Video: Testis histology

Take a guess – how many sperm cells do you think the testes produce each second? Ten? A hundred? How about 500? What? Still too little? A thousand? Yep – now we're getting somewhere. The testes actually produce 1500 sperm each second. At this rate, just under 130 million sperm are produced each day. Holy moly! Why so many and how on earth does the testis make this happen? In this tutorial, we shall explore the cells and intricate microstructure of the testes which makes all of this possible. Welcome to our video tutorial on the histology of the testes.

During this tutorial, we'll cover all the structures seen in this histological slide – a trichrome stain section of the testis. We'll start with the tunicae, which surrounds the testes. We'll then discuss the internal lobular microstructure of the testes including the many cell types and how to distinguish between them. After we have gained an understanding of the histology, we'll finish with some clinical notes. Let's waste no time and get right into it.

So we all know that the testes are a pair of primary sex organs of the male which are located in the pubic sub-region of the pelvis along with the rest of the male reproductive components. They have two functional roles, which are sperm production and testosterone secretion. Today, we're going to see exactly how the microanatomy of the testis fulfills this function.

The testes are tightly surrounded by coverings which are known as tunicae. There are three of these – the outermost of which is referred to as the tunica vaginalis. This is a dual-layered covering derived from the abdominal peritoneum. It is composed of an outer parietal layer and an inner visceral layer. As is common with histological sections of the testis, the outer parietal layer is not present in our image, but if it were, it would be here separated from the rest of the testis by a small space known as the cavity of the tunica vaginalis.

The visceral layer of the tunica vaginalis, on the other hand, is present, firmly adhering to the underlying tunic which we will look at at a moment. The visceral layer of the tunica vaginalis is formed from mesothelium, which is a simple squamous epithelium which otherwise lines our abdominal cavities and some organs.

The next layer is the tunica albuginea. We can see in the illustration that this is a whitish layer, which is the result of it being rich in collagen. We can also see this layer clearly in our trichrome section. Collagen stains bluish-purple with this kind of stain which makes identifying the tunica albuginea very easy. It is a thick layer which provides strength and elasticity to the underlying softer tissue of the testis. It's worth noting that in this section, the albuginea has separated from the testes, but this is due to processes of tissue preparation. This tunica is closely adhered to the testis in a living individual.

The innermost layer covering the testis is known as the tunica vasculosa and is the least well-defined of the three mentioned. The tunica vasculosa is a thin layer rich in vascular tissue and contains a plexus of blood vessels which we can see here.

Along the posterior border of the testis, the tunica albuginea and vasculosa project internally to form the mediastinum testis. This is a mass of fibrous connective tissue and acts as a sort of gateway for the entrance and exit of blood vessels. The mediastinum testis also houses the rete testis, which we will cover in a minute. We can see in this slide that the mediastinum has been stained the same dark blue color as the tunica albuginea which demonstrates that it, too, is largely composed of collagen fibers and, therefore, provides structural support to the substance of the testis.

If we zoom in a little, we can also see the lumens of several relatively large blood vessels here and here. These are branches of the testicular artery, which infiltrate the tunica vasculosa within the mediastinum to supply the blood to the testis. You may also notice extensions of the connective tissue of the mediastinum testis which appear to extend deep into the testis. These are known as connective tissue septa and have a really important job in bringing nutrients to the inner testis. If we zoom into one example of a septum here, we can see that the septae are composed of collagen fibers from the tunica albuginea which stain blue and we can also see vessels of the tunica vasculosa embedded within the collagen.

If we zoom back out to this slide, we can see that the septa appear to serve another purpose in defining subunits of the testis which are known as lobules. In reality, there are roughly 250 to 300 of these lobules in each testis with those in the center being generally larger than those at the superior and inferior extremities. Don't get confused that only a few lobules are visible here. Remember that this is only a 2D section and these lobules are spread across the entire 3D structure of the testis.

But what are the point of these lobules and what is contained in them? Each lobule contains one to four seminiferous tubules. Of course, as we were looking at the testis in section, it appears that there are much more than that, right? Well, yes, that is correct, but take note of the name. These are highly convoluted seminiferous tubules, meaning, they are folded repeatedly upon themselves within the lobule. So what we are seeing here in our image is several sections through the same group of seminiferous tubules.

