Pulsatile secretion of prolactin and oxytocin during nursing in the lactating rat

The secretory profile of prolactin and oxytocin in response to suckling stimuli by litters was studied in unanesthetized and urethane-anesthetized lactating rats. Serum prolactin levels were determined by radioimmunoassay. Oxytocin released at milk-ejection reflex was monitored by the changes in the intramammary pressure and/or the characteristic pup's reaction associated with the milk ejection. Serum prolactin concentrations began to rise earlier than the first milk ejection in unanesthetized rats, but they were never elevated without the appearance of milk ejections in urethane-anesthetized rats. Pulsatile fluctuation in serum prolactin levels at 6-15 min intervals was observed in the nursing period when 10 pups were suckling continually. The intermittent milk-ejection reflex occurred not always but preponderantly (64-91%) when the serum prolactin levels were at the nadir of the fluctuation. Injection of an estimated dose of oxytocin released at each milk ejection (1 mU) did not change the serum prolactin levels. These results indicate that the mechanism for prolactin release may be more susceptible to the effects of anesthesia than that for oxytocin release in response to the suckling stimuli and that the release of both the hormones is pulsatile in nature and be influenced by a common biological clock during the nursing period.     

Pineal peptides in the regulation of milk-ejection reflex in lactating rats

The effects of pineal peptides (mol. mass 1-4 kDa) intranasal infusions on some parameters of milk-ejection reflex were investigated. Peptides were extracted from dairy-cattle pineal glands. Pineal peptides increase body weight, levels of water intake, plasma prolactin concentration and milk yield in rats when infusing daily in dose 1 microgram/kg from the third day of lactation. On 9th and 12th days of lactation during 1-hour nursing seance the significantly greater number of reflective milk ejections were found. When 1 hour before the peptides infusion rats were intraperitoneally injected by rabbit antiserum to oxytocin (200 microliters at dilution of 1:20) the effects of pineal peptides were significantly less expressive or were absent at all. Using enzymimmunoassay it was demonstrated that there were greater increasing of oxytocin content in pineal gland in suckling-induced oxytocin release from neurohypophysial system in the chronic pineal peptides-treated female rats compared with control. This effect was absent when rats were injected by oxytocin antibodies. These data suggest that pineal peptides can participate in forming of reflect oxytocin release pattern. This pattern is initiated by suckling and is limited by oxytocin content in blood.     

Changing characteristics of the milk-ejection reflex during pregnancy, lactation and after weaning in the rat

The pattern of reflex milk ejection during suckling was investigated in anaesthetized Wistar rats at various stages of pregnancy, lactation and after weaning. Milk-ejection responses were measured using intramammary pressure recordings, and the amount of oxytocin released was estimated from log dose-response lines compiled from the mammary responses to exogenous oxytocin. The number of rats showing intramammary pressure responses to oxytocin increased on Day 22 of pregnancy (the day of parturition) and decreased at 8 days after weaning. The dose-response lines from pregnant animals were shallow, but steepened and shifted to the left during lactation and after weaning. Reflex milk-ejection responses during suckling were detectable in primigravid animals, indicating that birth of the litter and previous suckling experience are unnecessary for the immediate functioning of the reflex. Reflex milk-ejection responses improved during early lactation (such that the frequency and the amount of oxytocin released at each response were maximal at Day 10 of lactation), and subsequently declined in late lactation. Although the frequency of responses in animals 2 and 4 days after weaning was similar to that in late lactating animals, the amount of oxytocin released at each response had risen again to mid-lactation values. In animals undergoing a second pregnancy and lactation the pattern of change in the milk-ejection responses was similar to that of primiparous animals.     

Sodium induces oxytocin release from superfused rat pituitary

The effects of various osmotic agents on the release of oxytocin were examined in a superfusion system. Oxytocin was released significantly from the rat pituitary by superfusion with medium of an osmolality of 350 mOsm/kg H2O adjusted with NaCl, regardless of the presence of the rat hypothalamus. Media adjusted to an osmolality of 350 mOsm/kg H2O with sucrose, glucose, urea or mannitol had no effect on oxytocin release from the hypothalamo-pituitary complex. Medium containing excess Na2SO4 induced significant release of oxytocin from the pituitary without the hypothalamus. The administration of tetraethylammonium chloride had no oxytocin secretion. These data suggest that oxytocin release from the pituitary is influenced by the level of sodium ion rather than the osmotic pressure.     

