Involvement of intramural prostaglandin E2 in prenatal patency of the lamb ductus arteriosus

Release of prostaglandin E2 (PGE2) was studied in isolated ductus arteriosus preparations from immature (103 or 104 days gestation; term, 147 days) and near-term fetal lambs. Mature preparations produced measurable amounts of the compound in most cases and the release rate was 19 +/- 2 pg/(100 mg wet weight X min) at a PO2 of 3-8 Torr (1 Torr = 133.3 Pa). PGE2 release increased with the PO2 of the medium, peak values (about 125 pg/(100 mg X min)) being attained at 106-276 Torr when the oxygen-induced contraction was still submaximal. Experiments in which tissues were either contracted with excess potassium or relaxed with CO proved that PGE2 formation is independent from the contractile state. PGE2 was also released from ductus preparations lacking the adventitia, the intima, or both; however, release values were maximal when the adventitia was preserved. The magnitude of the intrinsic tone in these stripped preparations was inversely related to the rate of PGE2 formation. Reduced glutathione increased PGE2 release from the mature ductus, whole or stripped, and also relaxed hypoxic preparations; both effects were reversed by concomitant treatment with indomethacin. PGE2 synthesis tended to be greater in the immature than the mature ductus, maximal values (115 +/- 27 pg/(100 mg X min)) being observed at 6-8 Torr. We conclude that the ductus arteriosus is endowed with an enzyme system for the synthesis of PGE2 whose function accords with an effector role of the compound in the regulation of tone. These findings, together with the potent relaxation exerted by PGE2 at low PO2, indicate that the locally generated prostaglandin is well suited for keeping the ductus patent in the fetus.     

Ductus arteriosus: Developmental response to oxygen and indomethacin

It has been suggested that ineffective constriction in response to an increase in PO₂ is the primary cause for delayed closure of the ductus arteriosus in preterm infants. We studied the isometric contractile effects of increased PO₂ and indomethacin on isolated rings of lamb ductus arteriosus from animals of different gestational ages (87 to 147 days, term is 150 days). Rings from animals less than 110 days have a significantly smaller oxygen-induced contraction (2.53 ± .30 g/mm², n = 16) when compared with rings from animals near term (4.59 ± .69 g/mm², n = 9). Oxygen contracted rings from all gestational ages contract further upon addition of 1 μg/ml indomethacin. Rings from animals less than 110 days have a significantly larger indomethacin induced contraction (1.10 ± .17 g/mm², n = 16) than vessels near term (0.52 ± .12 g/mm², n = 9). Inhibition of prostaglandin production in rings less than 110 days results in a total combined oxygen and indomethacin induced tension that is not significantly different from the oxygen or oxygen and indomethacin induced tension developed in rings from animals near term. This is consistent with the hypothesis that, early during gestation, endogenous prostaglandins inhibit the vessel's ability to contract in response to oxygen. These observations are also consistent with the ability of indomethacin to constrict the patient ductus arteriosus in pre-term infants.     

Formation of prostacyclin (PGI2) by the ductus arteriosus of fetal lambs at different stages of gestation.

Prostaglandins appear to play a role in maintaining patency of the ductus arteriosus during gestation. Prostacyclin (PGI2) is the major product of prostaglandin biosynthesis in the lamb ductus arteriosus. This factor is both a vasodilator and a potent inhibitor of human platelet aggregation. We used inhibition of platelet aggregation as a sensitive bioassay to measure PGI2 generation in rings of ductus arteriosus from fetal lambs. Mechanical manipulation accelerated the rate of PGI2 released from the tissue 10 to 50 times. Tranylcypromine, an antagonist of prostacyclin synthetase, suppressed production of PGI2 by rings of ductus arteriosus. Rings from immature animals (98-103 days gestation, term is 150 days) released significantly more PGI2 (190 +/- 28 ng/g wet weight/ 20 min, n = 9) than did those from near term animals (136-146 days; 106 +/- 23 ng/g wet weight/20 min, n = 10). The capacity of the ductus arteriosus to generate more PGI2 earlier in gestation is consistent with the observation that vessels from animals less than 110 days gestation have a significantly larger indomethacin induced contraction than do vessels near term.     

