Degradation rate of acetylcholine receptors inserted into denervated vertebrate neuromuscular junctions

Many studies exist on the effect of denervation on the degradation of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (nmj). These studies have described the behavior of either the total population of junctional receptors at different times after denervation, or of the receptors present at the time of denervation (referred to as original receptors). No experimental studies yet exist on the degradation rate of the receptors newly inserted into denervated junctions. In the previous studies, the original receptors of mouse sternomastoid muscles were found to retain the slow degradation (t 1/2) of approximately 8-10 d of innervated junctional receptors for up to 10 d after denervation before accelerating to a t 1/2 of approximately 3 d. The total junctional receptors, on the other hand, showed a progressive increase in degradation rate from a t 1/2 of 8-10 d to a t 1/2 of 1 d. To reconcile these earlier observations, the present study examines the degradation of new receptors inserted into the nmj after denervation. To avoid possible contamination of the data with postdenervation extrajunctional receptors, we used transmission electron microscope autoradiography to study only receptors located at the postjunctional fold of the nmj. We established that the new receptors inserted into denervated junctions have a t 1/2 of approximately 1 d, considerably faster than that of the original receptors and equivalent to that of postdenervation extrajunctional receptors. Both original and new receptors are interspersed at the top of the junctional folds. Thus, until all the original receptors are degraded, the postjunctional membrane contains two populations of AChRs that maintain a total steady-state site density but degrade at different rates. The progressive increase in turnover rate of total AChRs therefore reflects the combined rates of the original and new receptors, as earlier postulated by Levitt and Salpeter (1981).     

Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.     

Redistribution of acetylcholine receptors during neuromuscular junction formation in Xenopus cultures

At the adult neuromuscular junction, acetylcholine (ACh) receptors are highly localized at the subsynaptic membrane, whereas, embryonic myotubes before innervation have receptors distributed over the entire surface. Thus sometime during development, ACh receptors accumulate to the nerve contact area. This nerve-induced receptor accumulation can be reproduced in Xenopus nerve-muscle cultures, which provides us with a unique opportunity to investigate the underlying molecular mechanism of this event. Anderson and Cohen (1977) have shown that nerve-induced receptor accumulation is, at least partly, due to migration of pre-existing receptors. It is, thus, plausible that freely diffusing receptors in the membrane are trapped at the nerve-contact region and form clusters. We tested this diffusion trap model. First, receptors in the background region are indeed predominantly mobile and those in the cluster are immobile. Second, the diffusion of receptors in the membrane is fast enough to account for the rate of receptor accumulation. Third, when receptors were immobilized by a lectin, Concanavalin A, the nerve no longer induced receptor accumulation. Thus the diffusion trap model seems adequate to accommodate these observations. Aside from this diffusion mediated mechanism, it is conceivable that newly formed receptors are preferentially inserted at the nerve contact site and these new receptors become immobilized at the site of insertion. To test this hypothesis we stained new receptors separately from old ones and quantitatively compared their distribution. For this purpose we developed a method to quantify fluorescence micrographs. We found that the ratio between old and new receptors was similar at all nerve-induced clusters examined and at the diffusely distributed region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of the neuromuscular junction in the chick embryo: The number,distribution, and stability of acetylcholine receptors

The number, distribution, and stability of skeletal muscle acetylcholine receptors during development of the neuromuscular junction in the chick embryo were studied. The distribution and turnover of receptors labeled with ¹²⁵I-labeled α-bungarotoxin were determined by quantitative autoradiography on individual teased muscle fibers. Each posterior latissimus dorsi muscle fiber, which in the adult is singly innervated, has a high density of acetylcholine receptors at a single spot from embryonic Day 10 through hatching. The spots stain more intensely than elsewhere for acetylcholinesterase and are assumed to be end plates. The receptors at these spots are presumed to be junctional receptors. The junctional receptor density remains constant at 10⁴/μm² from embryonic Day 14 through adult life, although the area of the junction increases 40-fold. In contrast, the extrajunctional receptor density drops precipitously from 250/μm² on Day 14 to only 10/μm² on Day 19. This decrease in extrajunctional receptor density can be prevented by chronic paralysis with curare. The rate of autoradiographic grain loss from junctional and extrajunctional regions after a pulse injection of ¹²⁵I-labeled α-bungarotoxin indicates that both classes of embryonic receptors turn over at the same rate ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 30}\text{hr}$).     

High-resolution localization of acetylcholinesterase at the rat neuromuscular junction

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.     

Regeneration of the active zone at the frog neuromuscular junction

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.     