Spontaneous milk ejection during lactation and its possible relevance to success of breast-feeding.

In a woman suckling twins it became apparent that both suckling-induced and precisely timed, spontaneous bursts of milk ejection were occurring. Observations on days 14, 28, 56, and 112 of lactation disclosed highly significnat increases in intervals between episodes of spontaneous milk ejection. Furthermore, at all stages of lactation the interval between a feed and the next episode of spontaneous ejection was significantly longer than the interval between spontaneous ejections. The decrease in frequency of episodes of spontaneous milk ejection during lactation may be related to the decreasing release of prolactin in response to suckling. Spontaneous milk-ejection episodes are felt only when the breast is full and may signal its readiness for a further suckling episode. Such bursts of milk ejection may stimulate the suckling response in babies, suggesting that rigid three- or four-hour feeding regimens may be unphysiological and pose a threat to the success of breast-feeding in the early postnatal period.     

Oxytocin receptors in the mammary gland and reproductive tract of a marsupial, the brushtail possum (Trichosurus vulpecula).

Previous studies of marsupial lactation have shown that the milk-ejection reflex changes in sensitivity, being greater in small mammary glands sucked by small pouch young and lesser in larger glands supplying milk to larger young. The involvement of oxytocin receptors in these changes was examined in the brushtail possum Trichosurus vulpecula. Oxytocin receptors were measured in the mammary glands, uterus, and medial vaginal sacs by radioreceptor assay, using [3H]oxytocin as radioligand. In the mammary gland, a single oxytocin binding site was found with an affinity and receptor concentration of 0.81 +/- 0.41 l/nmol and 10.2 +/- 4.8 pmol/g tissue respectively (SD, 10 possums). Competitive displacement curves with related peptides and analogs showed the following order of specificity: d(CH2)5[Tyr(Me)2,Thr4,Tyr9-NH2]-vasotocin much greater than vasotocin greater than oxytocin = Arg-vasopressin greater than mesotocin greater than [Thr4,Gly7]-oxytocin = Lys-vasopressin greater than [deamino-Pen1, O-methyl-Tyr2, Arg8]-vasopressin greater than isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. [3H]Oxytocin did not bind to vasopressin receptors in the thoracic aorta. The concentration of oxytocin receptors was very high in small mammary glands (18.6 pmol/g tissue in a 2-g gland) and decreased logarithmically as the size of the mammary gland increased. It is suggested that the changes in the sensitivity of milk ejection to oxytocin is related to the concentration of mammary oxytocin receptors. The presence of oxytocin receptors in both uterus and median vaginal sacs extends previous observations and supports the hypothesis that in marsupial parturition, the uterus and medial vaginal sacs respond as a single functional unit to oxytocin.     

Hypodypsia and selective dysfunction of osmoreceptors in the hypernatremia syndrome

We studied a patient with the rare syndrome of chronic hypernatremia associated with a frontal expansive process. The pituitary function was evaluated during dynamic tests bearing on radioimmunoassay of serum neurophysins levels. A test of water restrictionloading was performed during which urine appeared diluted (190-200 mOsm/kg) while the degree of serum osmolality was high (310-317 mOsm/kg). An hemodynamic stimulation resulted in a significant increase in serum neurophysins (from 3.5 +/- 0.3 to 5.5 +/- 0.2 ng/ml). After one intravenous injection of 2 mg nicotine, vomiting was observed, followed by a sharp rising of serum neurophysins levels (from 3.2 +/- 0.5 to 10.6 +/- 0.2 ng/ml). During hypertonic saline infusion, serum osmolality increased from 270 to 310 mOsm/kg, while neurophysins showed no significant change. Such results evidence a selective impairment of the hypothalamic-neurohypophyseal response to osmotic stimuli, with intact mechanisms of non-osmotic stimulation. In this patient, natremia was brought back to normal values by adequate water supply.     

