Equine anatomy from the seminiferous tubules to the ductus deferens.
Keywords: seminiferous, tubule, testis, testes, epididymis, ductus, deferens, caput, corpus, cauda, spermatozoa, spermatozoon, sperm, efferent, stallion, Leydig, spermatogonia, Sertoli
For gross images of the testis and adnexa, see the LORI entry on reproductive aspects of stallion anatomy. This entry addresses the applied histology of the testis and its adnexa.
This is a substantial entry and should be viewed as a project in progress. Images will be added or replaced with time. The entry will improve under the scrutiny of colleagues.
Because of their combined use in common conversation, the author uses a combination of Latin and English terminology in this entry. For example, the terms caput, corpus and cauda are used in preference to head, body and tail. Although the term vas deferens appears about 35 times more frequently than the term ductus deferens in literature searches, this is no doubt due to its prevalence in human oriented literature. The Nomina Anatomica Veterinaria uses the term ductus to the exclusion of vas deferens. Therefore the term ductus deferens is used in this entry. In addition, eponyms are capitalized; a gradual editorial change throughout LORI. The author begs the understanding of purists.
Overview
Figure 1. A simplified overview of the testis, epididymis and ductus deferens. The cranial aspect of the testis lies on the left side of the diagram. Size available: 2478 x 1645px
Although seminiferous tubules join major rete tubules as shown, this image is a simplification. In reality, loops of seminiferous tubules actually end throughout the testes, joining tubules of the rete system throughout the testis. The word "rete" is derived from the Latin word for net. This is appropriate because the rete tubules form a net-like system throughout the testis. Small rete tubules then run centripetally to major rete ducts as shown. Larger rete ducts then course cranially within a poorly defined mediastinum, to the efferent duct system.
For gross images of the testis and adnexa, see the LORI entry on reproductive aspects of stallion anatomy. This entry addresses the applied histology of the testis and its adnexa.
This is a substantial entry and should be viewed as a project in progress. Images will be added or replaced with time. The entry will improve under the scrutiny of colleagues.
Because of their combined use in common conversation, the author uses a combination of Latin and English terminology in this entry. For example, the terms caput, corpus and cauda are used in preference to head, body and tail. Although the term vas deferens appears about 35 times more frequently than the term ductus deferens in literature searches, this is no doubt due to its prevalence in human oriented literature. The Nomina Anatomica Veterinaria uses the term ductus to the exclusion of vas deferens. Therefore the term ductus deferens is used in this entry. In addition, eponyms are capitalized; a gradual editorial change throughout LORI. The author begs the understanding of purists.
Overview
Although seminiferous tubules join major rete tubules as shown, this image is a simplification. In reality, loops of seminiferous tubules actually end throughout the testes, joining tubules of the rete system throughout the testis. The word "rete" is derived from the Latin word for net. This is appropriate because the rete tubules form a net-like system throughout the testis. Small rete tubules then run centripetally to major rete ducts as shown. Larger rete ducts then course cranially within a poorly defined mediastinum, to the efferent duct system.
Although the mediastinum carries a large central vein (see figure 4), the mediastinum is not nearly as obvious as it is in ruminants. Interestingly, there is no central artery in an equine testis; arterial supply is served via several peripheral arteries.
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The rete is best developed towards the cranial pole of the testis where the total production of spermatozoa accumulates. As discussed later, the rete actually originates as part of the mesonephric system, not the primitive gonad. Therefore, rete tubules penetrate the testis during early embryonic development.
The label "Anastomosing rete tubules" in the illustration draws attention to the fact that parts of the rete tubule system fuse in that area. These rete tubules form sinus-like cavities (see figure 28) both inside and outside the tunica albuginea. The sinuses in turn, become efferent ducts in both the testis and caput epididymis. Downstream, the efferent ducts become a single tube, the epididymis.
Orientation of testis adnexa within the scrotum
In primates and ruminants the longitudinal axes of the testis is vertical. In those animals, the caput epididymis lies dorsal to the testis, the cauda lies ventral to the testis. In horses and dogs the testes "swing" in a caudal direction during embryonic development so that the cauda comes to lie caudal to the testis, roughly on the same plane as the caput. In cats, camelids and pigs that movement is even greater, so that the cauda lies above the caput. See these approximations in figure 2.
In all animals the ductus deferens runs medial to the epididymis. In horses, this is on the dorsal aspect of the testes (see figures 2, 3 and 4). In the rare instance that one should perform a vasectomy on an equid (in zoo animals for example) this should be born in mind; a ventral paramedian incision is required to access the ductus on the correct side of the vaginal cavity. See this entry on vasectomy for clarification.
For castration, local anesthetic is often injected directly into the testes to provide additional analgesia during general anesthesia. Indeed, studies show that blood pressure and heart rates remain lower in subjects so treated. See Haga et al. 2006. Similar effects have been shown in dogs and cats. The artifact created by local anesthetic is clearly visible in this image.