The Schwann cell at the neuromuscular junction

Synapses obtained in vitro in a system of co-culture of muscle cells and neurons are of embryonic type. We prepared a monoclonal antibody (6.17) which recognizes a molecule synthesized by Schwann cells and used it to show that the main characteristics of maturity (decrase in number of synapses, appearance of junctional folds, and suppression of butyrylcholinesterase expression) are under the control of Schwann cells. In addition, Schwann cells have the capacity to aggregate the acetylcholine receptors in myotube cultures.     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.     

Effect of castration and testosterone administration on the neuromuscular junction in the levator ani muscle of the rat

Summary The ultrastructure of the neuromuscular junction (n.m.j.) of the androgen-sensitive levator ani muscle was studied in normal adult male rats, in 8-month-old rats castrated at the age of one month and in castrated rats treated with testosterone propionate (TP). Castration does not result in significant changes of the n.m.j. The density of synaptic vesicles and the postsynaptic junctional folds remain practically normal in spite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in preand postsynaptic structures. There is slow progressive depletion of synaptic vesicles, appearance of cisternae and coated vesicles in axon terminals, and coalescence of coated vesicles with the plasma membrane. Coated vesicles are also found inside Schwann cells and among junctional folds. Dense core vesicles appear both in the axon terminals and in the postsynaptic area. Collateral sprouting of terminal axons with the formation of new immature junctions is observed. After 35 days of TP administration depletion of synaptic vesicles continues. Glycogen -particles, mostly freely dispersed, occasionally seen in axon terminals 7 days after TP administration, subsequently increase in number. In the endplate zone of the muscle fibre increased protein synthesis is indicated by a rapid increase in ribosomes and irregularly located myofilaments and myofibrils. The appearance of n.m.j. after testosterone administration resembles that described after nerve stimulation; the degree of change is however less pronounced.The authors wish to acknowledge the skillful technical assistance of Mrs. L. Vedralová     

Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

The turnover of acetylcholine receptors labeled with ¹²⁵I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≥ 5}\text{days}$) and different than the rate of extrajunctional receptor degradation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.     

Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction

Exposure of sternomastoid muscles excised from 16-day embryonic rats to medium depleted of Ca²⁺ or containing high concentrations of KCl leads to extensive loss of aggregates of acetylcholine receptors newly formed at the motor end plate region. Upon restoration of Ca²⁺ or removal of excess KCl, receptor accumulations reappear in the central regions of about one-third of the muscle fibers. This susceptibility of junctional AChR aggregates lasts only a short while during development of the neuromuscular junction. By the time of birth, end plate receptor aggregates have become resistant to these treatments.     

Formation and maturation of subneural apparatuses at neuromuscular junctions in postnatal rats: a scanning and transmission electron microscopical study

We examined the morphodifferentiation of subneural apparatuses at neuromuscular junctions with scanning and transmission electron microscopy (SEM and TEM) in the sternothyroid muscle of postnatal rats. As evidenced with SEM, primitive synaptic troughs found at birth were smooth cup-like depressions 5-6 micron in diameter. At the 5th postnatal day, low sarcoplasmic ridges appeared in the depression which successively grew and upheaved to remodel the depression into anastomosed gutters during the next 10 days. Subneural apparatuses attained almost the adult form by the 30th day, though synaptic troughs were smaller in size and exhibited a less complex pattern. At birth, the depression contained a few mostly pit-like or elongated oval invaginations:incipient junctional folds. By the 15th day, junctional folds rapidly developed, resulting in about an 18-fold increase in number per endplate with the parallel differentiation of slit-like junctional folds of adult form. At the 30th day, junctional folds were mostly slit-like, though pits still coexisted in a small proportion. As a shape factor, we measured the ratio of the length of the folds to their maximum width (L/W); the folds with L/W less than 2 were defined as pits, those with 2 less than or equal to L/W less than 5 as short slits, and those with L/W greater than or equal to 5 as long slits. At birth, pits occupied about 67% of the total number of the folds per endplate, which decreased to about 14% at the 30th day. Concomitantly, long slits remarkably increased from about 3 to 38%. Short slits increased from about 30 to 50% during the first 10 days but remained almost unchanged thereafter. The maximum L/W ratio was 12 at the 15th day and exceeded 20 after the 30th day. These quantitative data and the finding that pits were often closely associated with each other and also with a slit in a serial fashion indicate that the adjacent pits may fuse to each other and to the preformed slits. With TEM, a few incipient junctional folds were found at the 5th day, which extended into the subneural sarcoplasm with a depth less than 0.4 micron. At the 15th day, junctional folds increased both in number and in the maximum depth of about 0.8 micron. There also occurred a number of basal lamina-containing vacuoles identical in many respects to the transversely sectioned profiles of incipient junctional folds.(ABSTRACT TRUNCATED AT 400 WORDS)     

