SPERMATOGENESIS

Seminiferous epithelium ( 20-3 to 20-5 )

The seminiferous epithelium can be classified as a stratified epithelium with rather unusual characteristics not found in any stratified epithelium of the body.

In this stratified epithelium, somatic columnar Sertoli cells interact with mitotically dividing spermatogonia, meiotically dividing spermatocytes and a haploid population of spermatids, undergoing a differentiation process called spermiogenesis .

Several cross sections of seminiferous tubules are illustrated in 20-3 . Note that the random arrangement of seminiferous tubules within each lobule generates different geometric profiles.

A more detailed view of the seminiferous epithelium is seen in 20-4 . Different location and structural types of nuclei can be appreciated:

• 1.

  The nuclei of spermatogonia and Sertoli cells are closely associated with the seminiferous tubular wall.

• 2.

  Overlying the spermatogonial cell population are the primary spermatocytes . Their nuclei are larger and clumps of chromatin represent the meiotic chromosomes.

• 3.

  Close to the lumen are the early spermatids with a round light nucleus and the late spermatids with a cylindrical-shaped condensed nuclei.

An electron micrograph (see 20-5 ) shows the basal lamina and fibrillar components of the seminiferous tubular wall and the nuclear characteristics of two Sertoli cells, spermatogonia and primary spermatocytes. Note how Sertoli cell cytoplasmic processes extend between the spermatogenic cells.

The next step is to understand why each spermatogenic cell progeny occupies a specific space in the seminiferous epithelium

Basal and adluminal compartments ( 20-6 ; see 20-2 , 20-4 and 20-5 )

Sertoli cells are columnar cells extending from the basal lamina to the lumen of the seminiferous tubule (see 20-2 and 20-5 ). They behave as bridge cells between the intertubular space and the lumen of the seminiferous tubule, and as nurse cells , supporting the survival of spermatogenic cells.

The apical and lateral plasma membranes of Sertoli cells have an irregular outline; they provide niches and crypts to house the developing spermatogenic cells (see 20-6 ).

At their basolateral domain, Sertoli cells form tight junctions with adjoining Sertoli cells. Most epithelia have tight junctions at the apical domain. Therefore, tight junctions at the basolateral domain of Sertoli cells represents an exception to the rule.

If you consider that molecules and fluids in a standard epithelium follow the apical-to-basal transport direction, the transporting direction in the seminiferous tubules is reversed: it is basal-to-apical. In fact, the source of fluid and nutrients is not in the luminal space but rather in the inter-seminiferous tubular space.

Basolateral inter–Sertoli cell tight junctions subdivide the seminiferous epithelium into:

• 1.

  A basal compartment .

• 2.

  An adluminal compartment (see 20-4 ).

The spermatogonia cell population is housed in niches within the basal compartment. This location provides ample access to nutrients and signaling molecules derived from vessels in the inter-seminiferous tubular space.

Inter–Sertoli cell tight junctions are components of the so-called blood-testes barrier . This barrier protects developing spermatocytes and spermatids, located within the adluminal compartment, from autoimmune and genotoxic reactions.

The spermatogenic cell progeny ( Primer 20-A )

Somatic Sertoli cells represent the stable cell population of the seminiferous epithelium. Spermatogenic cell progenies—spermatogonia, spermatocytes and spermatids—are transient .

Primer 20-A illustrates relevant aspects of a mammalian spermatogenic cell sequence:

• 1.

  At puberty, a spermatogonial stem cell (SSC) , derived from a primordial germinal cell (PGC) in the fetal testes, divides by mitosis to generate two daughter cells. One daughter cell initiates a spermatogenic cell sequence. The other cell becomes a SSC with self-renewal capacity and able to soon initiate another spermatogenic cell sequence .

  We have seen in Chapter 3 , Cell Signaling | Cell Biology | Pathology, that stem cells can self-renew and give rise to another stem cell and a cell entering a terminal differentiation pathway. The same rule applies to SSCs.

• 2.

  After cell division, all spermatogenic cells remain interconnected by intercellular bridges because cytokinesis is incomplete.

• 3.

  Spermatogonia, spermatocytes and spermatids complete their proliferation and differentiation sequence in a timely manner. Each spermatogenic cell cohort proliferates and differentiates synchronously .

• 4.

  After puberty, SSCs periodically give rise to spermatogenic cell progenies to ensure the continuous production of sperm.

We will study later how spermatogenic cell progenies overlap along a segment of a seminiferous tubule and generate distinct combinations of spermatogenic cell cohorts called cell associations.