Incidentally, a "closed" castration technique was used to prevent post operative evisceration in this case. This was fortuitous because the parietal vaginal tunic is still present in this specimen, showing an intact vaginal cavity. That would not have been the case if open castration had been used. In open castrations, the parietal vaginal tunic is transected as the incision is made through the scrotal skin, into the testis. In closed castrations, the scrotal skin is incised but the parietal vaginal tunic is stripped away from the tunica dartos to ensure that vaginal cavity remains intact. This allows for easy closure of the inguinal canal, preventing post-operative evisceration.
Puberty
During fetal development, Sertoli cells aggregate around clusters of germ cells in the genital ridges. These cells are known as gonocytes (the precursors of spermatogonia) and in linear clusters, they form gonadal cords. The testes, so formed, descend into the scrotum within the vaginal pouch. Gonadal cords, persist up to puberty when they become canalized and forming seminiferous tubules.
Remember to double click all the images in LORI to appreciate the detail under discussion.
Figure 5. Gonadal cords in a specimen taken from a ten month old Standardbred colt. Size available: 2496 x 1113px
Inside the cords, Sertoli cells surround gonocytes, shielding them from the effect of retinoic acid that is circulating in the interstitium. Retinoic acid is an oxidation product of Vitamin A. It is known to induce maturation of cells throughout the body, especially in the bone marrow and testes. In the testes, retinoic acid initiates the division of gonocytes during puberty. After puberty, retinoic acid controls the maturation and division of spermatogonia themselves. Were it not for the protection from retinoic acid afforded by the Sertoli cells, gonocytes would undergo meiosis prematurely. In the gonadal cords therefore, there is no meiosis or mitosis; only solid aggregations of Sertoli cells and gonocytes.
During the gradual process of puberty, gonadal cords canalize under the effect of testosterone from Leydig cells in the interstitium, forming seminiferous tubules. That effect is seen in figure 6. Seminiferous tubules do not mature simultaneously throughout the testis. Maturation usually starts at the center of the testis, progressing towards its periphery. Areas of canalized tubules can even be seen macroscopically as lighter areas of tissue on the cut surface of a testis.
Figure 6. Maturation of seminiferous tubules in a 10 month old peripuberal colt. Size available: 2384 x 1471px
Although a central-to-peripheral pattern maturation pattern appears to be common, testes may also mature in no fixed pattern or even peripheral-to-central in some individuals. The Standardbred colt samples for figure 5 was such an example. Because meiosis was not present in solid areas of the testes of this colt but was seen in canalizing areas, the author was assured that this was indeed a physiological effect and not an artifact due to injection. See figure 7.
Figure 7. Peripheral-to-central maturation of seminiferous tubules in a 10 month old peripuberal colt. Size available: 1362 x 921px
In view of the fact that puberty initiates meiosis and at that time, the blood-testis-barrier is first tested against haploid cells, it is not surprising to see occasional infiltrations of lymphocytes within a peripuberal testis. Perhaps this formalizes the uniquely privileged immune system in testes. To the author's knowledge, this phenomenon has not be discussed in the literature. These infiltrations were visible in figure 7 and in sections from other peripuberal testes.
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In peripuberal testes, where spermatogenesis is just beginning, the typical cell associations of spermatogenesis seen in mature testes are not yet evident. In canalizing tubules the process is irregular. In that regard, see figure 8, a section from the Standardbred colt mentioned above. The division between active seminiferous tubules and non-canalized cords is obvious
Figure 8. Division between active seminiferous tubules and non-canalized cords in a 10 month old peripuberal colt. Size available: 1919 x 1157px
The apoptosis that normally occurs amoung dividing spermatogonia in mature stallions is also noticeable in peripuberal testes. See figure 9. Pyknotic nuclei commonly seen amoung immature spermatocytes substantiated that impression. Also in figure 9, notice the inactive tubule lying adjacent to one that is beginning to show meiosis.
Figure 9. Active and inactive tubules in a peripuberal stallion; apoptosis and the onset of meiosis. Size available: 2314 x 1268px
The mature stallion
Before the intricacies of spermatogenesis are illustrated and discussed, readers may be interested to see an overview of the seminiferous epithelium from a healthy six year old Morgan stallion taken during the breeding season. This is provided in the form of a virtual microscopy section. Cells in and around the seminiferous epithelium in mature stallions
What follows is an introduction to some of the cells visible in figure 10, a virtual microscopy section. The concept of stages of spermatogenesis is also introduced but a substantial explanation follows later in the entry. After the ensuing discussions, one may wish to peruse the section again from a renewed perspective.
Figure 10. Click here (NOT on the image) to view the virtual microscopy section. Once the image appears in your browser window, use the scale above the image to select the desired magnification. Magnification may also be selected by placing the cursor over the image and rolling the mouse wheel. To move within the image, left click and drag to a position of interest.
