Spermatogenesis

Spermatogenesis is a complex and synchronized process of cell division and differentiation of germ cells resulting in potential motile haploid male gamete, spermatozoa, in the seminiferous tubule (Fig.

Fig. 20.1 Spermatogenesis. [The Spermatocytogenesis involves both mitotic and meiotic divisions. In mitotic divisions, the spermatogonium is transformed into primary spermatocytes through spermatogenesis. The formation of secondary spermatocytes and spermatids occurs through first and second meiotic divisions under spermatocytogenesis. In spermiogenesis, the rounded spermatids are transformed into elongated spermatids and, finally, spermatozoa (Sing: spermatozoon) through morphological changes. The diploid cells are presented as ‘2n’ and haploid as ‘n’]

form spermatids. The spermatids undergo metamorphosis to become mature spermatozoa and release into the tubular lumen.

Spermatogenesis can be divided into three main stages: Spermatocytogenesis, spermiogenesis, and spermiation. Spermatocytogenesis involves the cycles of both mitotic and meiotic cell divisions, resulting in the formation of spermatids from spermatogonia. The mitotic divisions of spermatocytogenesis have a dual role; firstly, it helps the renew stem cells to produce spermatogonia and maintain a steady pool. Secondly, the spermatogonia become primary spermatocytes to produce mature spermatozoa. The meiotic cycles of spermatocytogenesis involve two cell divisions that reduce the chromosome number to yield haploid round spermatids. The spermatids then undergo metamorphic changes to become mature spermatozoa through spermiogen­esis. In spermiation, the mature spermatozoa are released into the lumen of seminiferous tubules from the germinal epithelium.

The seminiferous tubule contains a variety of cells involv­ing different stages of spermatogenesis. Thin cytoplasmic bridges connect all these cells until spermatozoa are formed.

The spermatogenesis is under the endocrine control of testos­terone, follicle-stimulating hormone (FSH), and oestrogen.

20.1.1 Spermatocytogenesis

Spermatocytogenesis is a proliferative phase in which many germinal epithelial cells are multiplied through a series of mitotic divisions followed by meiotic divisions to produce early stages of haploid gamete (Fig. 20.1). The spermatocytogenesis starts before puberty in most animals and birds, and the time required to complete the mitotic division is generally 21 days in the bull.

20.1.1.1 MitoticDivisions

Development of Spermatogonial Stem Cells (SSCs) The SSCs are the undifferentiated spermatogonia developed from primordial germ cells (PGCs). SSCs have the capabilities of self-renewal and can convert into pluripotent stem cells. Further, they can transfer the genome from one generation to the next, and they are the only adult stem cells of this kind. The PGCs migrate to the genital ridge and are differentiated into gonocytes during 10.5 days post­fertilization in mice. The replications of gonocytes are arrested at the G0/G1 stage of the cell cycle. The mitotic capability of the gonocytes is resumed after birth, and they start migrating centre to the periphery of the seminiferous tubule and reach the basement membrane to develop spermatogonia. The transformation of gonocytes into the spermatogonia required 2 months after birth in pigs and goats, 3 months in sheep, and 4 months in cattle. A particular microenvironment is essential for the survival and develop­ment of SSCs. It is called ‘niche’, made by Sertoli cells, peritubular myoid cells and Leydig cells. Sertoli cells provide nutrients to the SSCs and germ cells. The Sertoli cells derived growth factors (glial cell line-derived neurotrophic factor, GDNF and basic fibroblast growth factor, bFGF) are essen­tially required for the self-renewal property of SSCs.

Proliferation of Spermatogonia The spermatogonia are classified into two broad categories, type A and type B spermatogonia. The type A spermatogonia has a prominent round nucleus with condensed chromatin and peripheral nucleoli. It may also contain a nuclear vacuole. Cytologi- cally, type A spermatogonium has two types, Ad (dark) and Ap (pale) spermatogonia. The Ad spermatogonia don’t have proliferative capabilities under normal circumstances. They are considered the testicular stem cells, but when the sper- matogonial concentration is severely reduced (as in radia­tion), the Ad spermatozoa may show proliferative capability.