Each tubule is approximately 150 to 250 microns in diameter and about 50 centimeters in length giving a combined length of 540 meters of tubule in each testis. This layer results in the seminiferous tubules having a vast internal surface area. Why is this important? Well, it is in these tubules that the magic happens. Yep, this is where all the millions of sperm are made each day. You see, the convoluted seminiferous tubules provide a comfortable environment for spermatogenesis, which is the fancy word for the differentiation of a germ cell into a mature sperm cell. Let's now take a closer look at the cells and tissues around and within the seminiferous tubule to get a better idea of the histology here.

If we zoom in a little bit, we can see that the seminiferous tubules are enclosed by a basement membrane which supports the epithelium of the seminiferous tubule and separates it from the surrounding connective tissue. Note that we're still looking at trichrome-stained sections which gives the basement membrane a bluish-purple colour making it easy to identify. Within the lamina propria of the basement membrane are smooth muscle cells known as peritubular myoid cells, which can be defined by their spindle-shaped appearance. Their exact role is not well-defined; however, it's thought that these cells help to transport mature sperm cells through the tubules by contracting in a peristaltic manner. They also provide additional structural support to the tubules and have a role in the regulation of spermatogenesis.

If you look between the seminiferous tubules, you will see what's known as intertubular or interstitial connective tissue. Among this intertubular tissue, you will find specialized cells known as interstitial endocrine cells, commonly referred to as Leydig cells. As you can see in the image, they are mostly found in clusters among the connective tissue. These cells are characterized by having a central nucleus with lots of surrounding cytoplasm.

As the name interstitial endocrine cell suggests, the function of Leydig cells is to secrete a hormone, namely, testosterone which is responsible for the development of secondary male sexual characteristics and also regulates spermatogenesis. It also affects bone mass, fat distribution, muscle growth, and red blood cell production. These cells are most active during the fetal development of the genital tract in utero and then remain dormant or quiescent until puberty and early adulthood. Conversely, their number decreases with age.

If you have a sharp eye, you'll also notice other cells among the intertubular connective tissue. These are fibroblasts and can be identified by their spindle-shaped nuclei. These function to create the fibrous tissue which makes up the bulk of the intertubular connective tissue.

Let's now turn our attention to the internal structure of the seminiferous tubules which are lined with a specialized epithelium known as spermatogenic epithelium or germinal epithelium. It consists of two types of cells – large non-dividing sustentocytes or Sertoli cells and dividing cells involved in the process of spermatogenesis. Sustentocytes, also known as Sertoli cells, sustentacular cells, or even nurse cells are columnar epithelial cells stretching from the basement membrane to the lumen in the center of the tubule. Although we can't see this using light microscopy, adjacent Sertoli cells are joined by tight junctions creating a diffusion barrier, specifically, the blood-testis barrier.

Yes, I know. Identifying Sertoli cells amongst the thousands of other cells in this slide is not the easiest task, but just remember, that Sertoli cells have an ovoid or triangular morphology and large cytoplasm with one or two large nucleoli which are the stained dots here.

Sertoli cells function to nurse spermatogonia or immature sperm cells through the spermatogenic process. You see, spermatogonia are rather fussy and won't differentiate unless they are nursed. Each Sertoli cell can only nurse a fixed number of spermatogonia – think of it as one nurse being in charge of one ward with a fixed number of patients. So this means that's Sertoli cell number is an important factor in the large variation of sperm count and testicular size between different men. This number is defined during development but can decrease due to lifestyle factors such as smoking.

The Sertoli cell creates a sort of conveyor belt for the differentiation of sperm where spermatogonia reside near the basement membrane and fully differentiated spermatozoa near the lumen. Spermatogonia are undifferentiated male germ cells found at the base of Sertoli cells just on top of the basement membrane. There are three types of spermatogonia. Type A cells with dark nuclei which act as a reserve of stem cells and divide occasionally to form additional stem cells as well as type A cells with pale nuclei which undergo mitosis to produce type B cells.

Type B cells undergo mitotic division to give rise to primary spermatocytes. Primary spermatocytes are diploid cells, meaning they have two copies of each chromosome. They are formed when a diploid spermatogonium, which resides in the basal compartment of the seminiferous tubules, divides mitotically resulting in two diploid primary spermatocytes. Each primary spermatocyte then moves into the adluminal compartment of the seminiferous tubules. Primary spermatocytes have large round nuclei and are larger than spermatogonia. They are positioned roughly halfway between the basement membrane and the lumen. Their nuclei can take euchromatin or loose genetic material, which stains lighter and gives the nucleus a granular appearance.

