ENDOCRINE SYSTEM

Objectives

1. to identify the following on your slides:

2.	to know which hormones the secretory cells of the endocrine system produce, the functions of these hormones and how their synthesis is regulated.

 

Blue Histology

Slides

		D-131	Adrenal, monkey (H&E)
		D-133	Adrenal (Masson stain)
		D-134	Thyroid (Masson stain)
		D-137	Parathyroid (H&E)
		D-138	Thyroid (H&E)
		D-139	Pituitary, cat (H&E)
		D-140	Pituitary, cat (polychrome stain).

Optional slides

		D-125	Adrenal Gland (Chromaffin stain)
		D-132	Adrenal, cat (H&E)
		D-135	Thyroid, monkey (PASH)
		D-136	Thyroid (H&E)
		D-141	Pituitary, rat (H&E)

* * * * *

THE PITUITARY

Prologue

The pituitary has two fundamental divisions, the adenohypophysis derived from oral ectoderm and the neurohypophysis which is an extension of the brain. Each division has three parts; pars distalis, pars intermedia, and pars tuberalis in the former and median eminence, infundibulum and pars nervosa in the latter. These are labeled in the picture, below, of your slide D-140, sectioned sagittally. Unfortunately, there are some synonyms to confuse you. The anterior pituitary/anterior lobe is also called the pars distalis and the pars nervosa is named the posterior lobe. Also, the infundibulum and pars tuberalis together make up the pituitary stalk as a gross anatomical structure.

An important landmark is the series of cysts filled with colloid. These are the remnant of the embryonic cleft. In animals and the human child, the glandular portion of the pars intermedia often appears as a narrow band posterior to the embryonic cleft. In the adult human the cleft is reduced to a series of cysts filled with colloidal material. The glandular tissue of childhood pars intermedia degenerates and becomes vestigial.

The epithelial cells of the adenohypophysis can be grouped into three classes on the basis of staining characteristics; acidophils, basophils and chromophobes. Their colors are due to the presence of stainable secretory granules in the cytoplasm. Each of these three classes is heterogeneous. The basophils include gonadotrops, thyrotrops, corticotrops and residual, melanotrops but these individual cell types are difficult to distinguish by conventional stains. Acidophils include mammotrops and somatotrops, again indistinguishable here. Chromophobes lack granules and stain poorly, hence their name. They may include stem cells and/or stages of acidophils and basophils that have released their granules and not had time to gain them back.

The neurohypophysis is white matter, with axons, glial cells but no neuron cell bodies. The axons descend from two nuclei farther up in the brain, the paraventricular nucleus and the supraoptic nucleus. Their neurons synthesize the peptide hormones oxytocin and antidiuretic hormone (= ADH = vasopressin). Each neuron synthesizes only a single type of hormone but both nuclei have neurons for both. The hormones are packaged into secretory granules and transported down the axons to the pars nervosa. Here they accumulate in the expanded end of the axons which lie up against capillaries. When the neuron fires, its hormone is released as though it were a neurotransmitter and is and picked up by the capillary. The ends of the axons are visible in suitable preserved preparations as packets of secretory granules and are called Herring bodies. This name also applies to clusters of granules that pile up along the axon as they migrate down (in the infundibulum as well as the posterior lobe.

* * * * *

Slide descriptions

D-140 Pituitary, cat (polychrome stain).

Place the slide under the microscope so that the stalk is towards you and examine it under low power to orient yourself. Locate the seven parts listed in the picture above.

Examine the adenohypophysis at low power. Acidophil and basophils cells are distributed nonuniformly. In some regions acidophils predominate, in others, basophils (illustration).  The tissue is highly vascularized and a blue connective tissue capsule surrounds the gland.

Go to high power. The cells are in clusters surrounded by a delicate, blue-stained connective tissue stroma with many capillaries. Three types of parenchymal cells can be distinguished (illustration). Acidophils have a red-orange-colored cytoplasm since their secretory granules take up this stain. (Focus up and down to see the secretory granules) If the section is through the center of a cell, a weakly stained portion of the cytoplasm next to the nucleus can sometimes be seen. This is the location of the Golgi apparatus. The basophils have a blue or purple cytoplasm due to the large number of "basophilic" secretory granules within, and the location of the Golgi apparatus can similarly be detected. Finally, the chromophobes seem to lack a stainable cytoplasm, and the nuclei of adjacent cells cluster closer together owing to the smaller size of these cells.

The pars intermedia is poorly represented, as mentioned above. You will have no difficulty finding the cysts surrounded by a ring of lining cells . Their function, if any, is not known. A few basophils of the pars intermedia may perhaps be seen where they have migrated a short way into the pars nervosa. Do not worry too much about this tissue which almost does not exist on your slide. The optional slide D-141 demonstrates the pars intermedia in a more revealing species.