Important! This virtual section uses Adobe Flash. Chrome is a great browser but it makes the use of Flash difficult. Google does this to promote HTML5. If you have problems viewing the virtual image, use Firefox or Opera browsers. On Apple platforms, use Safari. After 2020, HTML5 will be adopted universally and this site will be amended accordingly.
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Spermatogonia:
A complete discussion of spermatogonia is complex and beyond the scope of this entry. Suffice to say that these are diploid cells formed from gonocytes during puberty. After puberty, they form a population of spermatogonia along the basement membranes of the seminiferous tubules. Figure 11 shows spermatogonia undergoing mitosis (arrows); multiplying along the basement membrane of a semiferous tubule.
Image 11. Spermatogonia undergoing mitosis along the basement membranes of seminiferous tubules. Size available: 514 x 428px.
From these spermatogonia, a sub-population is recruited to become spermatozoa at intervals typical of the species in question. In the case of the stallion, spermatogonia are recruited from the same location in a seminiferous tubule at intervals of 12.2 days. In the parlance then, a stallion has a seminiferous cycle of 12.2 days.
Figure 12 shows two forms of spermatogonia present along the basement membranes in adjacent areas of the same seminiferous tubule. From known associations between cells during spermatogenesis (see later) these were identified as B-2 and A-2 spermatogonia.
Figure 12. Two forms of spermatogonia present along the basement membranes in adjacent areas of the same seminiferous tubule. Size available: 1556 x 872px.
A complete discussion of spermatogonia is complex and beyond the scope of this entry. Suffice to say that these are diploid cells formed from gonocytes during puberty. After puberty, they form a population of spermatogonia along the basement membranes of the seminiferous tubules. Figure 11 shows spermatogonia undergoing mitosis (arrows); multiplying along the basement membrane of a semiferous tubule.
From these spermatogonia, a sub-population is recruited to become spermatozoa at intervals typical of the species in question. In the case of the stallion, spermatogonia are recruited from the same location in a seminiferous tubule at intervals of 12.2 days. In the parlance then, a stallion has a seminiferous cycle of 12.2 days.
Figure 12 shows two forms of spermatogonia present along the basement membranes in adjacent areas of the same seminiferous tubule. From known associations between cells during spermatogenesis (see later) these were identified as B-2 and A-2 spermatogonia.
Light-staining spermatogonia are recruited from constantly renewing populations on the basement membranes of seminiferous tubules. Although data are not certain in this regard, there are probably five successive generations of spermatogonia. After several successive A sub-type generations, two successive B generations form. B type spermatogonia have dark staining nuclei as shown here. The second B generation then divides to form preleptotene spermatocytes.
Sertoli cells:
The recruitment, growth and fate of spermatogonia are controlled by Sertoli cells.
Sertoli cells play an important role in recruiting resting spermatogonia into an unforgiving, one-way path to spermatogenesis. The path is unforgiving because most of the unsuspecting recruits will actually undergo programmed cell death (apoptosis) and be phagocytosed by the Sertoli cells i.e. the very cells that have sustained them. Why this act of stepmother-infanticide occurs is not known but is presumed to be a result of genetic infirmity amoung the huge population of spermatogonia that are recruited from the basement membrane. Although good examples of apoptosis do occur occasionally (see figure 9) apoptosis are not common in histological sections. At the light level anyway, phagosomes containing remnants of spermatogonia are not seen either. Readers may wish to convince themselves of these facts by searching for apoptic spermatogonia in the virtual slide. This phenomenon may reflect the phagocytic efficiency of Sertoli cells.
Sertoli cells:
The recruitment, growth and fate of spermatogonia are controlled by Sertoli cells.
Sertoli cells play an important role in recruiting resting spermatogonia into an unforgiving, one-way path to spermatogenesis. The path is unforgiving because most of the unsuspecting recruits will actually undergo programmed cell death (apoptosis) and be phagocytosed by the Sertoli cells i.e. the very cells that have sustained them. Why this act of stepmother-infanticide occurs is not known but is presumed to be a result of genetic infirmity amoung the huge population of spermatogonia that are recruited from the basement membrane. Although good examples of apoptosis do occur occasionally (see figure 9) apoptosis are not common in histological sections. At the light level anyway, phagosomes containing remnants of spermatogonia are not seen either. Readers may wish to convince themselves of these facts by searching for apoptic spermatogonia in the virtual slide. This phenomenon may reflect the phagocytic efficiency of Sertoli cells.
The cell walls of Sertoli cells are virtually impossible to discern using light microscopy. This is because their walls are in extremely close association with the cells that surround them. In the main image of figure 13, the author has attempted to illustrate Sertoli cell outlines by applying a transparent gray layer over two adjacent Sertoli cells.
Note that spermatogonia, spermatocytes and spermatids are surrounded by Sertoli cells. Even sperm are initially surrounded by crypts in the apical region of Sertoli cells. Then, during the last days of spermiogenesis (the process of forming sperm from secondary spermatocytes) they hang by tenuous cell-to-cell connections with the Sertoli cells, within the lumens of seminiferous tubules. In humans and primates it is known that a single Sertoli cell can sustain between 10 and 25 germ cells and up to three spermatozoa. The author is unaware of similar data in stallions.