In contrast, the Ap spermatogonia have the proliferative and self-renewal capabilities. Several types A spermatogonia remain as resting type A spermatogonia (intermediate spermatogonia), which later differentiated into type B spermatogonia by mitotic cell division. The type B spermatogonia contain central nucleoli with dispersed chro­matin and no nuclear vacuole. The type B spermatogonia undergo mitotic division to form primary spermatocytes. Generally, one type A spermatogonium can produce 12 pri­mary spermatocytes. The primary spermatocytes then enter meiotic divisions to develop spermatozoa.

The spermatogonial subpopulations vary with species and breeding seasons. Three types of spermatogonium have been found in bull, type A spermatogonium, intermediate sper­matogonium, and type B spermatogonium. In stallions, A₁, A₂, A₃, B₁, and B₂ spermatogonia are found. Usually, type A spermatogonia are more available during the non-breeding season, whereas type B spermatogonia are abundant in the breeding season.

20.1.1.2 Meiotic Divisions

The diploid primary spermatocytes are larger than spermatogonia and contain highly condensed chromosomes with coarse chromatin.

First Meiotic Division In the first meiotic division, primary spermatocytes generate haploid secondary spermatocytes. The first mitotic division is also called reductional division, as there is the reduction of chromosome number and the separation of homologous chromosomes. The reshuffling of genetic material also occurs during this stage. The first mei- otic division is testosterone dependent. Hence, before puberty, secondary spermatocytes are not developed. In hypophysectomized animals, the presence of secondary spermatocytes is also limited. Secondary spermatocytes are spherical cells with interphase nuclei.

Second Meiotic Division It is called equational division, in which the separation of the daughter chromatids is occurred to form haploid spermatids from secondary spermatocytes. The spermatids are round and mitotically inactive. The sec­ond meiotic division is testosterone independent and requires less time. Spermatids are transformed into spermatozoa through metamorphic changes.

20.1.1.3 Immune Protection OfSpermatids

The spermatids are autoimmunogenic as recombination of genetic materials occurs during their production through meiosis. Due to the recombination of genetic materials, the haploid gamete may differ from the parent somatic cells and are susceptible to auto-immune attack. The blood-testis bar­rier (BTB) provides an immune-privileged environment to the spermatids for their survival by eliminating the somatic parent cells’ humoral or cellular immune components. The temperature of the testes is also an essential criterion for meiosis as DNA polymerase and recombinase enzymes require a particular temperature for their action. Therefore, the descent of descending testes and its thermoregulation favour spermatogenesis.

20.1.2 Spermiogenesis

The transformation process of spermatids into spermatozoa is called spermiogenesis (Figs.

Fig. 20.2 Spermiogenesis. [Three distinct phases of spermiogenesis, viz. Golgi phase, Acrosomal phase or cap phase, and Maturation phase are presented where structural changes of various organelles occur]

of the Sertoli cells under the influence of FSH. The Sertoli cells provide the nutrients, enzymes, hormones, and other substances required for spermiogenesis (the role of the Sertoli cells in spermatogenesis has been discussed in detail in an earlier chapter). It commonly takes 17 days in the bull. The entire spermiogenesis process is usually divided into four phases: Golgi phase, cap phase, acrosomal phase, and matu­ration phase.

20.1.2.1 Golgi Phase

In this phase, the nucleus is compressed with tightly packed chromatin. The nucleus is transcriptionally inactive. The structure of the nucleus occurs with one side oval and the opposite one narrower. In birds, the nucleus is long or cylin­drical. It is spiraled in the passerine group of birds. The granules of the Golgi vesicles of spermatids are merged at the oval side to form a pro-acrosomal vesicle. The centrioles and mitochondria migrate to a position opposite to acrosomal vesicles. The nucleus and acrosome together form the head of the sperm. The proximal centriole gives rise to the attachment point for the tail, and the distal centriole gives rise to the developing axoneme. The mitochondria combine to form a mid-piece. The structure axoneme, composed of a group of microtubules, is initiated to develop. The spermatid becomes extended, and the elongation process begins. At the end of this phase, the elongated part is turned into the mid-piece of the sperm. The mitochondria are helical and more elongated than non-passerine birds in passerine birds.

20.1.2.2 Cap Phase

The acrosomal vesicle flattens to form a distinct cap to cover almost half of the nucleus. It is conical or tapering shaped in the bird. The acrosomal vesicle is made of an outer acrosomal membrane and an inner acrosomal membrane. It contains lysosomal enzymes like hyaluronidase and proteases that help in fertilization. The distal centriole forms the axoneme or flagellum that projects away from the nucleus to the lumen of the seminiferous tubule (Fig. 20.2).