Influence of opioid peptides on release from vasopressin and oxytocin neurones of the hypothalamoneurohypophyseal system: effects of naloxone in the conscious rat

We examined the effects of acute and chronic treatments with naloxone on release of vasopressin and oxytocin from the hypothalamoneurohypophyseal system (HNS) in conscious, chronically instrumented Long-Evans rats. Plasma concentrations of vasopressin-associated neurophysin and oxytocin-associated neurophysin were evaluated before and during an intravenous infusion of 18% saline at 100 microL.kg-1 body weight.min-1 for 60 min. Acute treatment with naloxone (2.75 mumol/kg, intravenous) did not measurably alter basal plasma osmolality or vasopressin-associated neurophysin concentration, but it caused a three-fold rise in basal plasma oxytocin-associated neurophysin concentration (16 +/- 2 to 46 +/- 3 fmol/mL, p less than 0.005). Chronic treatment with naloxone (13.75 mumol/day, subcutaneous pellets) increased plasma osmolality (292 +/- 1 to 300 +/- 2 mosmol/kg H2O, p less than 0.01) by day 5, but it had no measurable effects on basal vasopressin- or oxytocin-associated neurophysin concentration. There were also no significant differences in plasma sodium concentration (144.8 +/- 1.1 vs. 142.2 +/- 1.4 mequiv./L) under both conditions. Acute and chronic treatments with naloxone accompanied by salt loading produced a five- and four-fold decrease in the rates that plasma concentration of vasopressin-associated neurophysin changed with plasma osmolality, compared with untreated salt-loaded control rats. For oxytocin secretion from the HNS, both treatments accompanied by salt loading substantially decreased the threshold for changes in relation to plasma osmolality; the rise in plasma concentration of oxytocin-associated neurophysin was similar at all levels of hyperosmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prostaglandins on milk ejection.

Prostaglandins (PGs) of type F2 alpha, E1, and E2 have been reported both, to inhibit or to facilitate posterior pituitary oxytocin release in lactating animals and women, and to suppress or to stimulate the mammary myoepithelium. Prostaglandin-induced milk ejection in women and cows has been attributed to central oxytocin release, but no oxytocin blood levels were determined. Moreover, for lactating cows, sows, rabbits, guinea pigs, and rats a direct PG effect on the mammary myoepithelium resulting in milk ejection has been suggested. On the other hand, PGs were found to antagonize the milk-ejection response to oxytocin in rabbits and rats. The mechanisms involved in PG synergism or antagonism of oxytocin-induced milk ejection are not understood. Studies in lactating rats showed that blood pressure active PG doses of F2 alpha, E1, and E2 largely inhibited the intramammary pressure response to oxytocin. Whereas the oxytocin-antagonistic action of PGF2 alpha was not affected by adrenergic blockers (phenoxybenzamine, propranolol), the anti-oxytocin effects of PGE1 and E2 were eliminated after alpha-receptor blockade while the activity of oxytocin increased. Under beta-receptor or alpha- plus beta-receptor blockade, the oxytocin-inhibitory effects of PGE1 and E2 were almost abolished. Mechanisms of PG-induced inhibition of the oxytocin response may involve mammary vascular changes and/or alterations in myoepithelial activity of cyclic adenosine-3,5-monophosphate (c-AMP), cyclic guanosine-3,5-monophosphate (c-GMP), and phosphodiesterase (PDE). It seems unlikely that PGs bring about significant posterior pituitary oxytocin release in rats.     