Sertoli cells are simply marvelous. To reiterate, they support and select generations of spermatogonia undergoing mitosis to form primary spermatocytes. It is only as the selected spermatogonia become pre-leptotene spermatocytes, entering meiosis, that their surface antigens are recognized by Sertoli cells as beginning to differ from diploid cells. They are then admitted through the tight cell junctions into the adluminal compartment. After the process of selective destruction of most spermatogonia, Sertoli cells allow only the chosen few into that compartment.
Spermatogenesis consists of three phases: spermatocytogenesis, meiosis (including its mitotic phase) and spermiogenesis, which includes spermiation. The author has cut and pasted cells from images of various seminiferous tubules, inserting them into this image at equal magnification. Figure 14 shows their relative sizes, their appearance under light microscopy and their DNA content.
This sequence is never seen in reality because all of these cell types are never found simultaneously in a single cross section of a seminiferous tubule. The reason for this becomes clear later.
Size available: 844 x 742px.
Legend: Sc.gen = spermatocytogenesis. Sp.genesis = spermiogenesis
A quick reminder:
Meiosis consists of a meiotic phase followed by a mitotic phase. The DNA content of each new spermatocyte doubles in preparation for the pairing of homologous chromosomes. Then, double-DNA chromatids (including those of the X and Y chromosomes) cross over and exchange DNA for genetic diversity. All of this is accomplished during leptotene, zygotene, pachytene and diplotene. After the exchange of chromosomal information in diplotene, the paired homologous chromosomes (still containing double the amount of DNA as a somatic chromosome) separate into single chromosomes. During diakinesis, the primary spermatocyte divides into two cells with half the number of chromosomes of a normal somatic cell, essentially making them haploid cells (n). However, these cells still contain twice the amount of DNA of haploid spermatids because their DNA was doubled in the very first stage of meiosis.These are secondary spermatocytes. Note their size in figure 14 and convince yourself that their nuclei are indeed twice the volume* of the spermatids. Secondary spermatocytes then undergo the mitotic phase of meiosis. In somatic cells, doubling of DNA for mitosis is a time consuming process. However, during spermatogenesis, doubling of DNA has already occurred during leptotene. As a result, this mitotic event far more rapid during spermatogenesis that somatic cell division. Therefore secondary spermatocytes divide into spermatids in a very short time. This explains why secondary spermatocytes are only visible for single stage in equine spermatogenesis. In some species, secondary spermatocytes are seldom visible on histology. Diakinesis is also a rapid transitional phase. In fact it is not even long enough to be part of a single discrete stage of spermatogenesis in stallions. *Using the scale on this image and the formula relating radius to volume (V=4/3πrcubed), the author measured the approximate volume of the nuclei of primary and secondary spermatocytes, then spermatids. As expected, the volume of the nuclei halved in each successive stage. |
| More on the amazing Sertolis: Sertoli cells produce androgen binding protein and bind testosterone at the high concentrations required for spermatogenesis and various other processes. They also aromatize testosterone into estrogen for important roles it performs in spermiation and later, to regulate fluid absorption in the head of the epididymis. Sertoli cells regulate the secretion of FSH and therefore its effect on the division of spermatogonia by producing the hormone inhibin. Sertoli cells also produce anti-Mullerian hormone and activin. Other hormones have been identified as being of Sertoli origin as well. Sertoli cells also secrete that complex tubular fluid mentioned in the previous paragraph i.e.the torrent that tears spermatozoa from the nurturing grip of Sertoli cells. Admittedly this fluid will be changed considerably by the epididymis, but consider how specialized it must be to sustain the transcriptionally helpless spermatozoa en route to the outside world! Finally, bear in mind the chameleon-like nature of Sertoli cells with regard to the immune system: Although Sertoli cells are diploid and extend into the haploid environment of the tubule, they do not convey that immunological fact to the environment between the tubules. On the contrary, amoung their many talents, Sertoli cells also have a local immunosuppressive effects in the testes. Indeed, allografts in the interstitial tissue are usually well tolerated, while the same grafts are vigorously rejected elsewhere in the body, even in the epididymis. This is interesting because it indicates that immunological protection is less profound in the epididymis than the testis. |
More on the cells of spermatogenesis
Note the pre-leptotene spermatocytes in figure 15. These cells are beginning to undergo chromosome contraction and double their DNA. They have larger and darker nuclei than spermatogonia but the chromosomes in their nuclei have yet to develop the ribbon-like appearance of leptotene. The reader may recall that pre-leptotene spermatocytes are the first cells that are eligible to pass through the tight cell junctions of Sertoli cells en route to the adluminal compartment. The wonder of inter-cell signalling that makes this possible is not yet understood.