Turning to the pituitary stalk, the pars tuberalis is a thin layer of adenohypophysis surrounding a core of nervous tissue (illustration). It contains a few basophils but its main significance is in its portal veins (high mag.) that carry hormone releasing factors from the median eminence to the pars distalis. Shut your eyes and list the five segments of this portal system (starting with the arterioles in the median eminence). The hypothalamo-hypophyseal portal veins show up well. They run straight in the plane of section and have bright orange erythrocytes in them. These vessels break up into the sinusoids that surround the acidophils and basophils of the anterior lobe.

Move the slide over to the adjacent nervous tissue in the stalk. It differs markedly from the adenohypophysis in cellular composition (illustration). The bulk of tissue is made up of nonmyelinated axons running more or less in parallel down the stalk. There are no neuron cell bodies in the pituitary. The nuclei that you see are of glial cells which, in this gland, are called pituicytes.

The pars nervosa is similar in appearance and composition to the stalk except that the axons do not run so straight. The Herring bodies are poorly preserved in this slide. Their granules have lysed and the bodies look like amorphous bluish blobs. This is how they frequently look on slides (and in pictures in text books) but do not worry, they are preserved the way they should be on the next slide.

D-139 Pituitary, cat (H&E)

Retrace the features that you looked at in the last slide, now stained with H&E stain. D-139 differs from D-104 in two significant ways;

1. H&E does not distinguish acidophils from basophils. Both types of cells look orangish (illustration). Thus, the terms acidophil and basophil are misnomers. The granules of both types are acidophilic. The stain that differentiates them is PAS (a component of the polychrome stain for D-140). It stains the hormones that are glycosylated and not those that are simple peptides without sugar moieties. Since this leads to some cells ending up purplish while others are orangish, the conventional appellations of basophil and acidophil have been inappropriately extended to them. Collectively, the two together are called "chromophils". The chromophobes are easily distinguished from the chromophils. They continue to stain palely.

So, which of the pituitary hormones are glycoproteins?

2. The Herring bodies are excellently preserved. Scan the pars distalis carefully at high power to find them as oval shaped packets of yellowish granules. Each packet represents the granule-filled end of an axon next to a capillary (which may not be caught in the section). Herring bodies tend to be clustered so scan around (second image). What hormones do their granules contain?

* * * * *

THE ADRENAL

 

D-131 Adrenal, monkey (H&E)

Examine this slide by holding it up to the light. Distinguish the cortex and medulla (if the medulla shows up on your slide). If your slide is cut so far from the midline that it does not show any medulla obtain another slide. The masses of adipose tissue on the outside of the gland are remnants of the copious fat, which typically surrounds the kidneys and adjacent structures . The adrenal cortex has three distinct zones that stain differently: 1) zona glomerulosa, 2) zona fasciculata, and 3) zona reticularis, progressing from exterior to interior (illustration). The zones are variable in thickness, depending on the plane of section. They are traversed by a large number of radially oriented capillaries. These capillaries are enlarged and are best called sinusoidal capillaries but, sometimes, just sinusoids. They take their origin from capsular arteries (which you can see) and run straight through the cortex to the medulla where they become a network of even larger medullary sinusoids.

The layers of the cortex can be visually distinguished in two ways. One is by the orientation of the columns of cells (and hence of the capillaries between them). In the outer zona glomerulosa the cells are arranged in gently curled columns (illustration). The columns straighten out into almost perfectly radial alignment in the zona fasciculata and then become more disorganized again in the zona reticularis.

The layers also differ in the appearance of the cells. The cells of the middle zona fasciculata are largest and palest. A sharp eye looking at high magnification can see that the cytoplasm of these cells looks foamy (X 1000). This is due to the numerous droplets of steroid hormone which filled their cytoplasm until they were leached out during slide preparation. The cells of the two adjacent zones look darker because they synthesize hormones less actively and contain substantially less of them in their cytoplasm.

In order to appreciate the geometrical relationship of the secretory cells to the capillaries it is necessary to look at sections made both parallel to the capillaries (i.e. perpendicular to the surface of the gland) and transversely. In the former case a capillary may be followed in a straight path from near the capsule to almost the boundary with the medulla. The cells form columns one or two cells wide between capillaries. Cross sections give a very different picture, of a sheet of cells perforated by very regularly distributed round capillaries (illustration). You may be able to see both orientations on your slide. However, some of the sections show large areas of cross sections of the sinusoids but few longitudinal sections.

As the blood leaves the zona reticularis it enters the sinusoids of the medulla and bathes the cells there. From there it collects in medullary veins, which are much larger than the sinusoids. If your group has an insatiable curiosity, discuss the several possibilities that this arrangement of the blood supply offers for control of hormone secretion.