Differential effects of prostaglandin F2 alpha on oxytocinergic and non-oxytocinergic neurones in the paraventricular nucleus of the lactating rat

The effects of the prostaglandin F2 alpha (PGF2 alpha) given into the third cerebral ventricle on the unit activity of neurosecretory neurones in the paraventricular nucleus (PVN) were studied in urethane-anesthetized rats. The firing activity of PVN neurones was recorded extracellularly and 50 neurones were antidromically identified as neurosecretory neurones. Thirty of them were classified oxytocinergic neurones because they gave a burst of action potential 12-15 sec before reflex milk ejection and the remaining twenty PVN neurones which showed no response prior to reflex milk ejections were regarded as non-oxytocinergic ones. Twenty-five (83%) of the30 oxytocinergic neurones increased in the firing rate following the intraventricular (IVT) injection of PGF2 alpha (500ng in 1 microliter of isotonic saline) and the responses lasted for about 20-30 min. The remaining 5 (17%) oxytocinergic neurones showed no response in the firing rate to IVT PGF2 alpha. Fifteen (75%) of the 20 nonoxytocinergic neurones decreased in the firing activity in response to IVT PGF2 alpha, and the remaining 5 (25%) of them showed no response. IVT injection of isotonic saline (1 microliter) did not affect the firing activity of both the oxytocinergic and nonoxytocinergic cells. The intramammary pressure was slightly increased by the IVT administration of PGF2 alpha. These findings indicate that IVT PGF2 alpha has a differential action on oxytocinergic and non-oxytocinergic neurones in rats.     

Spinal pathway of the milk-ejection reflex in the rat

The effects of lesions of the spinal cord on the milk-ejection reflex evoked by suckling were studied in urethane-anesthetized lactating rats. All lesions were made between C6 and C7 vertebrae and milk ejection was monitored by recording intramammary pressure. In the first experiment on the rats with bilateral lesions, a 3-h suckling test with 5 pups on each side was performed. Eleven (84.6%) of 13 rats with the section of the dorsal funiculus (Group 2), and 12 (85.7%) of 14 rats with the combined section of the dorsal and ventral funiculi (Group 4) displayed regular milk ejection. The incidence of milk ejection in both groups was not significantly different from 81.8% (9 rats) of the 11 sham-operated rats (Group 1). In contrast, none of the 12 rats with bilateral section of the lateral funiculus (Group 3) displayed milk ejection and the incidence of milk ejection was significantly lower than that in Group 1. In the second experiment on the rats with unilateral section of the lateral funiculus, bilateral suckling with 10 pups (5 pups on each side) and unilateral suckling (both ipsilateral and contralateral to the lesion) with 5 pups were consecutively performed in the 10 rats. Milk ejection was induced in 50% by contralateral suckling and in 100% by bilateral suckling, and the incidence was significantly higher than that (0%) observed during ipsilateral suckling. A significant difference in the incidence of milk ejection was also observed between contralateral and bilateral sucklings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 synthesis in the inner medullary collecting duct of the rat: implications for vasopressin-dependent cyclic AMP formation

The effects of osmolality on prostaglandin E2 (PGE2) biosynthetic capacity and the interaction between endogenous PGE2 synthesis and vasopressin (AVP)-dependent cyclic AMP generation were examined in papillary collecting ducts (PCD) microdissected from collagenase-digested rat kidneys. Increasing medium osmolality with NaCl:urea (1:2 molar ratio) progressively increased PGE2 synthesis in PCD up to 1,500 mOsm. Addition of NaCl:urea or NaCl alone were equally effective in stimulating PGE2 biosynthetic capacity in PCD. In contrast, addition of urea alone had a much smaller stimulatory effect on PGE2 synthesis. Inhibition of endogenous PGE2 synthesis with naproxen (10(-5)M) suppressed AVP-dependent cAMP formation in PCD when incubated in 300 mOsm medium but had no effect when incubated in 1,500 mOsm medium. Addition of 2.5 X 10(-5) M PGE2 also suppressed AVP-dependent cAMP formation in PCD only when incubated in 300 mOsm medium. The present study suggests that the PCD is a site of active PGE2 synthesis that is modulated by osmolality. Our results do not support the concept that endogenous PGE2 antagonizes vasopressin action via inhibition of AVP-dependent cAMP formation.     