A voltage clamp study of the glutamate responsive neuromuscular junction in Drosophila melanogaster

The point and single electrode voltage clamp methods have been used to study the characteristics of junctional currents in Drosophila melanogaster larvae muscle fibers and the modulation of these currents by excitatory amino acids, short and long chain n-alkanols, and pentobarbital. The decay phase of junctional currents in Drosophila was found to be dominated by cooperativity in transmitter binding associated with reverberation, that is, repeated binding of transmitter with receptors as the transmitter molecules diffuse away from the active region. The current decay does not directly reflect the closure of ion channels and is qualitatively similar to the decay of miniature end-plate currents at the mouse neuromuscular junction after poisoning of acetylcholinesterase by paraoxon. In Drosophila an increase in membrane hyperpolarization both slows the time course of current decay and increases the degree of reverberation. The application of excitatory amino acids including glutamate, N-methyl-D-aspartate, quisqualate, and kainate causes a significant decrease in the amplitude of the junctional currents, a prolongation of the decay time course, and a reduction in reverberation of transmitter. The height of junctional currents is also diminished by the n-alkanols ethanol, pentanol, and octanol and by the barbiturate pentobarbital; ethanol also hastened the time course of decay of the currents.     

Development of rat soleus endplate membrane following denervation at birth

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.     

Histochemistry of 11-beta-hydroxysteroid dehydrogenase in rat submandibular gland. Effect of cortisol stimulation.

Synopsis The activity pattern of NAD/NADP-linked 11-hydroxysteroid dehydrogenase (HSD) in the submandibular gland of the rat was re-evaluated using several control experiments. The incubation time needed for the initial appearance of red and blue formazans was used to investigate the activity of NAD-dependent 11-HSD in control and cortisol-treated rats. The following results were obtained. (1) Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH 7.2) for up to 20 min preserved morphological integrity and maximal enzyme activity. The substantivity of formazans was enhanced. (2) The substantivity of Nitro BT was highly variable. The implication of this forin situ localization of enzymes was analysed. (3) Pretreatment with acetone and application of phenanthroline was necessary to avoid a false positive reaction caused by alcohol dehydrogenase. (4) No diffusion of 11-HSD was noticed within 30 min of incubation, nor was rediffusion of reduced intermediates seen. (5) With either NAD or NADP as coenzyme, 11-HSD was localized in the striated, intralobular, interlobular, interlobar, and main duct. (6) 11-HSD was found to be primarily NAD-dependent. (7) With DMF or DMSO as solvent, the rate of utilization of substrates was as follows: Cortisol=11-hydroxyandrostendione>11-hydroxyprogesterone. Aldosterone was utilized very poorly, if at all. (8) After injection (i.p.) of a single pharmacological dose of cortisol, the activity of NAD-linked 11-HSD was significantly increased 24 h later. (9) NADH-tetrazolium reductase was not inhibited by levamisole. (10) Distinct NADPH-tetrazolium reductase activity was localized in the apical part of cells (or cell membranes) of the interlobar ducts and the main duct.     

Transients in acetylcholine receptor site density and degradation during reinnervation of mouse sternomastoid muscle

The degradation rates of acetylcholine receptors (AchRs) were evaluated at the neuromuscular junction during and just after reinnervation of denervated muscles. When mouse sternomastoid muscles are denervated by multiple nerve crush, reinnervation begins 2-4 days later and is complete by day 7-9 after the last crush. In fully innervated muscles, the AChR degradation rate is stable and slow (t1/2 approximately 10 days), whereas after denervation the newly inserted receptors degrade rapidly (t1/2 approximately 1.2 days). The composite profile of degradation, which a mixture of the stable and the rapid receptors would give, is not observed during reinnervation. Instead, the receptors inserted between 2.5 and 7.5 days after the last crush all have an intermediate degradation rate of t1/2 approximately 3.7 days with standard error +/- 0.3 days. The total receptor site density at the endplate was evaluated during denervation and during reinnervation. As predicted theoretically, the site density increased substantially, but temporarily, after denervation. An analogous deleterious substantial decrease in density would be expected during reinnervation, without the intermediate receptor. This decrease is not observed, however, because of a large insertion rate at intermediate times (3000 +/- 700 receptor complexes per micro m2 per day). The endplate density of receptors thus remains relatively constant.     