Compare the nuclei of pre-leptotene primary spermatocytes to those of the leptotene/zygotene spermatocytes within the tip of a bend in a seminiferous tubule.
Figure 15. Pre-leptotene primary spermatocytes and leptotene/zygotene spermatocytes within the tip of a bend (see ring) in a seminiferous tubule. Size available: 1734 x 979px.
Compare the nuclei of pre-leptotene primary spermatocytes to those of the leptotene/zygotene spermatocytes within the tip of a bend in a seminiferous tubule.
Leptotene spermatocytes:
Primary spermatocytes in leptotene < Greek: lepto (fine) & tene (ribbon or band) are characterized by having chromosomes that may indeed resemble a cluster of fine ribbons in the imagination of an histologist. More prosaically, they resemble thick, dark wet-mops.
Because the cell combination in figure 16 is in stage II of spermatogenesis, it is possible to say with some assurance that the ringed cells are primary spermatocytes in the leptotene stage of meiosis. If the resolution of the image permitted it, the shortened chromatids of each chromosome could be seen. The chromatids would also be thicker than otherwise, having just doubled their DNA content.
Figure 16. Stage II of spermatogenesis. The ringed cells are primary spermatocytes in the leptotene stage of meiosis. Size available: 1356 x 591px.
Zygotene, pachytene and diplotene spermatocytes:
Again, using the cell combinations present in a cross section of a seminiferous tubule and the expected appearance of various stages of spermatocytes in meiosis, one is able to identify cells with some certainty. In that manner, primary spermatocytes in the zygotene, pachytene and diplotene stages of meiosis have been identified in figure 17.
Primary spermatocytes in the zygotene stage of meiosis are present with those in diplotene in only one stage of spermatogenesis in stallions i.e. stage III. The lower tubule in this image is in stage III, explaining the presence of both zygotene and diplotene cell types in that cross section.
A return to the virtual slide at the beginning of this entry will convince the viewer that spermatocytes in the pachytene stage of meiosis are very common in cross sections of the testis. This is because of the long duration of that stage of meiosis. In fact, in stallions, seven of the eight stages feature pachytene spermatocytes (See Fig. 18 below).
Seasonality and the effect of aging:
Although spermatogenesis declines during short-day seasons, all tubules show active spermatogenesis throughout the year in domestic stallions. The same appears to be true of donkeys, E. Przewalski and probably other equids (although data are scarce). This is in contrast to many wild ruminants where spermatogenesis may cease altogether in the non-breeding season.
Although spermatogenesis and the fertility of spermatozoa is maintained in stallions during short day seasons, there is a substantial decrease in spermatozoa production. Apparently there a relative shift in the numbers of one type of spermatogonium compared to another during seasonal transitions but that phenomenon is beyond the scope of this discussion.
As a stallion ages and maturity approaches (at four to five years of age) the diameter of seminiferous tubules and spermatozoa production continues to increase. The number of Leydig cells in the interstitium increase as well and continues to do so, even into old age.
An explanation of the stages of spermatogenesis:
Figure 18: The stages of spermatogenesis in stallions. Modified and used with permission. Authors: Drs L. Johnson and D. Varner TAMU. Size available: 1500 x 1115px.
Figure 19. Various stages of spermatogenesis. Size available: 2146 x 1140px.
Notice the difference between the various cross sections of seminiferous tubules. Although this holds little practical importance for practitioners, a knowledge of the stages of spermatogenesis it is potentially important for researchers. Failing all else, the author enjoys understanding why all the tubules in a cross section of a testis do not have an identical appearance. In some of the tubules shown in figure 19, spermiation is approaching, in others, only spermatids are present.
Notice that within each tubule, there is a set number of fixed cell associations based on the recruitment of spermatogonia, stage of meiosis, development of spermatids and finally, spermiation. Figure 18 shows the eight sets of fixed cell associations referred to in stallions. By convention, each set of associated cells is referred to as a stage, with a Roman numeral used for each stage. Staging of spermatogenesis is not unique to equids. In other animals, fixed stages of spermatogenesis are also seen. For example, six in humans, eight in dogs, eight to 12 in various ruminants and 12 to 14 in mice and rats respectively.
In stallions, at any point along a seminiferous tubule, one of the eight sets of cell associations (cell stages) is seen repeatedly, every 12.2 days. These are "assembly lines" for spermatozoa where new spermatogonia initiate the assembly of spermatozoa from the basement membrane to the lumen by starting new divisions. Some tasks in the assembly of spermatozoa take longer to complete (e.g. pachytene) than others (e.g. diplotene) so those tasks may be seen in more than one set of cell associations (stages) but each cell association changes every 12.2 days according to the drum-beat from the spermatogonia below. As mentioned earlier, that drum beat is known as the seminiferous cycle. Also to reiterate, a stallions has a seminiferous cycle of 12.2 days.