Formation of prostacyclin (PGI2) by the ductus arteriosus of fetal lambs at different stages of gestation

Prostaglandins appear to play a role in maintaining patency of the ductus arteriosus during gestation. Prostacyclin (PGI₂) is the major product of prostaglandin biosynthesis in the lamb ductus arteriosus. This factor is both a vasodilator and a potent inhibitor of human platelet aggregation. We used inhibition of platelet aggregation as a sensitive bioassay to measure PGI₂ generation in rings of ductus arteriosus from fetal lambs. Mechanical manipulation accelerated the rate of PGI₂ released from the tissue 10 to 50 times. Tranylcypromine, an antagonist of prostacyclin synthetase, suppressed production of PGI₂ by rings of ductus arteriosus. Rings from immature animals (98–103 days gestation, term is 150 days) released significantly more PGI₂ (190 ± 28 ng/g wet weight/ 20 min, n=9) than did those from near term animals (136–146 days; 106 ± 23 ng/g wet weight/20 min, n=10). The capacity of the ductus arteriosus to generate more PGI₂ earlier in gestation is consistent with the observation that vessels from animals less than 110 days gestation have a significantly larger indomethacin induced contraction than do vessels near term.     

Developmental response to indomethacin: a comparison of isometric tension with PGE2 formation in the lamb ductus arteriosus

We studied the effects of oxygen and indomethacin on the isometric contractile response and the production of PGE2 in isolated rings of lamb ductus arteriosus from animals of different gestational ages (100 to 144 days; term is 150 days). Rings of ductus arteriosus from animals less than 110 days released significantly less PGE2 than did rings from animals greater than 120 days. The indomethacin-induced increase in muscle tension in relation to the decrease in endogenous PGE2 production in preparations from animals less than 110 days gestation was greater than in animals older than 120 days. These findings do not support the hypothesis that immature animals have a larger indomethacin-induced contraction due to an increased production of PGE2 earlier in gestation. They are, however, consistent with a decreased sensitivity to PGE2 in the more mature animals; they also support the hypothesis that the decreased effectiveness of indomethacin on the ductus arteriosus from later gestation animals reflects primarily a decrease in the sensitivity of the vessel to PGE2 during development.     

Maturation of the contractile response of the Emu ductus arteriosus

The avian embryo has a pair of ductus arteriosi that allow the blood to bypass the pulmonary circulation prior to the initiation of lung ventilation. Our objective was to characterize the factors regulating DA tone during the later stages of development in the emu embryo. We examined in vitro the reactivity of the emu ductus from day 39 through 49 of a 50-day incubation. Steady state tension was not altered by the COX inhibitor indomethacin or the nitric oxide synthase inhibitor l-NAME. However, prostaglandin E₂ (PGE₂) produced a significant relaxation. Norephinephrine and U-46619 produced strong significant contractions in the emu DA and the adrenergic response matured with development. The contractile response to oxygen matured as the embryo developed with significant oxygen-induced contraction on days 45 and 49, but not on day 39 of incubation. The Kv channel inhibitor 4-aminopyridine induced the contraction of the day 48–49 ductus of similar magnitude as the oxygen-induced contraction. The oxygen-induced contraction was reversed by the reducing agent DTT and the electron transport chain inhibitor rotenone. These results suggest that while the emu DA responds to PGE₂, locally produced PGE₂ are not the important regulators of vessel tone. Additionally, relaxation upon addition of the mitochondria electron transport chain inhibitor rotenone suggests that the mitochondria might be acting as vascular oxygen sensors in this system through the production of reactive oxygen species to stimulate the oxygen-induced contraction in a similar fashion to mammals.     