Now that you have learned the basic aspects of the organization of a mammalian testis and the general aspects of a spermatogenic cell progeny, the next step is to understand the characteristics of different spermatogenic cells types in the seminiferous epithelium with respect to the Sertoli cell.

Sertoli cells (see 20-6 )

Sertoli cells are the predominant cell type of the seminiferous epithelium until puberty . After puberty, they represent about 10% of the cells lining the seminiferous tubules. Sertoli cells are postmitotic after puberty . No mitotic cell division is observed in the adult testes. In elderly men, when the population of spermatogenic cells decreases, Sertoli cells again become the major component of the epithelium.

Members of the spermatogonia progeny, interconnected by intercellular bridges, complete the mitotic amplification cycle, translocate from the basal compartment to the adluminal compartment and initiate the meiotic cycle as primary spermatocytes.

Inter-Sertoli tight junctions unzip and re-zip to enable the massive migration of interconnected cells.

The cytoskeleton of Sertoli cells (microtubules, actin microfilaments and the intermediate filament vimentin) facilitates the displacement of differentiating spermatogenic cells farther away from the periphery of the seminiferous tubule and closer to the lumen.

How can Sertoli cells be identified in a histology preparation?

The most useful cell identification parameter is the Sertoli cell nucleus . The cytoplasmic processes of a Sertoli cell are tortuous and difficult to resolve with the light microscope. The Sertoli cell nucleus is located at the base of the cell, near the basal lamina. It displays indentations and a large nucleolus with associated heterochromatin masses (see 20-6 ).

The cytoplasm contains smooth and rough endoplasmic reticulum, mitochondria, lysosomes, lipid droplets, an extensive Golgi apparatus and a rich cytoskeleton.

The functions of Sertoli cells are:

• 1.

  To support, protect and nourish developing spermatogenic cells.

• 2.

  To eliminate by phagocytosis excess cell portions, called residual bodies , discarded by spermatids at the end of spermiogenesis .

• 3.

  To facilitate the release of mature spermatids into the lumen of the seminiferous tubule by actin-mediated contraction, a process called spermiation .

• 4.

  To secrete a fluid rich in proteins, lactate and ions into the seminiferous tubular lumen.

Sertoli cells respond to follicle-stimulating hormone (FSH) stimulation and express androgen receptors . Androgens acting through Sertoli cells stimulate spermatogenesis by a still-undefined mechanism (see Box 20-A ). FSH regulates the synthesis and secretion of androgen-binding protein (ABP) .

ABP is a secretory protein with high binding affinity for the androgens testosterone and dihydrotestosterone . The androgen-ABP complex, whose function is unknown at present, is transported to the proximal segments of the epididymis.

We come back to this aspect later in this chapter and in Chapter 21 , Sperm Transport and Maturation.

Note that although both ABP and the androgen receptor have binding affinity for androgens, they are distinct proteins. ABP is a secretory protein , whereas the androgen receptor is a cytoplasmic and nuclear protein with DNA binding activity.

Sertoli cells secrete inhibin and activin subunits (αand βsubunits) to regulate FSH secretion:

• 1.

  Inhibin (an αβ heterodimer) exerts a negative feedback on gonadotropin-releasing factor and FSH release by the hypothalamus and anterior hypophysis.

• 2.

  Activin (an αα or ββ homodimer) exerts a positive feedback on the release of FSH (see Chapter 18 , Neuroendocrine System).

Sertoli cells also secrete regulatory proteins required for spermatogonia cell differentiation (discussed later).

S ertoli cell–only syndrome (SCOS) is a clinical condition defined by germinal aplasia (absence of spermatogenic cells in seminiferous tubules). Seminiferous tubules are lined only by Sertoli cells. SCOS can be determined by congenital (including Y chromosome abnormalities) or acquired factors (see Box 20-B ).

Spermatogonia (see 20-4 and 20-6 )

Spermatogonia are diploid spermatogenic cells residing in a unique environment, or niche, directly in contact with the basal lamina in the basal compartment in association with Sertoli cells. They are located below the inter-Sertoli cell occluding junctions and therefore outside the blood-testes barrier.

Two major morphologic spermatogonial cell types can be observed:

• 1.

  Type A spermatogonia display an oval euchromatic nucleus and a nucleolus attached to the nuclear envelope (see 20-6 ). Subclasses of type A spermatogonia (with a dark nucleus, called A dark spermatogonium , and with a lighter nucleus, called A pale spermatogonium) are observed in human testes.

• 2.

  Type B spermatogonia have a round nucleus, masses of heterochromatin attached to the nuclear envelope and a central nucleolus (see 20-4 ).

