[1] 10w Sex differences in lung and adrenal neurosympathetic development in rabbits

[1] 205w To asSElSS the possible mechanism for the increased incidence and severity of the respiratory distress syndrome (ADS) in male versus female infants, we studied neurosympathetic development in. the lung and adrenal glands in male and female fetal and newborn rabbits. Tissue levels of catecholamines, norepinephrine (NE), epinephrine (E), and dopamine (DA) were measured using a sensitive radioenzymatic assay. Beta adrenergic receptor development in the lung was studied using the tritiated radioligand dihydroalprenolol. Neui'Qsympathetic innervation of the lung was assessEtd by measuring tissue NE levels, which increased gradually from 27-day fetuses to 8-day-oid newborns. Sex differences were not significant. Lung ,a-receptor number was significantly elevated in females as compared with males at each gestational age and throughout the neonatal period (p < 0.005). Adianal gland content of E, NE, and DA incieased exponentialt"y with ad*'vancing developmental age (each p < 0.005). Adrenal E was significantly (p < 0.01) elevated in female as compared with male fetuses, as was the proportion of E. Adrenal NE and DA were similar in male and female fetuses. Sex differences were not significant in the newborn animals. The significant relative delay in adrenal medullary and lung ,a-receptor maturation may relate to the male susceptibility to neonatal morbidity and neonatal ADS. (AM.

[1] 52w Neurosympathetic innervation of the lung was assessed by measuring tissue norepinephrine (NE) levels. Results are shown in Table I. As can be seen, there was a significant increase (p < 0.005) in tissue NE levels with advancing development. There were, however, no significant sex differences in either the fetal or neonatal groups.

[2] 121w Results of fetal and neonatal adrenal CAT content measurements, in nanograms per gland, are shown in Tables II and Ill. Adrenal NE, epinephrine (E), and dopamine (DA) contents all showed significant (p < (1.005) increases with advancing age; NE and E in-<:teased exponentially over both the fetal and neonatal periods studied. When analyzed for sex differences, however. only the fetal adrenal E content values were significantly different, with females showing consistent elevations over males (p < 0.01). Female fetuses also showed a significant increase in the mean ratio of E to total adrenal amine content (p < 0.025). Although neonatal male and female animals had similarly increasing adrenal hormone contents, as shown in Table IlL there were no significant sex differences.

[3] 147w Beta adrenergic receptors (BAR) were characterized in lung tissues prior to study of developmental changes. A representative saturation curve for 3 H-DHA shm•;ing total and nonspecific binding is shown in Fig. l. Binding was saturable over a concentration range of 0 to 20 nM DHA. The binding assay was shown to be linear over the range of protein concentrations used. The Scatchard plot is shown in the inset; linear regression analysis was used to estimate binding capacity (Xintercept) and dissociation constant (negative recip-rocal of slope). The highly linear Scatchard plot suggests a single class of binding sites; this was confirmed by a Hill eoefficient of 11 = 0.914:, calculated from the data shown. 20 Agonist binding competition curves using 3 H-DHA label are shown in Fig. 2. The order of displacement potency was isoproterenol > epinephrine 2: norepinephrine; this pattern is typical of {3, receptors (Fig. 2).

[4] 76w The changes in binding capac~ty with a(\vancing developmental age are shown in Table IV. As can be seen, there is a significant (p < 0.005) increase in binding capacity over time. Moreover, females showed consistent significant (p < 0.(){)5) increases over males. These differences were preserved thr~ughout the late fetal and early neonatal period. No differ.ences were noted in dissociation constant with respet:t to developmental age or sex. Mean Kd observed was ~.35 ± 0.95 nM.

[5] 75w The results of our studies demonstrate that indices of neurosympathetic and neurohumoral •development mature at different rates in males and females. Measurements of lung NE concentrations were used to demonstrate the ontogeny of innervation. 21 Results• showed significant increases in !"JE with advancing age. There were; however, no detectable differences between males and females. Pulmonary ,B.-receptors, on the other hand, showed both significant developmental increases and significant relatively accelerated increases in female over male littermate.s.