Secondary spermatocytes are haploid cells, meaning they have only one copy of each chromosome. These cells are very rare to see histologically as they differentiate quickly to form two spermatids; however, they have nuclei that is somewhat more compact. Spermatids are haploid cells which have a small dark-staining nucleus which is round in early spermatids and oval in later spermatids due to DNA condensation. Also, some spermatids will be slightly elongated which marks the appearance of the characteristic flagella or whip-like tail of the mature sperm cell.

Once the spermatids reach the lumen of the seminiferous tubule, they become known as spermatozoa which are the finished product of the Sertoli cell conveyor belt. These are haploid cells and can be identified by the characteristic tail-like flagella and tiny slit-like nucleus. If we look to our trichrome section for just a second, we can get an even better view of these mature spermatozoa with their tails facing the lumen of the seminiferous tubule just here. The process of a spermatocyte developing into a spermatozoa takes approximately two months. If we think about that, it really is amazing that roughly 1500 spermatozoa are created each second.

So that briefly describes the process of spermatogenesis, but if you'd like more information, check out our article on gametogenesis for a lot more information and detail on the early life of a sperm cell.

Once differentiated, sperm are not motile and rely on fluid flow caused by smooth muscle contractions to move through the lumen of the convoluted tubules. This fluid is secreted by Sertoli cells and is rich in nutrients to keep the sperm healthy on their journey out of the testis.

The next structure at which sperm arrive are the straight seminiferous tubules. Luckily for us, despite having a similar name, these tubules are easily distinguished from the convoluted type. The first thing we notice in this slide is that the straight tubules are typically smaller in diameter than the convoluted tubules and have a flattened lumen. Initially, the straight tubules are lined only with Sertoli cells. There are no spermatogenic cells and no spermatogenesis occurs here. Towards the distal ends of the straight tubules, the epithelium will begin to transition from consisting entirely of Sertoli cells to having a simple cuboidal epithelium which is also characteristic of our next structure – the rete testis.

The rete testis is located in the mediastinum testes and consists of a network of anastomosing ducts. These ducts are not surrounded by any smooth muscle and are lined with a simple cuboidal epithelium which appears very flat. These channels are relatively irregular in shape and function to condense the numerous seminiferous tubules into approximately 15 efferent ductules.

All right, that just about covers the histology for the testis. Now that we have an understanding of the normal histology, let's have a look at what happens when all is not as it should be in our clinical notes.

Sertoli cell-only syndrome, also called germ cell aplasia or Del Castillo syndrome, is a disorder of the seminiferous tubules where no spermatogenic cells are present in the epithelium, only Sertoli cells. This means that spermatogenesis cannot occur and sufferers of this condition are infertile. Most cases are idiopathic, meaning, the cause is unknown; however, some cases have a genetic link when there are mutations on the Y chromosome, particularly, in the azoospermia factor.

Sertoli cell-only syndrome patients have normal gross anatomy such as normal-sized testicles and normal levels of testosterone. Consequently, diagnosis will typically only occur after the patient unsuccessfully tries to conceive.

The first step of diagnosis is identifying that the patient is azoospermic, which means that there's a complete absence of sperm in the ejaculate. The second step is a testicular biopsy to confirm the absence of spermatozoa in the seminiferous epithelium. Treatment is only available for men with some spermatogenic cells present. In these cases, microsurgery is performed to extract the sperm. The retrieved sperm are tested for viability and sperm can then be used for in vitro fertilization.

One hundred and thirty million sperm a day – an enormous number which requires a highly specialized structure to produce it.

In this tutorial, we covered the histology of this specialized structure starting with the tunica surrounding the testis. These layers are the tunica vaginalis with its parietal and visceral layers and cavity in the middle which provides cushioning, the tunica albuginea which is rich in collagen and provides protection, and the tunica vasculosa which supplies blood to the external and internal surfaces of the testis. The tunica albuginea and vasculosa then enter the testis from the posterior aspect forming the mediastinum testis. From here, septa project out creating the septa testis which divide the testis into numerous lobules. These lobules are filled with convoluted seminiferous tubules – the home of spermatogenesis. These tubules have a columnar epithelium and are lined with spermatogenic and Sertoli cells. The interstitium is filled with Leydig cells. The sperm produced in the convoluted seminiferous tubules then travel via fluid flow caused by peristaltic contractions to the straight seminiferous tubules and then onwards to the rete testes.

That just about does it for this tutorial. I hope you enjoyed it and happy studying!