Testicular Descent

The testes develop initially in the upper lumbar region of the embryo and gradually migrate during fetal life through the abdominal cavity and over the pelvic brim. This descent is influenced by hormones secreted by the Leydig cells and involves two ligamentous structures: the suspensory ligament at the superior pole and the gubernaculum at the inferior pole of the testis. As the fetal body grows in size, the suspensory ligament elongates and the gubernaculum does not, so that the position of the testis becomes localized to the pelvis. Between weeks 25 and 28 of pregnancy, the testes migrate over the pubic bone, and reach the scrotum via the inguinal canal by weeks 35–40. As a result of their external position outside the body cavity, the temperature of the testes is approximately 2°C below body temperature, which is optimal for spermatogenesis.

The adult testes contain approximately 200 m of seminiferous tubules, forming the bulk of the volume of the testis. These tubules are the site of spermatogenesis. The round tubules are separated from each other by a small amount of connective tissue that contains, in addition to blood vessels, a few lymphocytes, plasma cells and clumps of interstitial Leydig cells. The tubules are lined by spermatogenic epithelium, which is made up of spermatogonia at different stages of maturation; a cross-section of any normal seminiferous tubule reveals four or five distinct generations of germ cells. The younger generation cells are on the basement membrane, and the more differentiated cells approach the lumen of the tubule. This growth pattern has a wavelike cycle with intermingling of different stages that lie close to each other; any single cross-section of the tubule does not always reveal all generations of spermatogenesis. The tubules rest on a delicate anuclear basement membrane that in turn lies on a connective tissue layer, the tunica propria. The supporting Sertoli cells, which are believed to nourish the germ cells, form a continuous layer connected by tight junctions. These large polymorphous cells have large, pale nuclei and abundant cytoplasm that extends from the periphery of the tubule to the lumen, stretching through the layers of developing germ cells. Mature spermatozoa can be seen attached to and surrounding the Sertoli cells prior to their release (Figure 3.3). A wave of spermatogenesis passes along the tubule, and the process of development from spermatogonium to spermatozoon takes approximately 65 days. A transverse cross-section through the human testis shows tubules containing cells at many different stages in spermatogenesis (in contrast to the rat testis, where every tubule has cells at the same stage). In humans, many seminiferous tubules can be seen that are apparently without spermatocytes and spermatids, a phenomenon that may contribute to the relatively poor efficiency of spermatogenesis.

Figure 3.3 Spermatogenesis in the mammal. Maturation and modeling of the male gamete is regulated by the Sertoli cell.

Modified with permission from Johnson [2018].

Sperm released into the lumen of the seminiferous tubules pass via the rete testis through the ductuli efferentia into the caput epididymis. They traverse the epididymis over a period of 2–14 days, undergoing a number of changes in preparation for fertilization, and are then stored sequentially in the cauda, vas, seminal vesicles and ampullae prior to ejaculation. The seminal vesicles, prostate and urethral glands add glandular secretions to the sperm at the time of ejaculation. Figure 3.4 illustrates the anatomy of the adult mammalian testis.

Figure 3.4 Anatomy of the adult mammalian testis.

Drawing adapted from a number of sources.

Spermatocytogenesis

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  At intervals after puberty, stem cells in the germinal epithelium of the seminiferous tubules (type A spermatogonia) replicate their DNA and divide by mitosis.

  
  
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  Each mitotic division produces two cells: another type A spermatogonium and a second cell, which enters a pool of undifferentiated spermatogonia – transient amplifying progenitor cells, Aa1, that transition without cell division into type A1 differentiating spermatogonia.

  
  
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  These A1 cells undergo five synchronized cell divisions to form A2, A3, A4, Intermediate (In) and B spermatogonia, which then move into the adluminal compartment and start their differentiation by entering into meiosis.

Spermiogenesis

Spermatid differentiation occurs in four stages (Figure 3.5):

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   1. Golgi phase

   
   
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   2. Cap phase

   
   
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   3. Acrosomal phase

   
   
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   4. Maturation phase.

Spermatid nuclei now contain haploid sets of chromosomes. Their autosomes continue to direct the synthesis of low levels of rRNA, mRNA and proteins as they enter into their prolonged phase of terminal differentiation, spermiogenesis. During this phase, round spermatid cells are converted into elongated sperm cells with a condensed nucleus and a flagellum. They do not divide again, either by mitosis or meiosis, but must differentiate to acquire functions that will allow them to traverse the female tract and fertilize an oocyte. This differentiation process takes approximately 2 weeks in most species and follows well-defined stages:

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   1. Spermatid DNA becomes highly condensed and somatic histones are replaced with protamines.

   
   
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   2. The acrosome, a sac containing enzymes necessary for oocyte penetration, is constructed from Golgi membranes.

   
   
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   3. Cytoplasmic reorganization gives rise to the midpiece, which contains mitochondria and associated control mechanisms necessary for motility.

   
   
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   4. The flagellar apparatus (tail) is formed, which will make the cells motile.

   
   
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   5. A residual body casts off excess cytoplasm.

Figure 3.5 Developing sperm. A = acrosome; An = annulus; Ax = axoneme; C = centriole; F = flowerlike structures; Fs = flagellar substructures; M = mitochondria; Mp = middle piece; Mt = manchette; Ne = neck; PP = principal piece; R = ring fibers; Sb = spindle-shaped body.

Courtesy of M. Nijs and P. Vanderzwalmen, Belgium.