[6] 482w The adrenal E content was higher in female than male fetuses, as was the proportion of E, suggesting an acceleration in the maturation of synthesis and perhaps in availahi1ity for secretion during adaptation to ex• trauterine life .. Epinepl:lrine, the}1nal p.r()duct of amine biosynthesis in the mawre adrenal.gland, is.secrt:ited in response to a variety of stimuli including delivery and umbilical cord damping, but separa.te .mate and female response data are not available,$ Steady-state adrenal E concentration data as presegtly. reported are not as useful as production rate or turn<>ver rate data for assessment of functional responsh•ity: thus It is known, however, that sexual differentiation of catecholaminergic systems in the rat central nervous system also is advanced in newborn females as compared with males. Tyrosine hydroxylase, the ratelimiting enzyme in NE biosynthesis, and dopa decarboxylase are more active in female than in male brains during the n.~onatal period and during adulthood. 23 Females likewise show increased uptake ofNE by cortical brain slices. 24 Females show increased resistance to the central sympatholytic effects of neonatally adminis-• tered 6-hydroK.ydopamine and accelerated recovery following neonatal administration. 25 Females also show better rearing performance than male counterparts, reflecting heightened excitability and learning ability? 6 Steady-state concentrations of amines, on the other hand, show marked regional variations throughout the central nervous system and are not necessarily reflective of the higher efficiency of the adrenergic system in females. 15 There is a great deal of evidence suggesting a role for adrenergic mechanisms in both the synthesis and release of lun1~ surfactant. Deflation stability studies in fetal rabbits p•erformed 3 hours after direct fetal injection of isoxsuprine, a potent beta sympathomimetic, showed significantly greater air retention in drugtreated than control lungsY In similar studies beta agonists have been shown to cause an increase in surface activity and an increase in phospholipid concentrations in tracheal washes. 10 Concomitant decreases in concentration of lamellar bodies in type II pneumocytes were set:n. These effects were blocked by propranolol. Similar effects were induced by muscarinic agonists and blocked by phenoxybenzamine or pro-pranoloL ll In vivo studies have confirmed these observations; E infusion into the chronically catheterized fetal sheep results in increased surfactant flux into tracheal fluid and a decrease in the rate of tracheal fluid production. 12 • 13 NE did not have a similar effect. The decrease in lung water following beta sympathetic stimulation was noted in the earlier studies as well. 10 Beta adrenergk agonists increased the rate of choline incorporation into disaturated phosphatidylcholine (DSPC), the major phospholipid in surfactant, and increased the total lung DSPC, suggesting an effect on surfactant synthesis. 14 Available information regarding glucocorticoid and adrenergic influences in lung maturation suggests a complicated, integrated response involving both pulmonary and neurosympathetic maturation. A role for glucocorticoids in ncurosympathetic maturation was first documented by Wurtman and Axelrod, 27 who demonstrated that glucocorticoids increased adrenal Incubations carried out as described in Methods.

[7] 247w medullary levels of phenylethanolamine-N-methyltransferase, the terminal enzyme in the pathway of epinephrine biosynthesis. Our observation of increased adrenal E content in female fetuses suggests a sex difference in the timing of this adrenal CAT enzyme induction. Sex differences iri early human fetal adrenal glucocorticoid content or cord blood cortisol and cortisone concentrations have not been observed.2H• 29 Torday et aF recently reported significant correlations of increasing amniotic fluid cortisol, lecithin-sphingomyelin (LIS) ratio, and saturated phosphatidylcholine (SPC) concentration with gestational age in humans. Female infants reached mature amniotic fluid cortisol values 2.5 weeks before males. Similarly the LIS ratio was accelerated by 1.2 weeks and SPC concentration by 1.4 weeks. Cheng et al. 30 have reported recently that !3-adrenergic receptor number in fetal rabbit lung increases with advancing gestation and that this maturation is accelerated in the fetus following maternal glucocorticoid administration. A similar effect was not observed following maternal treatment with the T3 analogue 3,5-dimethyl-3-isopropyl-L-thyronine (DIM IT) at a dosage that increased surfactant synthesis and release. Thus there is evidence to support the view that glucocorticoid-mediated pulmonary maturation in the fetus is mediated in part by stimulation of autonomic nervous system maturation. Glucocorticoid augments adrenal medullary epinephrine synthesis and secretion and stimulates pulmonary J3-adrenergic receptor number. Differences in the timing of neurosympathetic or pulmonary development may explain the male susceptibility to neonatal morbidity. Moreover, females may show a greater apparent response to glucocorticoid treatment through direct effects on pulmonary maturation or to accelerated neurosympathetic maturation or both.