After 57 days on the spermatogenesis assembly line, spermatozoa are produced at the lumen. They roll off the assembly line in that process known as spermiation. Adjacent to each assembly line, there is another assembly line, slightly ahead of behind the line in question. The presence of one assembly line being slightly ahead or behind an adjacent assembly line along a seminiferous tubule forms a sequence known as the spermatogenic wave. This concept is illustrated in figure 22.
| The reason for the existence of a spermatic wave is not clear and to the author's knowledge, seldom addressed. It has been suggested that spermatogenic waves reflect be the inability of a single tubule cross section to accommodate continual spermatogenesis, preventing tubular clogging. However the author has recorded very long sections of synchronous stages in the seminiferous tubules in rats, putting that suggestion into question. Bear in mind however, that the cross sectional area of a tubule increases as square of its radius therefore it is more efficient to supply nutrients to the contents of a small cross section than a larger one and intermittent spermatogonial division allows for a small tubule cross section. Finally, smaller tubule cross sections are geometrically more advantageous compared to large cross sections in terms of total spermatozoa production within a given volume of testes. |
Each stage of spermatogenesis is only visible for as long as the shortest cell stage in that section of the tubule. As mentioned earlier, diplotene is a very rapid transition, so any stage containing cells in diplotene is visible for very short time and consequently form a small proportion (about 3%) of all the tubule cross sections in a testis cross section By contrast a stage containing both pre-leptotene and pachytene spermatocytes persists for longer. Therefore this combination is seen in about 16% of all tubule cross sections. It happens to be stage VIII. See figure 20.
Figure 20. Stage VIII of spermatogenesis. Size available: 1546 x 969px.
| Notably, only a small portion of any tubule is in stage VIII of the spermatogenic wave at any time; about 16%. This is the only stage during which spermiation is occurring. Therefore spermatozoa are produced by a mere 16% of a testes at any given time; two testes giving an amazing 5 billion spermatozoa every day! |
Figure 21 is another example of a stage; in this case, stage II. It is visible in about 15% of all tubule cross sections.
Figure 22 shows an idealized spermatogenic wave. In this case, cell stages are numbered from left to right along the tubule. Bear in mind that a seminiferous tubule is usually but not always in the form of a loop that attaches to the rete testis at either end. Some tubules are blind-ended. A spermatic wave may move in one direction or the other, depending on where it lies in the tubule. In most animals only one stage occupies a single section of a seminiferous tubule. However, humans, primates and others have no clear spermatogenic waves. Indeed, several stages often occupy a single segment of a seminiferous tubule. That state of disorder is infrequently seen even in stallions. In figure 22 these irregularities are indicated by red exclamation marks and are seen in reality in figure 23.
Figure 22. An idealized equine spermatogenic wave. See discussion for detail. Size available: 2154 x 1518px.
Note the arrow indicating how retinoic acid (a metabolite of vitamin A) travels along a tubule, stimulating spermatogenesis. It moves between cells along the tubule via paracrine secretion. As discussed in the section on puberty, retinoic is known to be the primary trigger for spermatogenesis. Indeed, retinoic acid is that very drumbeat noted earlier in the recruitment of spermatogonia. Because retinoic acid initiates activity in each region of the tube, stage numbers are in reverse to the direction of retinoic acid travel.
Retinoic acid recruits a new wave of spermatogenesis in each section of a tubule; every 12.2 days. In doing so, it initiates the activity of at least a half dozen transcription reactions that lead to the recruitment of inactive spermatogonia
Apparently retinoic acid is not only taken up from the blood but is also formed by Sertoli cells. It has been stated that spermatogonia themselves produce retinoic acid but this seems unlikely; perhaps retinoic acid is present in spermatogonia merely as part of paracrine secretion along tubules.
| Interestingly, one can destroy most of the cells in a seminiferous tubule in mice, leaving only Sertoli cells and early type-a spermatogonia. If this is followed by retinoic acid treatment, all the spermatogonia respond to retinoic acid simultaneously obliterating spermatic waves. As a result, spermiation occurs simultaneously along the whole seminiferous tubule! |
The combined length of the seminiferous tubules in a stallion is over two kilometers (useful for pub trivia contests) therefore each testis contain many thousands of spermatogenic waves. Because the tubules are convoluted, different stages usually lie adjacent to one another in testes cross sections. This explains why a cross section in stage II lies adjacent to another in stage VI in figure 24.
Figure 24. A cross section in stage II lying adjacent to another in stage VI. Size available: 1903 x 1192px.
Many readers will be aware that Leydig cells are the primary source of androgens (predominantly testosterone) in males. They are also the most common cells in the inter-tubular (interstitial) areas in testes. Figure 25 shows a cross section of a tubule in stage V. Note the accumulation of lipofuscin in the Leydig cells in this image. Although lipofuscin is even seen in the Leydig cells of prepuberal stallions, it is generally interpreted as a sign of aging. Indeed, lipofuscin becomes more obvious in older stallions. Even macroscopically, testes become darker with aging.