Sperm modeling is probably regulated by the Sertoli cells, and the cells are moved to the center of the tubular lumen as spermatogenesis proceeds. The rate of progression of cells through spermatogenesis is constant and is not affected by external factors such as hormones. The timing of stored mRNA translation is a major point of control: for example, the protamine1 gene is transcribed in round spermatids, and the resulting mRNA is stored for up to 1 week before it is translated in elongating spermatids. Other mRNAs are stored for only hours or a few days, indicating that there must be a defined temporal program of translational control.

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  Sertoli cells are connected by tight junctions and act as nurse cells during spermatogenesis.

  
  
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  Tight junctions restrict the passage of substances from the blood to the lumen of the seminiferous tubules and therefore form a blood–testis barrier. This barrier protects sperm from antibodies circulating in the bloodstream.

Epididymal Maturation

Mammalian spermatozoa leaving the testis are not capable of fertilizing oocytes. They gain this ability while passing down the epididymis, a process known as epididymal maturation. The epididymis is divided into different regions: the caput is the upper third, formed by efferent ductules that are lined by pseudostratified columnar ciliated epithelium (such as is found in nasal and bronchial passages; patients with upper epididymal obstruction often have associated nasal or respiratory problems, as in mucoviscidosis, Young’s syndrome). The vasa efferentia tubules unite to form the single coiled tubule of the corpus, which has flatter, non-ciliated epithelium and microvilli on the luminal surface. It starts to form a muscular wall toward the cauda, where the lumen is wider, and spermatozoa can be stored prior to ejaculation.

During its journey through the different regions of the epididymis, the head of the spermatozoon acquires the ability to interact with the zona pellucida, with an increase in net negative charge. Many antigens with a demonstrable role in oocyte binding and fusion are synthesized in the testis as precursors, and then activated at some point in the epididymis either through direct biochemical modification, through changing their cellular localization or both. Examples of such antigen processing include a membrane-bound hyaluronidase, fertilin, proacrosin, 1,4-galactosyltransferase (GalTase) and putative zona ligands sp56 and p95. The terminal saccharide residues of membrane glycoproteins and lipids also undergo changes in their physical and chemical composition.

Although all the necessary morphological structures for flagellar activity are assembled during spermiogenesis, testicular spermatozoa are essentially immotile, even when washed and placed in a physiological solution. Spermatozoa from the caput epididymis begin to display motility, and by the time they reach the cauda they are capable of full progressive forward motility. Demembranation and exposure to ATP, cAMP and Mg²⁺ triggers movement, which suggests that the ability to move is probably regulated at the level of the plasma membrane. Transfer of a forward motility protein and carnitine from the epididymal fluid is believed to be important for the development of sperm motility. Since the osmolality and chemical composition of the epididymal fluid vary from one segment to the next, it may be that the sperm plasma membrane is altered stepwise as it progresses down the duct, and motility is controlled by an interplay between cAMP, cytosolic Ca²⁺ and pH. During maturation, the spermatozoa use up endogenous reserves of metabolic substrates, becoming dependent on exogenous sources such as fructose; at this point they shed their cytoplasmic droplet.

DNA Packaging in Sperm

The amount of DNA in the sperm nucleus (approximately 1 m in length) has to be packaged into a volume that is typically less than 10% of the volume of a somatic cell nucleus; a different mechanism of packaging is required, as illustrated in Figure 3.6.

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  Somatic cell DNA is packaged into nucleosomes by a process of primary compaction that uses histones. A 10-nm fiber is supercoiled into the 30-nm solenoid, and supercoiled again into loops (Figure 3.6A–D. These loops are the major structural form of interphase chromatin.

  
  
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  During spermatogenesis, DNA is initially packaged by histones (as in Figure 3.6A–D) but following meiosis, at the secondary spermatocyte stage of spermiogenesis, histones are replaced first by transition proteins and then by protamines (Figure 3.6G). The solenoid structure is replaced by torroids (doughnut shapes), which are in turn supercoiled into torroidal loops. This highly compacted structure shuts down transcription during spermiogenesis. The loop domains shown in Figure 3.6E and J represent the chromatin state in the interphase somatic cell nucleus (Figure 3.6E) and the sperm nucleus (Figure 3.6J). Sperm appear poised for transcription.

Figure 3.6 DNA packaging in somatic cells and spermatozoa.

Image adapted with permission from Ward, 1993.

Gene Expression during Spermatogenesis

Gene expression during spermatogenesis can be subdivided into two distinctive phases:

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   1. Prior to meiosis, all stages up to and including the completion of telophase: diploid cells.

   
   
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   2. During spermiogenesis: late secondary spermatocytes are haploid cells, and the developmental process from this stage onwards is referred to as spermiogenesis.

Genes are expressed from both the diploid and the haploid genomes. DNA transcription is often coordinated with mRNA translation into its protein product (e.g., histones, X-linked lactate dehydrogenase). In spermiogenesis, however, transcription may be shut down well before the protein appears, i.e., the mRNA is translated later (e.g., PGK2, acrosin). As spermatogenesis progresses, the transcripts encoding the same protein differ in size, due to alterations in the length of the mRNA polyA tail. (A similar phenomenon occurs in oogenesis, as described later.)

The ultimate aim of the male reproductive system is to parcel the male genetic package, a set of 23 chromosomes, into the head of a single spermatozoon, and deliver this to the female reproductive tract, in the right place at the right time. However, in order to fertilize the oocyte and initiate embryonic development, the spermatozoon must also contribute two epigenetic factors: an oocyte-activating factor and the centrosome or cell division mechanism (see Chapter 4).