Endothelin is a potent constrictor of the lamb ductus arteriosus

Endothelin was tested on isolated ductus arteriosus preparations from mature fetal lambs. At low PO2 (18-24 Torr; 1 Torr = 133.3 Pa), the compound constricted the vessel dose-dependently over the range from about 10(-10) to 10(-7) M. The contraction was sustained and did not subside even after an extended period of washing. Endothelin was also effective on tissues (PO2,217-231 Torr; indomethacin, 2.8 X 10(-6) M) that had been completely relaxed with CO (CO/O2 ratio, 0.28). CO treatment interferes with a cytochrome P-450 mechanism, which is considered crucial for the contractile response of the vessel to oxygen. These findings are consistent with a role of endothelin in the closure of the ductus arteriosus at birth.     

Developmental response to indomethacin: A comparison of isometric tension with PGE2 formation in the lamb ductus arteriosus

We studied the effects of oxygen and indomethacin on the isometric contractile response and the production of PGE₂ in isolated rings of lamb ductus arteriosus from animals of different gestational ages (100 to 144 days; term is 150 days). Rings of ductus arteriosus from animals less than 110 days released significantly less PGE₂ than did rings from animals greater than 120 days. The indomethacin-induced increase in muscle tension in relation to the decrease in endogenous PGE₂ production in preparations from animals less than 110 days gestation was greater than in animals older than 120 days. These findings do $\text{not}$ support the hypothesis that immature animals have a larger indomethacin-induced contraction due to an increased production of PGE₂ earlier in gestation. They are, however, consistent with a decreased sensitivity to PGE₂ in the more mature animals; they also support the hypothesis that the decreased effectiveness of indomethacin on the ductus arteriosus from later gestation animals reflects primarily a decrease in the sensitivity of the vessel to PGE₂ during development.     

Evidence against a role for lipoxygenase-derived products of arachidonic acid in the lamb ductus arteriosus

The effects of leukotrienes, the leukotriene antagonist FPL55712 (sodium 7-(3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-2-hydroxypropoxy)-4-oxo-8-propyl-4H-1-benzopyran-2-carboxylate), and inhibitors of arachidonate lipoxygenase and cyclo-oxygenase (compound BW755C, 3-amino-1-(m-(trifluoromethyl)-phenyl)-2-pyrazoline; ETYA, 5,8,11,14-eicosatetraynoic acid) were studied in an isolated preparation of ductus arteriosus from mature foetal lambs. Leukotrienes (LT) C4 and D4 produced a modest relaxation of the ductus but only at the highest concentrations tested (10(-7) to 10(-6) M) and under hypoxic conditions (PO2, 6--9 Torr (1 Torr = 133.322 Pa)). LTB4 had no effect at any concentration tested. BW755C (10(-6) to 10(-5) M) and FPL55712 (10(-5) M) contracted the hypoxic ductus; however, their action was abolished by pretreatment of the tissue with the cyclooxygenase inhibitor indomethacin (2.8 x 10(-6) M). Indomethacin-treated preparations were also unresponsive to ETYA 3 x 10(-5) M. The contraction of hypoxic tissues to either BW755C or FPL55712 increased further upon raising the oxygen tension of the medium (PO2 591--691 Torr). These findings indicate that leukotrienes and allied compounds formed from lipoxygenase-catalysed reactions do not contribute to prenatal patency of the ductus and are unlikely to have a role in its closure at birth. It is also confirmed that prostaglandin E2 is essential for keeping the vessel patent in the foetus.     

Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall

Nitric oxide (NO) and cyclooxygenase (COX)-derived prostaglandins are critical regulators of the fetal ductus arteriosus. To examine the interaction of these pathways within the ductus wall, the ductus arteriosus of term and preterm fetal mice was evaluated by pressurized myography. The isolated preterm ductus was more sensitive to NOS inhibition than at term. Sequential NOS and COX inhibition caused 36% constriction of the preterm ductus regardless of drug order. In contrast, constriction of the term ductus was dependent on the sequence of inhibition; NOS inhibition prior to COX inhibition produced greater constriction than when inhibitors were given in reverse order (36 ± 6% versus 23 ± 5%). Selective COX-1 or COX-2 inhibition prior to N(G)-nitro-l-arginine methyl ester (l-NAME) induced the expected degree of constriction. However, NOS inhibition followed by selective COX-2 inhibition caused unexpected ductal dilation. These findings are consistent with NO-induced activation of COX in the ductus arteriosus wall and the production of a COX-2-derived constrictor prostanoid that contributes to the balance of vasoactive forces that maintain fetal ductus arteriosus tone.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGF1alpha.

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE2, PGF2alpha, 6 keto PGF1alpha (and its unstable precursor PGI2). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2 and indomethacin. All the prostaglandins (except PGF2alpha) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE2, however, relaxed the vessel at concentrations below 10(-8)M. PGI2 and 6 keto PGF1alpha had approximately 0.001 and 0.0001 times the activity of PGE2. Although PGE2 has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE2 might make it the most significant prostaglandin in regulating the patency of the vessel.     

The response of the lamb ductus arteriosus to endothelin: developmental changes and influence of light

Endothelin-1 (ET-1) is a putative messenger of oxygen in the ductus arteriosus. Since the ability of the vessel to contract to oxygen increases with gestation, we wished to ascertain whether ET-1 action is also developmentally regulated. A corollary objective was to assess whether any gestational variation in the ET-1 contraction is due to a change in the ET(A)-mediated action or to a shift in the balance between opposing, contractile (ET(A) - mediated) and relaxant (ET(B)-mediated), actions. Experiments were performed with isolated ductal strips from preterm (0.7 gestation) and near-term fetal lambs. ET-1 contracted the ductus dose-dependently (10(-10)-10(-7) M) at both ages; however, the peak contraction was about double in magnitude at term. Regardless of age, ET-1 contraction was greater with preparations kept in the dark compared to those exposed to light. This effect of light was not seen after removing the endothelium or when treating the intact tissue with the ET(B) antagonist BQ788 (1 microM). In the dark, however, BQ788 did not modify significantly the ET-1 response at either age. We conclude that ET-1 becomes a stronger ductus constrictor with fetal age, conceivably by acting on ET(A) receptors. Hence, the concept of ET-1 mediating the oxygen contraction is further validated. Peculiarly, the ET-1 contraction is curtailed by light through a hitherto undefined ET(B) receptor-linked process.     

Ductus arteriosus responses to prostaglandin E1 at high and low oxygen concentrations

It previously has been suggested that prostaglandin E₁ (PGE₁) relaxes the ductus arteriosus in a low but not in an elevated oxygen environment. However, in the experiments reported here PGE₁ relaxed rings on fetal lamb ductus arteriosus at both low (14 to 20 torr) and high (680 to 720 torr) oxygen tensions. The threshold concentration for PGE₁ was 10⁻¹⁰ M in either PO₂ and the ED50's of PGE₁ relaxation in high and low oxygen were 8.5 ± 3.4 × 10⁻¹⁰ M and 5.5 ± 0.7 × 10⁻¹⁰ M respectively. The magnitude of the relaxation was greater for the oxygen contracted ductus arteriosus than for that exposed to low oxygen. It is suggested that earlier reports of the lack of response of the ductus arteriosus to PGE₁ in a high oxygen environment following relaxation in a low oxygen environment may be related to loss of response of the ductus arteriosus to repeated doses of PGE₁ rather than to differences in PO₂. Prostaglandin E₁ therefore may play a significant role in the regulation of ductus arteriosus tone in the elevated oxygen environment of the newborn as well as the low oxygen environment of the fetus.     