The acrosomal vesicle covers almost half of the nucleus, resulting in the head cap of the cell (spermatozoa). The acrosome acts like a lysosome and contains lysosomal enzymes, like hyaluronidase and proteases, which have a vital role in fertilization.

20.1.2.3 Acrosomal Phase

The nucleus of the spermatid begins to elongate, and the acrosome covers the majority of the anterior nucleus. A unique microtubular system called manchette is extended from the posterior portion of the nucleus. It contains a bunch of nine peripheral double microtubules and two single tubules in the centre. Some of the microtubules are developed into the post-nuclear cap. The tail is covered with the exten­sion of the cell membrane. During the acrosomal phase, spermatids are deeply embedded in the Sertoli cells, with their tails protruding towards the lumen of the seminiferous tubule.

20.1.2.4 Maturation Phase

In this phase, the manchette migrates towards the tail and begins to disappear. Mitochondria are assembled around the flagellum to form mid-piece. The mid-piece portion contains a large amount of adenosine triphosphate (ATP) to provide energy to the sperm. The manchette microtubules form the post-nuclear cap. The dense outer fibres cover the flagellum. It contains a bunch of nine peripheral double microtubules and two single tubules in the centre. The junction between the middle piece and principal piece annulus is formed. The spermatids are elongated and extrude the rest of the cyto­plasm in the form of a spheroidal lobule called residual body. The residual bodies are phagocytosed by the Sertoli cells. The secretory activity of the Sertoli cells (inhibin, ABP and interleukin-1 and 6) depends upon the elongation of

Fig. 20.3 Structure of fully developed spermatozoa

spermatids and residual bodies. A new Spermatogenic cycle begins after the degradation of residual bodies.

The remnant cytoplasm (after phagocytosed by the Sertoli cells) adheres at the neck region of the elongating spermatid in the form of a cytoplasmic droplet (CD). The mammalian CD is about 2 μm in diameter and composed of organelle- derived membranes and cytosol. The CDs are removed from the spermatozoa during its passage through epididymis. They are generally seen at the neck in the immature sperm of caput epididymis and migrate further, and retain at the end of mid­piece in the sperm in cauda epididymis. Their migration along the mid-piece indicates sperm maturation, and the retention of CD at the mid-piece is one of the vital sperm abnormalities. Phospholipid binding protein (PBP) present in the ampulla and seminal vesicles of the bull helps in the removal of CD. PBP is generally absent in accessory sex glands of boar; hence the spermatozoa of porcine ejaculate contain more CD than other species. The enzyme 15-lipoxygenase (15LOX) and the ubiquitin-proteasome help degrade cytoplasmic droplets. The CD also helps in sperm volume adaptation and protects the spermatozoa from the hypotonic challenge. There is a relationship between the presence of CD and the progressive motility of the spermatozoa. The sperm possess CD display progressive motility, and the sperm without CDs are mostly non-motile. The relationship between CD and motility can explain because CDs act as mitochondrial modulators and potentiate mitochondrial activity and membrane potential. Despite sev­eral beneficial roles, the sperm cytoplasmic droplet remains an enigma as the retention of CD in the ejaculated sperm is associated with infertility. The sperm with CD have poor binding capabilities with zona pellucida and a reduced preg­nancy rate. More recently, the CDs are considered the normal morphological occurrence in the human spermatozoa and the terminology ‘excess residual cytoplasm’ is designated as abnormal. The spermatozoa with ‘excess residual cytoplasm’ can now be considered immature spermatozoa without termi­nal differentiation.

The fully matured spermatozoa (Fig. 20.3) morphologi­cally contain five major components with various cellular organelles (Table 20.1). The head of spermatozoa has a shape characteristic of different species. The spermatozoa of