Regulation of spermatogonia cell function ( 20-7 )

Stimulated by follicle-stimulating hormone (FSH), Sertoli cells secrete GDNF (for glial cell line–derived neurotrophic factor), which stimulates SSC renewal and differentiation. GDNF binds to the GDNF family receptor α1 (GFRα1).

There is a balance between SSC renewal and spermatogonial differentiation. Maintenance of this balance determines an input-output equilibrium between the number of SSCs being produced (input) and the number of sperm released (output).

The transcription factor Plzf (for promyelocytic leukemia zinc finger) deters SSC self-renewal by blocking the gene expressing the c-kit tyrosine kinase receptor.

When SSCs are ready to start their self-renewal process, retinoic acid down-regulates the transcription factor Plzf, thereby unblocking the expression of the c-kit receptor, which becomes available for binding to the stem cell ligand. The ligand is bound to the plasma membrane of Sertoli cells.

There are two regulatory mechanisms of SSCs:

• 1.

  A paracrine regulatory mechanism exerted by the GDNF-GFRα1-RET complex and the c-kit receptor-stem cell ligand complex . By this mechanism Sertoli cells regulate SSC self-renewal and differentiation.

• 2.

  An autoregulatory mechanism , mediated by the retinoic acid–Plzf interplay , which modulates the expression of the c-kit gene. This mechanism determines whether SSCs undergo self-renewal.

SSCs have important implications for male fertility. SSCs are relatively quiescent and therefore resistant to radiation and cancer chemotherapy. Mitotically dividing spermatogonia, meiotically dividing spermatocytes and differentiating spermatids are sensitive to radiation and cancer chemotherapy. After cessation of radiotherapy or anticancer chemotherapy, SSCs can reestablish the spermatogenic developmental sequence. Postmitotic Sertoli cells are highly resistant to these therapies.

Failure of spermatogonia to undergo differentiation in humans results in a neoplastic transformation into carcinoma in situ, leading to testicular germ cell carcinoma in the adult.

Spermatocytes ( 20-8 )

Type B spermatogonia enter meiotic prophase immediately after completing the last S phase (DNA synthesis) and G ₂ phase of their cell cycle . This is the last round of major DNA synthesis in the lifetime of spermatogenic cells. Primary spermatocytes initiate meiotic prophase I with two times the amount of DNA per cell .

Spermatogonia B, which become primary spermatocytes, have a 4C DNA value, where 1C equals about 1.5 pg of DNA per cell. And each of its chromosomes consists of two identical chromatids .

How does the spermatocyte reduce an initial 4C DNA value and two chromatids per chromosome at the end of meiosis?

Spermatocytes initiate two successive meiotic cell divisions soon after they enter the adluminal compartment of the seminiferous epithelium, just above the inter–Sertoli cell occluding junctions. Therefore, meiosis occurs inside the blood-testes barrier.

A primary spermatocyte undergoes the first meiotic division (or reductional division) to produce two secondary spermatocytes (see 20-8 ). Just a small amount of DNA synthesis takes place to repair breakages during genetic crossing over.

The secondary spermatocytes rapidly undergo the second meiotic division (or equational division) . Each secondary spermatocyte gives rise to two spermatids, which differentiate into sperm without further cell division.

By the end of the first meiotic division, the original 4C DNA content of a primary spermatocyte is reduced to 2C in a secondary spermatocyte and each chromosome consists of two chromatids.

By the end of the second meiotic division, the 2C DNA content is reduced to 1C; the two chromatids separate to become chromosomes. The resulting spermatids are the haploid spermatids with one chromosomal set. Now, spermatids are ready to initiate a complex differentiation process, called spermiogenesis , to become sperm.

Because the first meiotic division is a long process (days) and the second meiotic division is very short (minutes), primary spermatocytes are the most abundant cells observed in the seminiferous epithelium.

Turn your attention to 20-8 to review the highlights of the meiotic process of the male and female gametes.

Note that in the female, a primary oocyte (with a 4C DNA content) completes the first meiotic division a t ovulation to produce a secondary oocyte (2C DNA content) and the first polar body .

When fertilization occurs , the secondary oocyte completes the second meiotic division to reach the haploid state (1C DNA content) and a second polar body is generated.

Keep in mind that female meiosis starts in the ovary during fetal development (see Chapter 23 , Fertilization, Placentation and Lactation). In contrast, male meiosis initiates at puberty.

Meiosis (Primers 20-B, 20-C and 20-D)

Meiosis is focused on chromosomal events and on establishing the conditions appropriate for sex determination.

