INTRODUCTION
Sperm is the motile male reproductive cell, which is highly specialized cell in human body. The term spermatozoon is derived from the ancient Greek word “sperma” (meaning seed) and “zoon” (meaning living being) and more commonly known as a sperm cell. It is the haploid male gamete cell.
There are three types of cells in our body, e.g. somatic cells, stem cells, and germ cells.
Spermatogonia are the immature germ cells. They divide several times during the process of spermatogenesis. The spermatogenic process is directed by genes located on the Y chromosome and takes around 70 days to complete from the spermatocyte stage.12−21 more days are required for the transport of sperm from the testis through the epididymis to ejaculatory duct.
A uniflagellar sperm cell that is motile is referred to as a spermatozoon, whereas a nonmotile sperm cell is referred to as a spermatium.
Human spermatozoa have some unique characteristics which are summarized below:
- Sperm cell is the smallest cells in the body in terms of volume
- These cells (adult sperm cells) do not grow or divide
- The sperm cells are the most polarized cells; head in front and tail at the rear part of the body
- They fulfill their function outside the body, in different individuals, e.g. in female genital tract
- Unlike somatic cells, the sperm head has a large nucleus but lacks large cytoplasm. Nucleus constitutes 65% of spermatozoa head
- The sperm cells are unique among mammals for presence of plenty of abnormal forms of spermatozoa in the ejaculate.
Most tightly compacted eukaryotic DNA that is present in mammalian sperm, at least sixfold more highly condensed than the DNA in mitotic chromosomes.1 To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin.2
This differs markedly from the builder DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored.
The centromeres are located centrally and telomeres peripherally. Folding of chromosomal p- and q-arms are flexible (Fig. 1). This specific chromosomal arrangement may be responsible for increased frequency of abnormal sperm shape and increased frequency of aneuploidy.
- It has been observed that sex chromosome and G-group (chromosome 21 and 22) are more susceptible to nondisjunction during spermatogenesis.
- Morphologically abnormal sperms (large head, round head, etc.) have either numerical or structural abnormalities of chromosomes.
ANATOMICAL SEGMENTS OF ADULT SPERMATOZOA (FIG. 2)
A mature human sperm cell has got the following parts—head, neck, and tail. Tail is again divided into—middle piece, principal piece, and end piece.
Head
It is oval in shape consisting of large nucleus and a dome-shaped acrosome present on the nucleus. In humans, sperm cells consists of a flat, disk-shaped head which is 5 µm in length and 3 µm in width and a tail 50 µm long.2 The tail flagellates, which propels the sperm cell (at about 1−3 mm/minute in humans) by whipping in an elliptical cone.3 The spermatozoon is characterized by a minimum of cytoplasm and the most densely packed DNA. The coverings of the sperm head from outside are:3
- Plasma membrane
- Outer and inner acrosomal layers of membrane
- Acrosomal sac containing enzymes
- Nuclear cap
- Nucleus.
Acrosome
This is found at the anterior tip of the sperm (derived from Greek term “akron” meaning extremity and “soma” meaning body). The acrosome forms a cap like structure called the acrosomal cap. This occupies the space between anterior half of the nucleus and the plasma membrane of the sperm tip. The acrosome arises from the golgi complex during spermatogenesis. The acrosome itself is bounded by a unit membrane. It consists of a number of hydrolytic enzymes such as acid phosphatase, hyaluronidase, and others. These enzymes help in tissue lysis (dissolving) and this facilitates the penetration of the sperm into the egg membrane. The enzymes are proteolytic and help in dissolving the egg membrane.
The membranes of late spermatids and spermatozoa contain a special small form of angiotensin-converting enzyme called germinal angiotensin-converting enzyme. The function of this enzyme in the sperms is unknown, although male mice in which the function of the angiotensin-converting enzyme gene has been disrupted have reduced fertility.
Sperm Nucleus
The nucleus occupies most of the available space of the sperm head. It is the shape of the nucleus that ultimately decides the shape of the sperm head. Structurally it is enveloped by a nuclear membrane. Sometimes, however the posterior part of nuclear membrane (towards the body of the sperm) is 4somewhat depressed to accommodate the proximal centriole. The nucleus consists of DNA as well as basic proteins. There is no nucleolus.
Function: Nucleus contain genetic information and half number of chromosomes. The acrosome releases a hyaluronidase enzyme which destroys the hyaluronic acid of the ovum and enters into the ovum.