One other feature of vasculature that you will have to hunt for on your slides of the adrenal, and still may not see satisfactorily, is medullary arteries (illustration). These small arterioles branch off from capsular arteries and run down the thickness of the cortex from the capsule to the medulla but gain their name from the fact that they deliver fresh blood to the medulla. Thus the cells of the medulla have a direct arterial supply instead of relying only on the blood that the cortical cells have washed their feet in (like the poor Australians in Adelaide who drink the water that the folks all along the Murray River have swum in (or worse)). If you cannot find a sure example wait for the next slide.  It has a trichrome stain so that you can see the small amount of collagen that surrounds these tiny arterioles.

Developmentally, as well as physiologically, the cortex and the medulla are distinct glands. The cortex derives from mesoderm, the medulla from neurocrest. Neurocrest is also the origin for the peripheral nervous system and in several ways the adrenal medulla resembles an organ that started out developing as a sympathetic ganglion and then switched to an endocrine developmental pathway half way along. The cells that would have become postganglionic sympathetic neurons neglected to send out axons or dendrites.  They did develop the enzymes for making catecholamines but just dump their secretions locally as endocrines. Postganglionic sympathetic neurons release norepinephrine (noradrenaline) but the adrenal medulla has two populations of secretory cells, one releasing epinephrine and the other norepinephrine. These two types of cells look very similar. Also, both are stained by the so-called chromaffin reaction specific for the adrenal medulla. (It stains catecholamines.) Therefore, both types are given the same name of chromaffin cells (illustration). They are innervated by preganglionic sympathetic axons.

This developmental just-so story ("How the adrenal medulla got its sympathetic ganglion-like characteristics") offers an explanation for another curious detail of the gland. The medulla has a number of ganglion cells scattered about (i.e. postganglionic sympathetic neurons). It is as though some of the developing neurocrest cells did not get the message to switch over to endocrine-type cells and developed as they normally would in a sympathetic ganglion. You can see a few of them on this slide and perhaps find one or two on the human slide D-133, if you look very hard in just the right spot. Like ganglion cells elsewhere they have large pale nuclei with a prominent nucleolus.

D-133 Adrenal, human (Masson)

D-125, D-132 and D-133 are slides of adrenal with other stains.  Look at them as you did D-131. D-133 is of interest as coming from a human. Be aware that it suffers from what I call the MacDonald hamburger defect. Its small medulla is sandwiched between oversized layers of cortex so that sections cut not close to the middle of the gland end up with only two outer crusts. Your slide may have almost no meaty medulla at all, except for veins. This slide, by the way, offers another chance of finding the medullary arteries of the adrenal.  If you still cannot find a decent example, so not worry about it. Also, if you look exactly in the place indicated above you may see a ganglion cell.

* * * * *

THE THYROID

 

D-138 Thyroid, human (H&E)

Under low magnification note that the thyroid gland is made up of round blank pink structures called follicles. Under higher power observe that a single layer of follicular cells form a simple epithelium around each one. The material inside the follicle is mostly the protein thyroglobulin but often is called "colloid" on histological slides.

Follicular cells have a double secretory function. They secrete thyroglobulin into the follicle in the manner typical of an exocrine process. Enzymes at the cell surface iodinate the tyrosine residues of the protein as it is secreted. The follicle thus becomes a reservoir for the substrate of thyroxin.

The follicular cells also synthesize and secrete thyroxin. They phagocytize the iodinated thyroglobulin from inside the follicle and break it down to form thyroxin, which the cells secrete basally into capillaries in a typical endocrine manner.

You can see evidence of this second process at super high power (illustration). The follicular cells have orangish inclusions in their cytoplasm which are digestion vacuoles of thyroglobulin, pretty neat, eh!

The amount of stroma between follicles is minimal. The scattering of red blood cells indicates the very extensive system of capillaries. Every follicular cell is close to one. Most of the flattened nuclei belong to endothelial cells, except for a few fibroblasts.

You are aware that the thyroid also contains a second type of secretory cell. This is the parafollicular that secretes the hormone calcitonin. These cells are located in the stroma outside of the follicles (as their name suggests) and look pale in comparison to follicular cells (as their alternative name, clear cell = C cell, suggests). Look around for them but do not be disappointed to find only doubtful candidates. There are specific stains for these cells but they are not represented in the slide set.

D-134 Thyroid (Masson stain)

Masson stain emphasizes stroma while still giving good views of cells. It shows that there is a small amount of collagen between adjacent follicles, but not much. Somewhat thicker seams penetrate the gland to provide a pathway for blood vessels. Also, the thin, nondescript capsule around the organ shows up well. The stain here does not provide any new insights about the follicular cells but it is a bit better than H&E for picking out possible parafollicular cells. Ignore the gouges in the colloid as artifacts.