Studies on closure of the ductus arteriosus. XII. effect of indomethacin and sodium salicylate in rats and rabbits

Administration of prostaglandin synthetase inhibitors to pregnant does and dams in late gestation was followed by contraction of the fetal ductus arteriosus when studied by the whole-body freezing method. In the rat this contraction was well established within 6 h and persisted up to 36 h following 15 mg/kg indomethacin p.o. No effect was observed in the 18 d rat fetus but fetuses at 20 d and 22 d of gestation responded significantly to indomethacin. Doses of indomethacin approaching clinical usage (2.5 mg/kg) also caused a positive response . The rat was found to be sensitive also to sodium salicylate and in the rabbit both indomethacin and sodium salicylate were effective. Exposure to prostaglandin synthetase inhibitors with resulting contraction of the ductus may seriously disturb cardiac function in the fetus.     

Prostaglandin biosynthesis and catabolism in the developing fetal sheep lung.

The prostaglandin biosynthetic and catabolic capacity of homogenates of lungs from fetal sheep of various gestational ages was measured. Prostaglandin biosynthesis was assayed by the deuterium-isotope dilution technique making use of mass fragmentography whereas prostaglandin catabolism was measured by the radioisotope-dilution method described previous (Pace-Asciak, C.R. and Rangaraj, G. (1976) J. Biol. Chem. 251, 3381-3385). Homogenates of lungs from fetuses of all ages tested (40 days to term) formed both prostaglandins E2 and F2alpha; although prostaglandin F2alpha was formed to a greater extent than prostaglandin E2 by the 40 days lung, prostaglandin E2 increased with increasing age until at term the ratio of both prostaglandins approached unity. Total prostaglandin biosynthesis (E2 + F2alpha) rose gradually with age (approx. 3 fold increase between 40 days and term). Prostaglandin F2alpha catabolism occurred mainly by the prostaglandin 15-hydroxy dehydrogenase pathway; this activity was detectable even at 40 days and remained unchanged up to 80 days. Prostaglandin catabolic activity rose sharply at 90 days (approx. 3 fold) with a maximum around 110 days (approx. 4 fold) decreasing back to 40 day levels by term (143 days). The increasing prostaglandin catabolic activity around 90-100 days in this species is discussed in relation to the hemodynamic changes in the lungs starting around this age and the appearance of surfactant. Prostaglandin catabolism might play an important role in the developing organ controlling steady state concentrations of prostaglandins during certain periods of organogenesis.     

Postnatal constriction, ATP depletion, and cell death in the mature and immature ductus arteriosus

After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1alpha/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1alpha/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age.     

Cyclooxygenase isoenzymes and patency of ductus arteriosus

Prenatal patency of the ductus arteriosus is maintained mainly by prostaglandin (PG) E(2). Accordingly, the vessel is endowed in its muscular component with a complete, cyclooxygenase (COX) and PGE synthase (PGES), system for the synthesis of the compound. COX1 is better expressed than COX2, particularly in the premature, but COX2 is more extensively coupled with microsomal PGES (mPGES). No evidence was obtained of either COX being coupled with cytosolic PGES (cPGES). Functionally, these data translate into a differential constrictor response of the ductus to dual, COX1/COX2, vs. COX2-specific inhibitors (indomethacin vs. L-745,337), with the latter being less effective specifically prior to term. This difference, however, subsides upon treatment with endotoxin and the attendant upregulation of COX2 and mPGES. Furthermore, when studied separately, COX1 and COX2 prove to be unevenly responsive to indomethacin, and an immediate and fast developing contraction of the vessel occurs only when COX2 is inhibited. Deletion of either COX gene results into upregulation of NO synthase, and a similar compensatory reaction is expected when enzymes are suppressed pharmacologically. We conclude that PGE(2) and NO can function synergistically in keeping the ductus patent. This arrangement provides a possible explanation for failures of indomethacin or ibuprofen treatment in the management of the prematurely born infant with persistent ductus. Coincidentally, it opens the way to new therapeutic possibilities being based on interference with the NO effector or a more selective disruption, possibly having mPGES as a target, of the PGE(2) synthetic cascade.     