Neck
It contains centrioles which are proximal centriole and distal centriole.
Function: Distal centriole gives rise to axial filament of the sperm which runs up to the end of the tail. Centrioles help the zygotic division by forming the first mitotic spindle. The posterior or the distal centriole is responsible for the formation of the microtubules of the sperm tail.
Tail
Middle Piece (Figs. 3A and B)
It is tubular structure in which mitochondria are spirally arranged. It has a pair of longitudinal fibers called beta fibers, surrounded by a ring of nine pairs of longitudinal fibers called alpha fibers. In human sperms, the alpha fibers of axial filament are accompanied on the outside by nine, much thicker fibers called gamma fibers or coarse fibers. The alpha, beta, and gamma fibers are the sites of various enzymes.
Alpha fibers have ATPase enzyme, while beta fibers have acetyl co-A succinate. These fibers are anchored to the distal centrioles. The fibers are surrounded by the mitochondria. Very often the mitochondria are fused together and form a spiral sheet that surrounds the axonemal fibers. Around the periphery of midpiece of the sperm is found a thin sheet of cytoplasm mainly composed of microtubules. This layer is called manchette.
Function: Middle piece is called power house of sperm because it gives energy to the sperm to traverse through the female genital tract.
Principal Piece
The principal piece which constitutes most of the length of tail consists of the central core made up of axial filaments with a 9+2 arrangement (2 central, 9 peripheral). The tail fibers are attached to each other by arms containing the protein dynein, which is an ATPase. Hydrolysis of ATP (adenosine triphosphate) in the adjacent mitochondria provides the energy for sperm motility, which is produced by a sliding action between the fibers in the sperm tail.
Surrounding this core is a fibrous tail sheath which often appears as semicircular ribs oriented at right angles to the long axis of the filament. Sometimes, they appear as helical coils. In human beings, two of the gamma fibers are fused with the surrounding ribs to form anterior and posterior columns extending throughout the length of the principle piece.5
This arrangement divides the principal piece into two functional compartments—one having three gamma fibers and the other containing four. This symmetry is thought to help in a more powerful stroke of the tail in one direction. This is called the power stroke. The end piece is a small tapering portion of the tail containing only the axial filament covered with cytoplasm and plasma membrane.
End Piece
This the terminal end of tail. Length is about 5 µm.
Sperms have no cytoplasmic organelles such as ribosomes and endoplasmic reticulum. There is no stored food in the sperm.4
MOLECULAR FUNCTIONS OF DIFFERENT SEGMENTS OF SPERMATOZOA
Functions of the Head
The plasma membrane which constitutes the outer coat of the head consists of a very unstable fatty acid which is known as polyunsaturated fatty acid (PUFA). 6PUFA has both helpful and unwanted functions in reproduction. The helpful function consists of facilitating fusion and disintegration of plasma and acrosin membrane leading to exocytosis of the enzyme acrosin. This happens when the sperm head comes in contact with zona pellucida at the time of fertilization. This procedure is known as “acrosome reaction” and “zona penetration” which allows the sperm head to enter into the perivitelline space.
Due to presence of PUFA (unstable fatty acid) excessive fluidity of plasma membrane is seen that is very specialized character of sperm. These may be responsible for premature disintegration and exocytosis of acrosome. This may happen when many leukocytes are present in seminal plasma, or due to presence of plenty of immature sperm cells, varicocele, and excessive centrifugation.
Under normal conditions, cytoplasmic syngamy occurs when the sperm head after zona penetration comes in contact with oocyte oolemma (outer coating of oocyte cytoplasm). Cytoplasmic syngamy has two important molecular events: (a) calcium oscillation, (b) cortical reaction. This happens due to oocyte activation through sperm head contact with oolemma.
Calcium Oscillation
Calcium oscillation occurs due to calcium influx from cytoplasmic organelles (rich in calcium stores). The primary effect of calcium oscillation within oocyte cytoplasm is removal of inhibitory factor for completion of meiosis-II which gets initiated with meiosis-I of oogenesis (during intrauterine life). In other words calcium oscillation induces maturation promoting factor (MPF) within oocyte cytoplasm, which is necessary for release of second polar body (completion of meiosis-II). Following meiosis-II, the oocyte nucleus is converted into a spindle (containing half of the genetic material) and forms the female pronucleus). Before the female pronucleus is formed, calcium oscillation also helps in formation of male pronucleus. This is an interesting step. The male pronucleus is formed primarily by removal of nuclear cap of the sperm head, replacement of the special type of sperm head protein—protamine by histone migrating from the oocyte nucleus. This is followed by assembly of a new nuclear envelop—formation of male pronucleus. Pronuclear chromatin condenses to form nucleolar precursor body (NPB). These are also known as nucleoli. Arrangement and synchrony of male pronuclear nucleoli with regard to nucleoli of the female pronucleus are significant markers of good or bad pronuclei (normal or abnormal fertilization)(Fig. 4).7
Function of Centriole in Sperm Neck
This helps in apposition of two pronuclei (male and female) by forming microtubules. The last stage of fertilization namely “nuclear syngamy” is completed by these microtubules.