There are also large numbers of macrophages in the interstitial spaces, with lipofuscin being present in those cells as well as Leydig cells. There is an interesting but poorly understood relationship between Leydig cells and macrophages in the interstitial spaces because both appear to have phagocytic and steroidogenic activity! In adult stallions, interstitial macrophages can form close membranous interdigitations with Leydig cells suggesting that they even become a functional syncytium. In figure 25 therefore, it is not clear if the lipofuscin resides in Leydig cells alone, macrophages with Leydig cell-like activity or both cell types.
After spermiation
Following spermiation, spermatozoa are transported towards the rete testis within the seminiferous tubules. They move along the tubules via the smooth muscle action of myoid cells (see figures 20 and 23). Eventually, these tubules join rete tubules as shown in figure 26. Close examination the rete tubules shows smooth muscle in their walls.
Rete tubules course around the central vein of the testis towards its cranial-dorsal extremity. Some rete tubules fuse as they approach the anterior aspect of the testis. This can be seen in figure 27. In one case, several rete tubes lead into an efferent duct. Simple cuboidal epithelium identifies the rete tubules and pseudostratified columnar epithelium, efferent ducts.
In most cases, rete tubes remain narrow until they reach the efferent ducts but occasionally rete tubes fuse into large, sinus-like collection sites with many criss-crossing trabeculae. One of these is shown in figure 28. An excellent scanning electron micrograph of such a sinus can also be seen in: Amann, R. P. et al. 1977 Connection between the seminiferous tubules and the efferent ducts in the stallion. Am.J. Vet. Res.38: 1571-1579.
Figure 28. Rete tubules and efferent ducts. In one case, efferent ducts have fused to form a sinus with trabeculae. The figure shows a white rectangle over the cranial portion of a testis, adjacent to the caput epididymis. This area included many rete tubules and efferent ducts. However, rete tubules and efferent ducts were also found outside the tunica albuginea. This tissue was obtained from a peripuberal stallion therefore spermatozoa were generally absent from the the duct systems. Size available: 2243 x 1589px.
Amazingly, seminiferous tubules and the rete-efferent systems develop from two extremities. Seminiferous tubules arise from the genital ridges (testes) while rete ducts and efferent ducts arise from the mesonephroses. In most cases these tubes meet end-to-end perfectly; the dream of every engineer on the chunnel project between England and France. In an embryo, the single tube that is the epididymis diverges from the mesonephric side into 10 to 15 efferent ducts and they in turn, diverge into hundreds of rete tubules. From the testis side, hundreds of seminiferous tubules straighten for a short distance then merge with ingrowing rete tubules. On rare occasions, perfect end-to-end anastomosis does not occur and after puberty, spermatozoa granulomas form in the affected areas, especially outside the testis where the interstitium is not as immunologically privileged as it is within the testis.
The epididymis
| Within the epididymis: steroids, polypeptides and the autonomic system: Fluid produced by the Sertoli cells is mostly absorbed in the efferent ducts and the initial segment of the caput epididymis, increasing the concentration of spermatozoa. Fluid absorption is strongly mediated by estrogens with principal cells showing clear evidence of estrogen receptors. In fact, estrogen receptors abound throughout the epididymis. In stallions, more estrogen is produced by the testes than testosterone. Estrogen up-regulates oxytocin receptors in the cauda epididymis and modulate spermatogenesis via FSH secretion. Surprisingly, estrogens also modulate Leydig cell and Sertoli cell function! Finally (but probably not finally...) estrogens are known to be important in libido and erectile function in some species; likely the case in horses as well. So much for testosterone and being a "stud"! Smooth muscle increases in bulk and tone as one approaches the cauda epididymis. Both vasopressin and oxytocin receptors increase in the cauda too. This explains why spermatozoa numbers in ejaculates can be increased by oxytocin treatment before semen collection. The function of vasopression in the epididymis is unclear, causing contraction of the cauda in some studies but not others. Although it may be an oversimplification to say so, there is general agreement that the parasympathetic system is largely responsible for erection and the sympathetic system, ejaculation. Therefore it is not surprising that sympathetic stimulation in rats is largely responsible for myoid cell contraction in the corpus and cauda epididymis. As expected, there is little or no contraction in the caput epididymis. Even after repeated ejaculation in rats, the spermatozoa in the caput are too immature for ejaculation. Similar data are not available in stallions but are presumed to be similar. The influence of the parasympathetic on epididymal function is not clear. |
In stallions, the epididymis is about three quarters the length of a football field (more beer trivia) and although reports vary slightly, the usual time taken for sperm to move through this distance varies from 9 to 11 days. Of that time, sperm spend just four days travelling through the caput and corpus. The remaining 5 to 7 days are spent in the cauda epididymis. The cauda epithelium secretes substances that promote quiescence and optimal storage conditions for spermatozoa, so it is essentially a storage organ. The caput and corpus hold only 20% of the extra-testicular spermatozoa while the cauda, approximately 65%. The remaining 15% are held in the ampullae and ducti deferentia. Despite the motility they may display in vitro, sperm in the caput and corpus are still immature and infertile. However, spermatozoa harvested from the cauda are indeed fertile, providing conception in both raw and frozen states.