[1] 136w Seventeen pregnant New Zealand white rabbits with known dates of conception were obtained from a com-merciai suppiier. Fetai studies were carnea our at ~ 1 and 31 days' gestation (term is 31 days) andneonatal studies at 3 and 7 days of age. At dre designated gestational ages the does were killed by intravenous administration of 300 mg of pentobarbital. Fetuses were delivered by cesarean section and killed immediately by decapitation; the left adrenal gland was removed through a midline abdominal incision. The chest was opened, and a 50 to i 00 mg portion of the right middle lobe of the lung was removed and rapidly weighed. Sex determination was by inspection of internaf gonadal development as previmtsly de8cribed. 5 Criteria included the size, shape, and degree ofgonadal descent as well as presence of a well-defined epididymis.

[2] 110w For n1easure-ment of tissue catecho!atnin_e (C,A T) concentrations the lung and adrenal tissues were immersed in iced O.lN perchloric acid \vith .r:> tnM reduced glutathione (GSH) and immediately homoge-ni7Prl with ;1 Tt>Hon-al::~.~~ hnmnaPni7Pr. HornnaPn::~tes --------•-----------o-~---------o----~-------o -----o--------_ were transferred to chilled 12 X 75 mm tubes and centrifuged at 2,000 g for W minutes. The supernatant was removed, quick frozen, and stored at -70° C for later CAT analysis. An aliquot was srored br protein determination. For lung ,8-adrenergic recept.or studies the remainder of the lung tissue was removed; dissected free of major airways, blood vessels, and-connective tissue: washed with iced homo-genizing buffer; and pooled with littermates of the same gender.

[3] 231w Pooled lungs were honiogenized (with a Tekmar tissuemizer for 3-to I 0-second bursts at high speed) in six Yolumes of iced 250 mM sucrose, 5 mM Tris, and 1 mM MgCl 2 , pH 7.4. The homogenate was centrifuged for 10 minutes at 3,000 g and the supernatant decanted and centrifuged at 40,000 g for ZO minutes, The supernatant was discarded .and the pellet resuspended in homogenizing buffer with the Tekmar Tissuemizer at setting 50 for a I 0-sccond burst, and centrifugedag;ain at 40,000 g for 20 minutes. The resulting homogenous pellet was resuspended to a final pn~ein com:entration of 3 to 5 mg/ml, separated into aliquots, <tnd quick frozen with dry ice and acetone. Membranes thus prepared were stored at 70° C until assay, usually within 1 week. Catecholamine measurement. Fifty-microliter duplicates of the perchloric acid extracts were used directly in a modified single-isotope radioenzymatic assay after Johnson and Peuler. 16 The basis of the assay is enzymatic methylation of the CATs to the 0-methyl derivatives, which are then separated by thin-layer chromatography, extracted, periodated, and counted in a liquid scintillation spectrometer. Sensitivity in perchloric acid was 2 to 5 pg of norepinephrine and epinephrine and 10 to 20 pg of dopamine per tube. The assay was shown to be linear up to 1,000 pg per tube. Adrenal extracts that contained large amounts of CATs were diluted prior to assay.

[4] 44w In separate preliminary experiments recovery of 14 C norepinephrine added prior to homogenization was evaluated. A portion of the supernatant was neutralized and separated by thin-layer chromatography, eluted, and counted in a liquid scintillation system as previously described. 16 Recovery was greater than 90%.

[5] 173w Corrections for CAT extraction recovery were not used in further experiments. Adrenergic receptor measurements. Direct binding studies were performed in duplicate in 50 mM Tris-HCl pH 7.4, IO mM MgC1 2 with concentrations of tritiated dihydroalprenolol (DHA, New England Nuclear, specific activity 45 to 50 Ci/mmole) ranging from 0.5 to 10 nM. Bound and free material were separated by rapid filtration and washing on Whatman GF/C glass fiber filters and were counted in a liquid scintillation system. Nonspecific binding was defined as the amount of DHA bound in the presence of 1 JA-M o, L-propranoloL The protein concentration of the membrane preparation was determined by the method of Lowry. 17 Binding capacity or receptor number, expressed as femtomoles DHA bound per milligram of membrane protein, and the dissociation constant Kd were determined by Scatchard 1 H analysis of the direct binding data. Competitive binding curves were determined urider similar assay conditions by addition of agonists over the designated concentration ranges (L-epinephrine bitartarate, L-norepinephrine • bitartarate, and L-isoproterenol HCI were purchased from Sigma Co.).