Hormonal regulation of cell junction permeability: Upregulation by catecholamine and prostaglandin E1

Summary By cellular activation with hormones, we test the proposition (Loewenstein, W.R.,Physiol. Rev. 61:829, 1981) that the permeability of cell junction is upregulated through elevation of the level of cyclic AMP. Cultured rat glioma C-6 cells, with -adrenergic receptors, and human lung WI-38 cells, with prostaglandin receptors, were exposed to catecholamine (isoproterenol) and prostaglandin E₁, respectively, while their junctions were probed with microinjected fluorescent-labelled mono-, di-, and triglutamate. Junctional permeability, as indexed by the proportion of cell interfaces transferring the probes, rose after the hormone treatments. The increase in permeability took several hours to develop and was associated with an increase in the number of gap-junctional membrane particles (freeze-fracture electron microscopy). Such interaction between hormonal and junctional intercellular communication may provide a mechanism for physiological regulation of junctional communication and (perhaps as part of that) for physiological coordination of responses of cells in organs and tissues to hormones.     

SUBSTRUCTURE OF AMPHIBIAN MOTOR END PLATE : Evidence for a Granular Component Projecting from the Outer Surface of the Receptive Membrane

End-plate membrane has been examined at amphibian myoneural junctions by means of transmission electron microscopy of thin tissue sections. The postjunctional membrane exhibits morphologically specialized dense, convex patches which are located superficially facing the axon terminal but do not extend into the depths of the junctional folds. In the specialized regions the plasma membrane is ~ 120 Å thick and trilaminar. The outer dense lamina is thickened by the presence in it of granular elements ~60–120 Å in diameter which are spaced semiregularly at ~100–150-Å intervals and which border the junctional cleft directly. In these regions the concentration of the granules is of the order of ~ 10⁴/µm², which is in the same range as the estimated concentration of receptor sites at other vertebrate cholinergic junctions. Filamentous projections can sometimes be seen extending from the granules to the overlying basement membrane, and in oblique views a reticular pattern may appear both in these patches and in the basement membrane. The cytoplasmic surface of the specialized membrane is covered with an amorphous and filamentous dense material whose distribution coincides with that of the granules visible in the outer layer and which may be connected to them across the membrane. In unosmicated specimens stained with permanganate and uranyl acetate the specialized regions exhibit the same morphological features but stand out sharply in contrast to adjacent areas of unspecialized membrane which appear only faintly. Such preparations are particularly useful in assessing the extent of the specialized membrane. It is proposed that the granules visible at the outer surface of the end-plate membrane represent acetylcholine receptors and that in amphibians, as in annelids, the receptors at myoneural junctions are concentrated into patches which occupy less than the total postjunctional membrane surface area.     

Synaptic current between neuromuscular junction folds

Measurements of membrane infoldings of vertebrate subsynaptic membranes were taken to evaluate the possible electrophysiological implications. The shapes of standard interfolds of different neuromuscular junctions were established from micrographs available in the literature. Electrical properties were estimated using published fibre membrane and myoplasm electrical values. Models of synaptic current pathways were designed taking into account the small size of the postsynaptic patch activated by a transmitter quantum. This analysis reveals a resistance "in series" between the ACh-sensitive interfold crest and the remainder of the muscle fibre. The calculated cytoplasmic resistance of an interfold is between 0.2 and 3 Mohms which is in the same range as the fibre DC input resistance. The calculated interfold resistance appears to be dependent on the fibre type, the age and the pathology. Functional roles of junctional folds and dendritic spines are discussed.     

The bovine tubouterine junction: general organization and surface morphology

The bovine tubouterine junction is composed of three parts (terminal tubal segment, transition region proper, uterine apex) and follows a sigmoidal course displaying a tubal and an uterine curvature. In the terminal tubal segment, 4–8 primary longitudinal folds and a system of lower secondary folds, ridges and chords project into the centrally located lumen. The transition region proper possesses a slit-like lumen because of the existence of a thick mucosal pad containing the first uterine glands. The longitudinal primary folds of the tube broaden, flatten and start to diverge when they reach the transition region proper. The mucosal pad and broadened folds are heavily vascularized. A system of lateral outpocketings with blind ends pointing in an ampullary direction develops between the primary and secondary folds, the ridges and chords of the terminal tubal segment and transition region proper. From the bottom of these outpocketings, short tubulo-alveolar crypts originate. The mucosa of the uterine apex forms low transversal ridges. The musculature of the bovine tubouterine junction is divided into a continuous circular or spiral intermediate layer, flanked by inner and outer longitudinal layers. The outer longitudinal layer is incomplete in the terminal tubal segment but increases in thickness to form a continuous stratum in the uterine apex. An inner longitudinal layer occurs only in the terminal tubal segment where it is best developed in the bases of the primary longitudinal folds. The simple columnar surface epithelium of the tubouterine junction contains ciliated and non-ciliated cells. The former undergo cyclical changes, and increase during estrus and postestrus. During proestrus, groups of non-ciliated cells display bulbous apical protrusions. During proestrus and estrus, circumscribed epithelial lesions expose the underlying basal lamina.