maturation exhibits some dependence on circulating T4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of adrenocorticotropin-(1-39) and precursor peptide secretory responses in the fetal sheep during the last third of gestation.

Although it is known that concentrations of immunoreactive ACTH increase during late gestation in fetal sheep plasma, the nature of the ACTH has not been well characterized. We used two-site immunoradiometric assays to separately measure high mol wt ACTH precursors (POMC and pro-ACTH) and ACTH-(1-39) in plasma of fetal sheep with chronic arterial and venous catheters. We compared the ratio of these peptides as a function of gestational age under basal conditions and in response to exogenous vasopressin and/or corticotropin-releasing hormone. Under basal conditions, the concentration of precursors was not changed throughout the last third of gestation; however, ACTH-(1-39) increased significantly approaching term. The molar ratio of precursors to ACTH-(1-39), therefore, decreased from 15.8 +/- 1.0 at 110 days to 7.9 +/- 0.6 at 140 days gestation. At all gestational ages, vasopressin and corticotropin-releasing hormone increased ACTH-(1-39) and precursors, albeit with different time courses. At 120 days gestation, arginine vasopressin plus CRH produced synergistic increases in ACTH-(1-39) and precursors, whereas the response was only additive at other ages. The present results indicate that the elevation in the resting plasma immunoreactive ACTH concentration that occurs near term is constituted by an increase in the concentration of ACTH-(1-39) relative to those of POMC and pro-ACTH, which may have further physiological significance. Also, CRH and AVP are potent stimulators of both ACTH-(1-39) and ACTH precursors.

Identification of epidermal growth factor-like activity in human male reproductive tissues and fluids.

Epidermal growth factor-like activity (EGF-LA) has been detected in human seminal plasma in concentrations of 5-150 ngeq/ml (36.4 +/- 2.1, mean +/- SEM), using a heterologous RRA with murine EGF. The samples were obtained from normal, subfertile and azoospermic men, aged 21-50 yr. No correlation was found between EGF concentration and age of donor, sperm count, sperm motility, or period of sexual abstinence before sample collection. High performance liquid chromatography of the seminal plasma resulted in a main peak of EGF-LA which eluted at 29% acetonitrile, compared to 33% for murine EGF. Microsomal membranes were prepared from several tissues from the human male reproductive tract and were tested for their ability to bind radioiodinated murine EGF. Specific EGF binding activity was detectable in testicular membrane preparations but was not detectable in membranes prepared from human prostate, seminal vesicle, epididymis, or spermatozoa. Endogenous EGF-LA was detectable in human testis, seminal vesicle, prostate, and epididymis.

Pro-opiomelanocortin mRNA levels fall in the fetal sheep pituitary before birth.

In order to clarify the corticotrophic capacity of the fetal sheep anterior pituitary in late gestation, we have measured the relative levels of messenger RNA for the ACTH precursor molecule pro-opiomelanocortin (POMC) in individual fetal sheep anterior pituitaries collected between 100 and 144 days of gestation. The mean relative POMC mRNA:poly(A)+ RNA ratio of the pituitary glands collected between 100 and 135 days (1.35 +/- 0.15) was significantly greater than the mean relative POMC mRNA:poly(A)+ RNA ratio of the pituitaries collected between 141 and 144 days (0.81 +/- 0.09). Northern blot analysis showed that a single band of RNA hybridized with the human POMC cDNA probe in adult and fetal sheep pituitaries. Our results do not contradict the hypothesis that an increase in basal ACTH concentrations after 140 days of gestation could reflect a change in the post-translational processing of POMC in the fetal sheep anterior pituitary.

Characterization of particle-associated choriomammotrophin and progesterone in ovine placentomes.

The subcellular distribution and compartmentalization of choriomammotrophin (CM) and progesterone within ovine placentomes was investigated using differential and density gradient centrifugation techniques. Approximately 67% of placental CM and 45% of progesterone was associated with subcellular particles. The 10,000 g particulate fraction contained the highest specific activity of both CM and progesterone (19.1 +/- 3.8 (S.E.M.) micrograms/mg and 71.5 +/- 9.2 pmol/mg protein respectively). This fraction was also shown to contain electron-dense granules with morphology similar to that of hormone-containing secretory granules isolated from other endocrine tissues. Particle-associated CM sedimented to a density of 1.051-1.054 g/ml in colloidal silica gradients and displayed physicochemical characteristics consistent with its storage in secretory granules. During in-vitro incubations, particle-associated CM was stable for up to 90 min, but dissociated when incubated in hypoosmotic medium. Particulate progesterone, which was also present in the CM-rich fraction and was stable for up to 90 min of incubation, was not affected by decreasing the osmolality of the incubation medium. These data suggest that ovine CM (but not progesterone) is stored within a population of secretory granules located within placentomes.

Parturition in goats: studies on the interactions between the foetus, placenta, prostaglandin F and progesterone before parturition, at term or at parturition induced prematurely by corticotrophin infusion of the foetus.

Relationships between foetal corticosteroid concentrations, utero-ovarian prostaglandin F (PGF) and maternal peripheral progesterone have been examined in detail in goats shortly before spontaneous parturition at term. Foetal corticosteroids increased during the last 13-11 days of gestation and particularly sharply during the last 3 days and even during advanced labour. About 24 h before parturition, acute releases of PGF were evident in the vein draining the pregnant uterine horn, and these corresponded closely to the time of luteal regression. Further release of PGF occured when progesterone declined to low levels, probably reflecting in the course of labour. The changes observed before premature parturition, induced by infusing ACTH into foetal goats, were similar except for the more rapid increase in foetal corticosteoid concentrations. Immature neonates born after ACTH treatment were viable, placental delivery was normal and lactogenesis occurred in the mothers indicating that the treatment promoted full expression of the critical perinatal events. The early, acute releases of PGF were ipsilateral to the ACTH-infused foetus and were luteolytic provided the corpora lutea were also on that side. Luteolysis failed or was abnormally delayed if the corpora lutea were contralateral and prolonged ACTH treatment of the foetuses in such cases caused foetal death probably because of premature failure of the placenta. Similar findings were noted if ACTH infusion of the foetus was accompanied by simultaneous progesterone treatment of the mothers in order block the induction of labour. It was suggested that placental changes occurring during foetal hypercortisolism might be caused by increased placental oestrogen synthesis and the effect of this on the foeto-maternal junction along with a stimulatory action on PG synthesis in the maternal placenta. Experimental disruption of the normal sequence of events, when labour was blocked by progesterone, proved to be lethal to the foetus if the loss of placental integrity progressed sufficiently. The chain of regulatory signals linking increased activity of the foetal adrenal with parturition thus appears to involve stimulation of oestrogen biosynthesis, PGF release from the maternal placenta and the start of physical changes at the placental junction. Provided the foetus and corpora lutea are ipsilateral, the early releases of PGF effect luteolysis and a withdrawal of progesterone from the maternal circulation. When progesterone concentrations are sufficiently low, labour is initiated and its progress reflected by further release of PGF. The control mechanisms, which also provide for the final maturation of the foetus, clearly enable a close synchronization of the various perinatal events which are essential for the transition from foetal to postnatal life.