The control of cardiovascular shunts in the fetal and perinatal period

The fetal circulation has two major vascular shunts, the ductus arteriosus and the ductus venosus. The ductus arteriosus connects the pulmonary artery with the descending portion of the aortic arch, hence shunting most of the right ventricular output away from the unexpanded lungs. The ductus venosus connects instead the portal sinus with the inferior vena cava and allows well-oxygenated umbilical vein blood to bypass the liver and reach the central circulation rapidly. Both blood vessels cease their function after birth and undergo permanent closure. It is now well established that prenatal patency of the ductus arteriosus is an active state sustained by a prostaglandin. A similar mechanism has been recently recognized in the fetal ductus venosus. Evidence is presented indicating that prostaglandin E2 and prostaglandin I2 are natural relaxants, respectively, for the ductus arteriosus and the ductus venosus. In addition, both vascular shunts share the dependence on an endogenous cytochrome P-450 mechanism to develop their contractile tone. This mechanism may be important in the normal process of shunt closure at birth. While broadening the knowledge of fetal cardiovascular homeostasis, advances in this field have important implications for the prevention and management of certain pathological conditions affecting the newborn.     

Lamb ductus arteriosus: Effect of prostaglandin synthesis inhibitors on the muscle tone and the response to prostaglandin E2

The prostaglandin synthesis inhibitors, indomethacin and eicosa-5,8,11, 14-tetraynoic acid (ETA), have been tested on the isolated lamb ductus arteriosus at low and high PO₂ levels. Both compounds produced a gradual contraction of the hypoxic vessel, and at equal doses the effect of indomethacin was stronger. The maximal tension output of the hypoxic tissue under indomethacin was equal to that of the oxygen-contracted control. ETA- and indomethacin-treated preparations contracted further upon transfer from a low to a high oxygen environment, and the response under indomethacin exceeded significantly control values. Control preparations were relaxed markedly by PGE₂ in low oxygen but showed little or no response in high oxygen. In contrast, preparations pretreated with the inhibitors retained their sensitivity to PGE₂ during exposure to high oxygen. The data are consistent with the idea that E-type prostaglandins play a role in the regulation of the intrinsic tone of the ductus arteriosus during foetal life. It is also suggested that the sensitivity of ductal muscle to E-type prostaglandins is controlled by the rate of endogenous prostaglandin formation.     

Prostaglandin biosynthesis and catabolism in the developing fetal sheep lung

The prostaglandin biosynthetic and catabolic capacity of homogenates of lungs fetal sheep of various gestational ages was measured. Prostaglandin biosynthesis was assayed by the deuterium-isotope dilution technique making us e of mas fragmentography whereas prostaglandin catabolism was measured by the radioisotope-dilution method described previously (Pace-Asciak, C.R. and Rangaraj, G. (1976) J. Biol. Chem. 251, 3381–3385).Homogenates of lung sform fetuses of all ages tested (40 days to term) formed both prostaglandins E₂ and F_2α; although prostaglandin F_2α was formed to a greater extent than prostaglandin E₂ by the 40 day lung, prostaglandin E₂ increased with increasing age until at term the ratio of both prostaglandins approached unity. Total prostaglandin biosynthesis (E₂ + F_2α) rose gradually with age (approx. 3 fold increase between 40 days and term). Prostaglandin F_2α catabolism occurred mainly by the prostaglandin 15-hydroxy dehydrogenase pathway; this activity was detectable even at 40 days and remained unchanged up to 80 days. Prostaglandin catabolic activity rose sharply at 90 days (approx. 3 fold) with a maximum around 110 days (approx. 4 fold) decreasing back to 40 day levels by term (143 days).The increasing prostaglandin catabolic activity around 90–100 days in this species is discussed in relation to the hemodynamic changes in the lungs starting around this age and the appearance of surfactant. Prostaglandin catabolism might play an important role in the developing organ controlling steady state concentrations of prostaglandins during certain periods of organogenesis.