The following are the major objectives of meiosis:

• 1.

  The homologous chromosomes pair together and exchange segments by a process known as crossing over , or recombination . Recombination of genes is fundamental to the genetic diversity of a species.

• 2.

  The end products of meiosis are four haploid spermatids with only one set of chromosomes. When the chromosomes of haploid egg and sperm combine at fertilization, the embryo regains the normal diploid number.

• 3.

  Males have one X and one Y chromosome. By the end of meiosis, half the spermatids get an X chromosome and half get a Y chromosome. The Y chromosome carries a gene named SRY (for sex-determining region of the Y chromosome) .

During fetal development, the SRY gene, encoding a transcription factor, determines the fetal gonadal tissue to become testes (see Chapter 21 , Sperm Transport and Maturation). In the absence of a Y chromosome, the fetus develops as a female.

Females have two X chromosomes. Upon completion of meiosis, all the eggs get one or the other X chromosome.

The first meiotic division is characterized by a long prophase , lasting about 10 days. A coordinated chain of events in the extended meiotic prophase I results in pairing and synapsis (Greek syn , together; hapto , to connect) of homologous chromosomes, creating bivalent chromosomes . Each bivalent consists of four chromatids, two sister chromatids for each member of the bivalent structure. If we count the number of chromatids per bivalent, we have a tetrad (Greek tetras , the number four). Genetic recombination takes place between the non-sister chromatids of each bivalent. The X and Y chromosomes normally cross over at one of their paired ends; the SRY gene is present in the opposite unpaired end (see 20-B).

During meiotic anaphase I, the chromosome number is reduced by one-half, when homologous chromosomes, each consisting of sister chromatids , separate. Meiosis I is a reductional division .

During meiosis II, sister chromatids segregate , following a similar mechanism observed in mitosis. Meiosis II is an equational division .

Precise cell cycle events, involving cyclin-dependent protein kinase–cyclin complexes, are required for the proper partitioning of genetic material during meiosis I and II.

Let us now examine the details of the long lasting prophase of meiosis I.

The prophase substages of meiosis I are as follows (see Primers 20-B and 20-C):

• 1.

  Leptotene: chromatin condenses to form visible thread-like chromosomes. The nuclear envelope remains intact.

• 2.

  Zygotene: chromosomes line up to form homologous pairs (bivalents or tetrads). Synapsis starts when homologous pairing takes place. The synaptonemal complex initiates its assembly (see Primer 20-D ).

  

• 3.

  Pachytene: non-sister chromatids of the synapsed homologous chromosome exchange parts or segments (crossing over) when chromosomal pairing and synapsis are complete.

  Gene recombination starts by DNA double-strand breaks (DSBs) , which take place at precise chromosomal sites. Crossovers along any given chromosome pair are evenly spaced over distances ranging from 300 nm to 30 μm. This regularly spaced distribution, known as crossover interference , involves the catalytic activity of topoisomerase II (TopoII) , an enzyme which breaks and rejoins double-stranded DNA. Crossover interference implies that crossover at a given chromosomal site prevents another crossover from occurring too close to it.

  A chiasma (Greek chiasma , two crossing lines; plural chiasmata ) forms where crossing over has occurred. Because of gene recombination, each bivalent is now different from its parent chromosomes, but the amount of genetic material is the same.

• 4.

  Diplotene: the synaptonemal complex begins to disassemble and the paired chromosomes begin to separate, or disjoin .

• 5.

  Diakinesis: chromosome condensation proceeds as chromosomes shorten. Homologous chromosomes separate further but are still joined by chiasmata . Chiasmata move toward the ends of the chromosomes by a process referred to as terminalization . The meiotic spindle begins to form. The nuclear envelope breaks down. Microtubules attach to the chromosomes at the kinetochore site.

After the prolonged meiotic prophase I, and upon completion of metaphase I, anaphase I and telophase I, t he homologous chromosomes, each consisting of two chromatids , separate into daughter cells, the secondary spermatocytes .

During the short second meiotic division (prophase II, metaphase II, anaphase II and telophase II), the sister chromatids of each chromosome are freed , as in mitosis, and are distributed to the haploid spermatids . Remember that no S phase occurs before meiosis II; the DNA has already replicated before meiosis I started (see 20-8 ).

Errors during meiosis I and II impact on developmental defects and causes of infertility. A review of Basic Concepts of Medical Genetics in Chapter 1 , Epithelium | Cell Biology, is recommended to strengthen the understanding of the basic concept of genetic abnormalities.