Interactions between oxytocin, glucagon and glucose in normal and streptozotocin-induced diabetic rats

Oxytocin has been suggested to have glucoregulatory functions in rats, man and other mammals. The hyperglycemic actions of oxytocin are believed to be mediated indirectly through changes in pancreatic function. The present study examined the interaction between glucose and oxytocin in normal and streptozotocin (STZ)-induced diabetic rats, under basal conditions and after injections of oxytocin. Plasma glucose and endogenous oxytocin levels were significantly correlated in cannulated lactating rats (r = 0.44, P less than 0.01). To test the hypothesis that oxytocin was acting to elevate plasma glucose, adult male rats were injected with 10 micrograms/kg oxytocin and killed 60 min later. Oxytocin increased plasma glucose from 6.1 +/- 0.1 to 6.8 +/- 0.2 mM (P less than 0.05), and glucagon from 179 +/- 12 to 259 +/- 32 pg/ml (P less than 0.01, n = 18). There was no significant effect of oxytocin on plasma insulin, although the levels were increased by 30%. A lower dose (1 microgram/kg) of oxytocin had no significant effect on plasma glucose or glucagon. To eliminate putative local inhibitory effects of insulin on glucagon secretion, male rats were made diabetic by i.p. injection of 100 mg/kg STZ, which increased glucose to greater than 18 mM and glucagon to 249 +/- 25 pg/ml (P less than 0.05). In these rats, 10 micrograms/kg oxytocin failed to further increase plasma glucose, but caused a much greater increase in glucagon (to 828 +/- 248 pg/ml) and also increased plasma ACTH. A specific oxytocin analog, Thr4,Gly7-oxytocin, mimicked the effect of oxytocin on glucagon secretion in diabetic rats. The lower dose of oxytocin also increased glucagon levels (to 1300 +/- 250 pg/ml), but the effect was not significant. A 3 h i.v. infusion of 1 nmol/kg per h oxytocin in conscious male rats significantly increased glucagon levels by 30 min in normal and STZ-rats; levels returned to baseline by 30 min after stopping the infusion. Plasma glucose increased in the normal, but not STZ-rats. The relative magnitude of the increase in glucagon was identical for normal and diabetic rats, but the absolute levels of glucagon during the infusion were twice as high in the diabetics. To test whether hypoglycemia could elevate plasma levels of oxytocin, male rats were injected i.p. with insulin and killed from 15-180 min later. Plasma glucose levels dropped to less than 2.5 mM by 15 min. Oxytocin levels increased by 150-200% at 30 min; however, the effect was not statistically significant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Respiratory and metabolic effects of decreased osmolality in conscious rats

We investigated the respiratory and metabolic effects of decreased osmolality, and the potential roles of angiotensin II (ANG II) and the subfornical organ (SFO) in mediating these effects, in conscious Sprague-Dawley (SD) rats. Gastric water loading was induced either by oral gavage or an externalized indwelling stomach tube (20 mL x kg(-1) distilled water at body temperature). Repeated measurements after oral gavage were obtained with and without water loading and with and without ANG II receptor block (saralasin, 1.3 microg x kg(-1) x min(-1) iv). At 15 min after water loading by oral gavage, ventilation (V, 1.14+/-0.08 L x kg(-1) x min(-1)) and tidal volume (10.7+/-0.6 mL x kg(-1)) were transiently higher (P < 0.05), at a time when plasma osmolality was decreased (-8+/-1 mOsm), compared with gavage tube alone (0.95+/-0.08 L x kg(-1) min(-1) and 9.1+/-0.7 mL x kg(-1), respectively). However, water loading via stomach tube did not stimulate V; only during the 60-s period of water infusion did V increase briefly, but this was due to increased respiratory frequency. Dye indicators demonstrated that oral gavage exposes upper airway and esophageal afferents to water, presumably accounting for respiratory stimulation. Lesions of the SFO did not affect respiration or metabolism. A decrease in osmolality, associated with both water loading techniques, caused a sustained increase in oxygen consumption (Vo2 ) and a decrease in the V/Vo2 ratio. ANG II receptor block reduced the Vo2 response and prevented the decrease in V/Vo2 following water loading by oral gavage, but did not affect the transient stimulation of V. Unlike larger mammals, decreased osmolality does not stimulate respiration in the SD rat.     

Vascular responses to plasma hypoosmolality in man.