* * * * *

THE PARATHYROID

 

D-137 Parathyroid (H&E)

Under medium power, examine the parathyroid tissue. Most of the parenchymal cells are pale-staining  chief cells = principle cells.  A few larger, darker-stained oxyphil cells can be found if you look hard. I search for them on the basis that, being larger cells, there is a bit of extra space between them and the surrounding nuclei.

Two minutes and you have seen all that is worth seeing of this gland. To be sure the parathyroid has interesting functional aspects but it has to be the most uninspiring looking gland ever slapped down on a histology slide.  The edges of the tissue on some of your slides show that the cells sometimes are clumped into small clusters. So what? Even the cluster of fat cells off to the left is more exciting to look at - and that is not saying much. Of course if you were Sherlock Holmes you might say:

	"Aha, Dr. Watson, I see that this tissue was taken from a young animal"
	"Amazing, Holmes, how did you deduce that?"
	"Elementary, my dear Watson, elementary; in an old animal half of the tissue or more gets replaced by fat cells."

So, what are those more interesting physiological curiosities of the parathyroid, you ask? Well, I ponder over why the C cells of the thyroid and the chief cells of the parathyroid with their complementary functions are located so close to one another and yet not in the same organ. Is it just one of those coincidences of nature that my detective hero, Hercule Poirot, assures us simply do not occur? Or, is there some adaptive reason. Maybe it was important for these hormones to be especially affected by levels of thyroxin and each in its own way got moved to a location close to the thyroid gland.

Well, that  may be dumb, but it is not as dumb as the way the stupid parathyroid looks. Only fibrocartilage is worse. . . . .Or the feeble Australian attempt at humor:

	"Aha, Dr. Watson, I see that you are wearing your red flannel underwear today."
	"Amazing, Holmes, how did you deduce that?"
	"Elementary, my dear Watson, elementary. You forgot to put your pants on."

* * * * *

Optional slides

D-141 Pituitary, rat (H & E)

The purpose of this slide is to show the pars intermedia in a species for which this structure is well developed. Also in the rat, the cleft from Rathke's pouch remains as a continuous potential space instead of being broken up into a series of cysts.

Before you begin on this slide make sure that you understand the geometry of the gland and of this section. This pituitary has been sectioned coronally here giving a cross section at the base of the stalk. The pars nervosa is the lightly stained inner portion. The pars distalis, the largest portion, curves around both sides. The pars intermedia (stained blue) is attached to the pars nervosa and separated from the pars distalis by the cleft (illustration). The connective tissue capsule surrounding the gland has been removed during processing.

Under high power, observe different cell types in the three compartments. The main purpose of this slide is to show the basophils in the pars intermedia (illustration), which are only residual in our species. What hormone do they secrete that humans apparently have little use for?

D-125 Adrenal Gland (Chromaffin stain)

Chromaffin cells are stained yellowish brown by the "chromaffin "reaction in clear contrast with the cortical cells. They are of two types: some secrete epinepherine and some norepinepherine. However, neither the chromaffin stain nor the other stains in your slide set allows you to distinguish them visually.

This is a good slide to look at the medullary sinusoids. They are extended and relatively large. The very largest vessels are considered to be veins; medullary veins.

 

D-132 Adrenal, cat (H&E)

Use this slide to look for the layers and structures that you could not see adequately in your other adrenal slides. It shows nicely the straight sinusoids separating columns of cells of the zona fasciculate.

D-136 Thyroid, Human (H&E)

Slide D-138, which you started off looking at, was cut especially thin. This made it very easy to see the tissue organization. D-136 is a more conventional, thicker wax embedded section (illustration). Look at it to see the appearance of human tissue and to appreciate the joys of the thin sections. Most histology is carried out on thicker wax sections such as this which is why most of your sections are of this sort.

D-135 Thyroid, monkey (PASH)

This slide demonstrates the location of the thyroid in the body. The section is transverse through the trachea at the level of the thyroid. The large circle in the center is the trachea flanked by the two darkly stained purple lobes of the thyroid.

Under low power you will immediately note the intense staining of the colloid with PAS (illustration). What does this tell you about thyroglobulin? The stain also allows you to visualize the basement membranes around the follicles. Follicular cells form a true epithelium. PAS is a poor stain for examining the cells themselves.

You might as well pause a moment and notice the orientation of the trachea vis a vis the esophagus. Next quarter when your professor of gross anatomy tells you that the cartilage free side of the trachea lies next to the esophagus you can say "Of course we know that, we have already taken histology". Think a minute so you can also say "and we also understand the physiological rational for this and we know that the thyroid lies near the top of the esophagus instead of in t he middle".

If, that is if, the parathyroid were shown in this section, where would it be located. Well, do you see it there or not?