Function of Tail
This helps the sperm to swim in the female genital tract. It is the main part of sperm that helps in movement through the female genital tract. The ability to move forward (progressive motility), which is acquired in the epididymis, involves activation of a unique protein called CatSper, which is localized to the principal piece of the sperm tail. This protein appears to be a Ca2+ ion channel that permits cAMP-generalized Ca2+ influx. In addition, spermatozoa express olfactory receptors, and ovaries produce odorant-like molecules. Recent evidence indicates that these molecules and their receptors interact, fostering movement of the spermatozoa towards the ovary (chemotaxis).
EXAMPLES OF SPERM ABNORMALITIES (FIG. 5)
- Head: Defects in shape and size—like large, small, tapering, pyriform, amorphous, and vacuolated (more than 20% of head surface is occupied by vacuoles). There may also be double head or combination defect.
- Neck and midpiece abnormalities: This consists of absence, non-inserted, fractured, bent and thin midpiece.
- Tail abnormalities: Tail abnormalities include short, multiple, hair pin, broken, coiled, and tail with terminal droplets.
- Cytoplasmic droplets in the head: Cytoplasmic content of the sperm head is much less than the nuclear DNA content. Cytoplasmic area greater than one-third of the area of the normal sperm head are considered abnormal.
SPERMATOGENESIS
This can be discussed under two broad headings:
- Molecular consideration
- Anatomic consideration.
Molecular Consideration
The origin of adult spermatozoa from spermatogonial germ cell passes through three molecular phases:
- Mitotic proliferation: Duplication of chromosomal DNA followed by cell division, to maintain pool of stem cells. Proliferation and differentiation of diploid spermatogonial germ cell occurs in this phase.
- Meiotic division:
- Duplication of chromosomal DNA, two cell divisions, results in haploid spermatogonia, to halve chromosome number.
- Genetic diversity.
- Phase of spermiogenesis (cytodifferentiation): This phase consists of a series of changes involving development of nuclear DNA, acrosomal cap, tail, and ultimately resulting in development of an adult spermatozoa.
These events collectively, approximately continue for 70 days. Within this long period there may be numerous opportunities for introduction of damage to the genome of male gamete. This knowledge provides the practical information that while performing intracytoplasmic sperm injection (ICSI) with spermatid or secondary spermatocyte, there may be a risk of injecting a damaged spermatocyte. This may lead to failure of fertilization or development of an abnormal embryo.
Proliferation and Differentiation of Diploid Spermatogonial Cell
In the testis, the spermatogonial stem cells proliferate and differentiate producing three types of spermotogonia:
- Population trying to differentiate towards adult spermatogonial cell—they are the precursors of future adult spermatozoa.
- Cells that are likely to undergo apoptosis.
During this phase, the spermotagonial cells are diploid, i.e. they contain two chromosomes each, and two chromatids (DNA strands) in each chromosome.
Phase of Meiosis
During this phase, the diploid proliferating and differentiating stem cells are converted to haploid gamete. This is a critical and unique event of genetic recombination. Primary spermatocyte (spermotogonial stem cells) with DNA content equivalent to two chromatids in two chromosomes replicate into four distinct chromatids (DNA strands) initiating meiosis (Fig. 6).
Chromosome segregation and crossing over of genes amongst DNA strands occurs during this phase. Crossing over is critical in gametogenesis—may lead to genetic defect and structural anomaly of sperm. Because, during this phase of crossing over, there may be loss or defect in the genetic material.
After chromosomal pairing and crossing over—the first meiotic division is completed, i.e. two secondary spermatocytes are formed. There is one chromosome and two chromatids (DNA strands) in each secondary spermatocyte. Therefore, the DNA content in each secondary spermatocyte is still diploid.