[6] 106w Preliminary nested analysis of variance (ANOVA) showed that the variance within litters for each pa- Table IV. Pulmonary ,B-receptor number in developing M and F rabbits (fmoles/mg protem\ Fetal Newborn Sex 27 days I 31 days l 8..days Male Female 93 ± 5.7* (4)t 113 :!: :~.5 (4) 167 ± 3 (4) 198 i (4) 213 ± 17 (2) 241 13 (2) 286 :!: 14 {3) 339 ± 20 (3) Assays carried out as described in Method&. Two-way A NOVA showed significant increases with advancing age (p < 0.005) and females significantly greater than males (p < 0.005). *Mean± SEM in fmoles/mg protein-'. tNumber of litters.

[7] 55w rameter studied was equal to or greater than the variance between litters. Data from individual litters was therefore pooled for two-way ANOV A, comparing the effects of sex and advancing developmental age on each parameter. 19 Fetal and neonatal groups were analyzed separately. Logarithmic transformation was applied to adrenal hormone contents prior to statistical manipulation.

[1] 7w 0002-9378/81/180199+06$00.60/0© 1981 The C. V. Mosby Co.

[2] 141w involved. Several hormones are known to influence lung maturation, including thyroid hormones, adrenal corticosteroids, and catecholamines. Thyroid hormone concentrations do not differ in cord sera of male and female premature infants. 6 Moreover, there is little evidence that thyroid hormones in physiologic levels (as contrasted with pharmacologic doses) influence lung maturation? In contrast, the acceleration of lung maturation and amelioration of RDS in response to antenatal glucocorticoid administration have been shown to be augmented in females in both human 8 and animal studies. 4 More recently adrenergic mechanisms have been shown to influence the synthesis and release of pulmonary surfactant, 9 -14 but sex differences have not been studied. In the central nervous system during development females have higher rates of synthesis and turnover of monoamines than males.i 5 Comparable studies in the peripheral nervous system have not been carried Out.

[3] 19w Our report describes studies of pulmonary neurosympathetic development and development of the :-.eptemi><>r Lt, J;IXJ \til .. ! Oh•.:tet.•(,\IH_'cqJ.

[4] 14w Table I. Pulmonary norepinephrine levels in developing rabbits (pg/rng protein) -~-~ ~~~--------0 -,ry--~--.--,"------F_e_taTl-------,-,,-.t---.-y••,------~--------,--1 _-_-------.----~---------------

[5] 38w ............. • -'-"".N-,_, , ,.,........ .... ~ nu}•' l:l ,fa,)•-~ Vewborn Male 133 :!: 15.7* 24J :+-31.3 490 ± 65 (12)t i17) (4) 1.099 ~ ; :W d)) 91!! 1-M (!)) Female 171 ± ~4.5 20'\ ±: :!8.5

[6] 8w Assays were carried out as described in Methods.

[7] 138w Two-way ANOVA showed significant effect of advancing age (p < 0.005) but no effect of sex. *Mean ± SEM in pg/mg protein. tNumber of animals. Tabie H. Adrenai CAT content in ferai rabbits at 27 and 31 days' gestation (nanograms per gland) CAT Male Female 27 Days E* 363 ± 36.8t (13):j: 430 ± 34.0 (20) NE § 97 ± 12.5 98 ± 10.9 DA § 8 ± 0.8 10 ± 1.07 31 Days E 929 ± 83.2 (13) 1,120 ± 66.8 (19) NE 278 ± 55.5 344 ± 44.0 DA 32 ± 7.1 34 ± 6.02 *Two-way AN OVA showed significant effect of advancing age (p < 0.005) forE and females> males (p < 0.01). tGeometric mean ± SEM. *Number of fetuses. §Two-way ANOVA showed significant effect of advancing age (p < 0.005). No significant sex difference.

[8] 18w Table III. Adrenai CAT content in neonatai rabbits at 3 and 7 days of age (nanograms per gland)

[9] 11w Male Female E* 2,831 ± 320.5t (15):j: 2,629 ± 124.9 (7)

[10] 73w 3 Days NE 1,003 ± 204.9 750 ± 218.9 DA 29 ± 24.1 27 ± 9.3 E 9,396 ± 1,011.5 (6) 9,536 ± 791.6 (6) 7 Days NE 2,020 ± 389.5 2,112 ± 281.3 DA 47 ± 23.2 51 ± 37.0 No significant sex differences were noted. *Two-way ANOV A showed significant effect of advancing gestation age for E, NE, and DA (all p < 0.005). tGeometric mean ± SEM. :j:Number of animals.

[11] 34w peripheral adrenergic nervous system in male and female rabbit fetuses. The results suggest that lung .B-receptor development but not innervation is accelerated in female fetuses. Adrenal medullary matmation is likewise accelerated in female fetuses.