The entire epididymis is lined with a pseudostratified columnar epithelium in which two cell types predominate; principal and basal cells. Figure 30a and 30b are cross sections of the caput epididymis from a 6 year old Morgan stallion.
Principal cells in the caput epididymis are very tall but gradually become shorter towards the cauda .
Principal cells control the pH and oxygen tension within the epididymal lumen and provide energy for sperm maintenance. They are also the main source of proteins in the epididymis. Indeed, more that 200 proteins have been identified within the epididymal lumen. Some arise from the testis but many from the epididymis itself. These proteins coat sperm and are important in sperm/oocyte interaction. They also protect sperm from the immune system of the female. Others bind to sperm to prevent premature capacitation.
Note that the free surface of most principal cell bear tufts of very long, microvilli. Although they appear to be cilia at low magnification they are indeed villi, not cilia. The point here is that spermatozoa transport along the epididymis is due to smooth muscle contraction, not cilial action. In that regard, a thin layer of longitudinally oriented smooth muscle cells surrounds the caput, corpus and proximal cauda, becoming thicker towards the distal cauda and very thick in ductus deferens. See figure 35.
At their tips, the secretory products of principal cells are voided into the lumen within membranous extracellular vesicles, termed epididymosomes (see figure 33). At the bases of principal cells, there are tight adhering junctions between adjacent cells. These separate the haploid environment of the ductus from the diploid surrounding tissue; an extension of the blood-testis barrier.
Principal cells are by far the predominant cell type but figure 30b shows that halo cells, apical cells and basal cells also populate the epididymis. In the stallion, the author was unable demonstrate narrow cells such as those seen in rats and other species.
| Under the effect of "cross-talk" between principal cells, basal cells and clear cells, bicarbonate absorption and hydrogen ion secretion occurs in the epididymis so that pH drops. This acidic environment prevents calcium channel activation that would otherwise trigger capacitation. It also maintains sperm quiescence. The fluid of the accessory glands are high in bicarbonate, increasing pH and reversing the quiescent state of sperm during ejaculation. |
Halo cells are clearly visible in figure 31, from the corpus epididymis of a peripuberal colt. Although it is not possible to discern the difference between types of halo cells with ordinary histology, they have been identified as T lymphocytes, helper T lymphocytes and monocytes; all parts of the immune system in the epididymis.
Apical cells secrete protons, maintaining a low luminal pH but the function of basal cells remains controversial. They are thought to be part of the blood-epididymis barrier and assist in cross-talk between principal cells. At best however, their function is poorly understood.
Principal cells in the caput epididymis (figure 32) are packed with vesicular secretory bodies containing protein. After breaking off into the lumen they will will form the epididymosomes seen in figure 33. Contrast has been enhanced in one region of the image to enhance the visibility of the secretory bodies. Long villi seen in most areas of the caput are absent in others.
Figure 32. Caput epididymis from a 6 yr old Morgan stallion. Note the principal cells packed with vesicular secretory bodies. Size available: 2562 x 1520px.
Clear cells (see figure 34) like apical cells, are responsible for pH balance and for phagocytosis of discarded midpiece droplets from sperm. To the author's eye however, there appeared to be relatively few clear cells in the epididymis, seeming too few to assist significantly with the task of midpiece droplet phagocytosis. Interestingly, phagocytosis of midpiece droplets is mainly attributed to principal cells, not clear cells. Yet even in clear cells, where millions of midpiece droplets are phagocytosed every day, phagosomes were not obvious. Colleagues may wish to comment on this puzzling finding.
| Although phagocytosis of spermatozoa themselves has been documented in various sections of the epididymis in more than one species, epididymal phagocytosis remains a contentious subject. Indeed, some authors have not been able to document phagocytosis of spermatozoa at all. Certainly, phagocytosis of defective sperm is not the common system for eradication of defective sperm that it was once thought to be. |
The ductus deferens
The ductus deferens shows many of the characteristics of the cauda epididymis with two remarkable differences; one is its relative absence of storage space and two, the massive development of smooth muscle required for ejaculation. Figure 35 shows a comparison between the smooth muscle layers of the cauda epididymis and the ductus.
The ductus deferens shows many of the characteristics of the cauda epididymis with two remarkable differences; one is its relative absence of storage space and two, the massive development of smooth muscle required for ejaculation. Figure 35 shows a comparison between the smooth muscle layers of the cauda epididymis and the ductus.
Although the epithelium in the ductus is more folded than the epithelium in the cauda, the epithelium of the ductus is similar to that in the epididymis, with principle and parabasal cells predominating. Villi are also present in some areas but not others. This suggests that metabolic activity still occurs within the ductus. This activity is probably related to sperm quiescence rather than maturation.
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