The second meiotic division (Fig. 7) starts where there is separation of two DNA strands in each chromosome—resulting in formation of four spermatids, each spermtaocyte having a haploid number of chromosome and a haploid DNA.
The diagrammatic representation of the entire process of spermatogenesis at molecular level is shown below.
Possible Problems Arising during the Phase of Meiosis
For comprehensive meiotic division to occur, meiotic cell contains many novel proteins and enzymes. These are essential for chromosome and DNA alignment, DNA breakage, recombination and DNA repair. Occasionally DNA repair mechanism in the phase of meiosis may be defective; there may 11be anomalies in chromosomal segregation and pairing and crossing over of genetic material. These defects may lead to germ cell differentiation arrest at either primary or secondary spermatocyte level.
Phase of Spermiogenesis: Cytodifferentiation
During this phase, maturation of spermatozoa starts. From the stage of secondary spermatocyte a round-shaped spermatid forms followed by elongated spermatid and finally an adult spermatozoa develops.
During these transitional phases, the specific changes which occur consist of:
- Elongation of nucleus and nuclear condensation
- Appearance of acrosomal sac containing proteolytic enzymes
- Formation of neck and differentiation of the terminal part into three distinct segments—midpiece, principal piece, and end piece. These changes occur during six different stages; the stages have been designated as SA-1 and 2, SB-1 and 2 and SC-1 and 2
- Cytoplasmic reduction.
Specific and Remarkable Changes during Spermiogenesis
- Head nuclear protein consisting of histone is replaced by protamine, producing a tightly compacted nucleus. Protamine is a stronger DNA protein compared to histone. Unlike all other somatic cells of the body where histone is present with the DNA in the nucleus, spermatozoa is the only cell which contains protamine to offer compactness of the sperm head nuclear DNA.5,6
- The adverse effect of displacement of histone and replacement by protamine may result in haploid genome damage (after secondary spermatocyte, the sperm cell genome becomes haploid).
- Repair capabilities during spermiogenesis phase is limited (unlike those during meiosis phase).
- In addition to nuclear DNA structuring, axoneme, outer dense fiber and protein (dynein) in midpiece, principal piece and end piece develop.
- Mitochondria develops on the sheath of midpiece as germ cell differentiation by spermiogenesis continues.
As a consequence of massive changes during spermiogenesis there may be tremendous load on “haploid” spermatozoa, leading to germ cell arrest or blockage—thereby causing infertility in many individuals.
Defects in synthesis of midpiece and tail mitochondria may result in structurally abnormal spermatozoa with poor motility. Also mutation in protein essential for compaction of sperm nuclear DNA may result in spermatozoa with abnormal head.11–1712
Minor genetic defects may not alter spermatozoa morphology but may lead to production of genetically defective spermatid. This will be a great concern for spermatid injection which is sometimes performed in the procedure of ICSI (ROSNI-round spermatid nuclear injection).
Difference of Initiation of Meiosis in the Male and Female Gamete
In female, meiosis starts at 12 weeks of intrauterine life, but remains arrested at meiosis-I. This is completed at puberty with onset of luteinizing hormone (LH) surge. In male, spermatogonial cells (the stem cells) remain at rest till puberty—meiosis and spermiogenesis start after puberty.
SPERMATOGENESIS—ANATOMICAL CONSIDERATION
Sperms develop and mature within seminiferous tubules. Seminiferous tubules consist of basement membrane and lumen (Fig. 8).
Basement Membrane
Basement membrane consist of two types of cells: Germ cells and Sertoli cells.
Sertoli cells are triangular-shaped cells with their apex projecting towards the lumen. The base of these triangular cells are situated peripherally. Apex of the Sertoli cells are interconnected by tight junction. This tightly interconnected apical junction forms “blood-testis barrier”. Blood-testis barrier when intact does not allow seminal antigens to pass into the systemic circulation (reticuloendothelial system) to produce self antibodies.
Germs cells lie in between the Sertoli cells, they are precursors of adult spermatozoa.
Lumen
Interconnected Sertoli cells divide the lumen of seminiferous tubules into two compartments:
- Basal compartment
- Adluminal compartment.
Significance of Two Compartments
- Basal compartment: In this compartment, maturation of early stages of spermatozoa occur. This compartment is in direct contact with interstitial cells containing Leydig cell, blood vessels, and lymphatics and therefore is directly exposed to immune phenomenon. But the tight interconnection of the apices of Sertoli cells, which forms the blood-testis barrier, prevents antigens to cross this barrier and prevents antibody formation. But when this blood-testis barrier is damaged as in infection, trauma, exposure to heat, and following vasectomy, antigens may crossover allowing antibodies to develop in reticuloendothelial system of the body. These antibodies then reenter the seminiferous tubules and damage the developing spermatozoa. Basement membrane contains myofibrils—which are under control of oxytocin. They help in forward sperm propulsion.