To study limb vascular responses to plasma hypoosmolality in man, we infused test solutions of hypoosmolar NaCl (145 mOsm/kg) and control solutions of isosmolar NaCl (290 mOsm/kg) into the brachial arteries of 14 mornotensive and 13 essential hypertensive patients. Limb blood pressures were monitored, limb blood flow was measured by indicator-dilution, and limb vascular resistance was calculated as mmHg/ml flow/min/100 cm3 limb volume. The infusions did not significantly change systemic plasma osmolality, sodium concentration, or blood pressure. Compared to control infusions, the hypoosmolar infusions decreased limb venous plasma osmolality and serum sodium concentrations by an average of 12 mOsm/kg and 7 mEq/1, respectively. Compared to control infusions, limb venous serum concentrations of potassium, calcium, magnesium, or blood hematocrit were not altered by the hypoosmolar infusions. In response to the hypoosmolar infusions, limb resistance increased by 28% in normotensives and by 26% in hypertensives. We conclude that the acute local vascular response to a small reduction in plasma osmolality in the limb of man is a large increase in vascular resistance. We found no evidence for abnormal responses to plasma hypoosmolality in essential hypertensives.     

Effects of changes in plasma hepatic-portal and systemic osmolality on plasma concentrations of arginine vasopressin in conscious newborn calves

Systemic plasma concentrations of arginine vasopressin (AVP) were studied in three groups of 10-15 day-old conscious newborn calves. Animals in the first group (control group) and in the second group (systemic-hypertonic-injected group) received respectively isotonic and hypertonic (8 mmol NaCl/kg body weight) saline injection into the right jugular vein. Animals in the third group were fitted with chronic mesenteric and hepatic-portal catheters and received a 1 h-hypertonic saline infusion (2 mmol NaCl/kg body weight) into the main mesenteric vein. In animals in the second group there were parallel increases in systemic plasma concentration of Na+ (from 148.0 +/- 2.6 to 177 +/- 8 mmol/l; P less than 0.01), osmolality (from 289 +/- 2 to 319 +/- 4 mOsmol/kg H2O; P less than 0.01) and systemic plasma concentrations of AVP (from 4.2 +/- 0.4 to 11.1 +/- 0.6 pmol/l; P less than 0.01) 10 min after the injection. There were no significant changes in control animals. Hypertonic saline infusion into the main mesenteric vein in the third group induced an increase in concentration of Na+ (from 147.3 +/- 2.0 to 165.0 +/- 5.0 mmol/l; P less than 0.01) and osmolality (from 288 +/- 5 to 315 +/- 10 mOsmol/kg H2O; P less than 0.01) in hepatic-portal vein plasma but did not alter systemic plasma osmolality or concentrations of Na+ and AVP. This study demonstrates that the relationship between plasma concentrations of AVP and systemic osmolality is operative in the newborn calf but does not support the hypothesis that hepatic portal osmo-receptors sensitive to hyperosmolality influence AVP release.     

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.     

Identification and characterization of DNA clones encoding group-II glycinin subunits

Summary DNA clones that encode the group-II subunits of soybean glycinin were identified and compared with clones for group-I subunits. The group-I clones hybridize weakly to those from group-II at low stringency, but fail to hybridize with them at moderate or high stringency. The genes for the group-II subunits are contained in 13 and 9 kb EcoRI fragments of genomic DNA in cultivar CX635-1-1-1. These fragments contain genes for subunits A₅A₄B₃ and A₃B₄, respectively. The larger size of mature group-II subunits compared with group-I subunits is correlated with a larger sized mRNA. However, the gross arrangement of introns and exons within the group-II coding regions appears to be the same as for the genes which encode group-I subunits. Messenger RNA for both groups of glycinin subunits appear in the seed at the same developmental interval, and their appearance lags slightly behind that of mRNAs for the a/a subunits of -conglycinin. These data indicate that the glycinin gene family is more complex than previously thought.Abbreviations bp base pairs - kb kilobase pairs - SDS sodium dodecyl sulfate Cooperative research between USDA/ARS and the Indiana Agric. Expt. Station. This work was supported in part by grants from the USDA Competitive Grants Program and the American Soybean Association Research Foundation. This is Journal Paper No. 10,078 from the Purdue Agricultural Experiment Station