- Adluminal compartment: Within the adluminal compartment, late stages of spermatozoal maturation continues. This is a sealed compartment and therefore not exposed to external or environmental trauma.
Final Maturation and Acquisition of Motility of Spermatozoa
This occurs through exposure of spermatozoa to many biochemical components while the sperm travels through the seminal pathway. The principal sites where the sperm acquires significant motility are—rete testis, epididymis, seminal vesicles, and prostate. The important biochemical constituents which provide additional sources of sperm vitality, motility, and integrity are—carnitine, acid glycerophosphate from epididymis, fructose and coagulase (from seminal vesicle), liquefying enzymes and acid phosphatase from prostate.
Effect of Temperature on Spermatogenesis
Spermatogenesis requires a temperature considerably lower than that of the interior of the body. The testes are normally maintained at a temperature of about 32°C. They are kept cool by air circulating around the scrotum and probably by heat exchange in a countercurrent fashion between the spermatic 14arteries and veins. When the testes are retained in the abdomen or when, they are held close to the body by tight cloth binders, degeneration of the tubular walls and sterility result. Hot baths (43−45°C for 30 minutes per day) and insulated athletic supporters reduce the sperm count in humans, in some cases by 90%. In addition, evidence suggests a seasonal effect in men, with sperm counts being greater in the winter regardless of the temperature to which the scrotum is exposed.
Endocrine Control of Spermatogenesis (Flowchart 1)
Just like ovulatory control, spermatogenesis has also an endocrine control with feedback mechanism between hypothalamic pituitary control from one side and testicular control from the other side. Follicle-stimulating hormone (FSH) is secreted from the pituitary which stimulates Sertoli cell within the seminiferous tubules and Sertoli cells in turn produce two factors:
- Inhibin which regulates the production of FSH from pituitary
- Androgen binding globulin (ABG).18
Androgen binding globulin transports testosterone produced by Leydig cells which exist outside the seminiferous tubules into the lumen of the seminiferous tubules allowing maturation of germ cells. LH also produced by pituitary stimulates Leydig cells to produce testosterone (5−10 mg per day). Testosterone on one side helps maturation of germ cells and on the other hand regulates production of pituitary LH through negative feedback mechanism. The stages from spermatogonia to spermatids appear to be androgen-independent. However, the maturation from spermatids to spermatozoa depends on androgen acting on the Sertoli cells in which the developing spermatozoa are embedded. FSH acts on the Sertoli cells to facilitate the last stages of spermatid maturation. In addition, it promotes the production of androgen-binding protein (ABP).18
Flowchart 1: Endocrine control of spermatogenesis.(ABG: androgen binding globulin; FSH: follicle-stimulating hormone; LH: luteinizing hormone).
In addition to endocrine control there are other paracrine procedures which help in spermatogenesis. These paracrine factors consist of— IGF-1, cytokines, proteins, and enzymes.19
The estrogen content of the fluid in the rete testis is high, and the walls of the rete contain numerous estrogen receptors (ER). In this region, fluid is reabsorbed and the spermatozoa are concentrated. If this does not occur, the sperm entering the epididymis are diluted in a large volume of fluid, and infertility results.
After production in seminiferous tubules under tight hormonal control, sperm have to pass through a long pathway to act in its final destination. Spermatozoa leaving the testes are not fully mobile. They continue their maturation and acquire motility during their passage through the epididymis. During its journey, it gets matured, motility as well as sperm becomes susceptible to different type of damage by free radicals.
Following the morphological transformation of nucleus in the testis, as spermatozoa transit through the epididymis, there occurs a stabilization of the chromatin through establishment of disulfide bond between the thiol rich protamines.20 Qualitative and quantitative modifications of the plasma membrane occurring in the lipidic composition21 and the absorption of specific proteins secreted by the epididymal epithelium result in changes of its electric charges. The lack of all this changes is associated with a decreased ability of epididymal spermatozoa to bind and penetrate the oocyte.22 Ejaculation of the spermatozoon involves contractions of the vas deferens mediated in part by P2X receptors for ATP and fertility is reduced in mice in which these receptors are knocked out.
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