Table 20.1 Spermatozoa and its organelles with major functions

bulls and humans have paddle-shaped heads. The spermatozoa of rodents have hook-shaped heads. The head contains an oval and flattened nucleus in which a nuclear membrane surrounds compact chromatin. The acrosome covers the anterior 2/3rd of the nucleus. The acrosome has an outer and inner acrosomal membrane. Hydrolytic enzymes like acrosin, hyaluronidase, zonalysin, esterase, and acid hydrolases are present within the acrosome. These enzymes are resealed during the acrosomal reaction and help to pene­trate the zona pellucida of the ovum during fertilization. The tail is made of the capitulum, mid-piece, principal, and termi­nal pieces. The capitulum lies at a depression in the posterior nucleus called the implantation socket. The anterior portion of the tail contains laminated columns that aid the flexibility of the neck. The axonemal components of the tail are made of 9 pairs of microtubules arranged radially around the central filaments. There are nine dense fibres surrounding the micro­tubular arrangements at the flagellum of spermatozoa. The mid-piece is composed of a mitochondrial sheath arranged in a helical pattern. The mid-piece contains a large amount of adenosine triphosphate (ATP) to provide energy to the sperm. The annulus is the junction between mid-piece and principal piece. The principal piece makes the major portion of the tail and connects with the terminal piece.

20.1.3 Spermiation

After the completion of spermiogenesis, the spermatozoa are released from the Sertoli cell into the lumen of the seminifer­ous tubules through a process called spermiation. The spermatozoa are arranged perpendicularly to the tubular wall and gradually expelled out into the lumen of the tubule. The release of spermatozoa is facilitated due to the attenua­tion of the slender cytoplasmic stalk connecting the spermatids with the residual body. Once the spermatids sep­arate from the residual body, it becomes spermatozoa. The breakage of the stalk results in proximal CDs in the neck region. The residual bodies are degraded by Sertoli cells.

20.1.4 The Final Maturation of Spermatozoa

The testicular spermatozoa are immature and non-motile. They gain motility and fertilizing capability during their transit through the epididymis. The epididymis cells have high metabolic, secretory, and endocytic activity regulated by androgens which facilitate the absorption of fluids, and secretion of ions, antioxidants, and proteins.

20.1.4.1 Epididymal Transit of Spermatozoa and Storage

The sperm move from the head of the epididymis towards the tail by the hydrostatic pressure gradient. Most mammals generally use transit time for 1-2 weeks (Table 20.2). The peristaltic contraction of the epididymis facilitates the sperm transit, which is controlled by testosterone, oxytocin, vaso­pressin, and PGF2α. Frequent ejaculation favours sperm transit. In bull, daily ejaculation can reduce the transit time by up to 3 days and increase sperm concentration. The rate of movement is highest at the head, followed by the body and

Table 20.2 Transit time of the spermatozoa from caput to cauda in epididymis

tail. In bull, it is 420 mm/2 h at the head, 64 mm/2 h at the body, and 25 mm/2 h in the cauda epididymis and vas deferens. The lowest speed at the tail of the epididymis increases the transit time and favours sperm storage. In the tail of epididymis, 50-80% of total spermatozoa are stored, which can be sufficient for ten successive ejaculations in stallions and bulls. At the cauda of the epididymis, the spermatozoa remain quiescent, and three to fivefolds can increase their metabolic activity upon ejaculation. The exact mechanism of this metabolic quiescence is unknown; how­ever, the presence of specific enzymes, proteins, and luminal pH may be the contributing factors. The epididymal spermatozoa can be viable up to 2-3 weeks in most mammals, and the unutilized spermatozoa are released into the urethra and excreted through urine.

20.1.4.2 Morphological and Biochemical Changes of Spermatozoa During Epididymal Transit

The spermatozoa undergo many morphological, biochemical, and functional changes during epididymal transit necessary for successful fertilization. The major changes are summarized in Table 20.3.

The sperm released from the head of the epididymis are rarely motile and gain a progressive motility pattern during their epididymal transit (Table 20.4). The factors that pro­mote this progressive motility are forward motility protein (FMP), the elevation of cyclic adenosine monophosphate (cAMP), and increased sperm pH. FMP alters the permeabil­ity of the sperm plasma membrane and allows the influx of Ca⁺⁺ inside the sperm. Cyclic adenosine monophosphate (cAMP) activates cAMP-dependent protein kinases. These cAMP-dependent protein kinases, in turn, activate phospho­protein phosphatase to phosphorylate multiple intra sperm phosphoproteins. The phosphorylation of intra-sperm proteins initiates flagellar movement, and sperm gain their motility.

20.1.4.3 Epididymal Proteins and Their Role in Sperm Maturation

During epididymal transit, the spermatozoa acquire some proteins required for protection against reactive oxygen spe­cies (ROS), gaining progressive motility and fertilizing capa­bility. The proteins are thought to be transferred from the epididymis through some membranous vesicular structure called epididymosomes. These epididymosomes transfer

Table 20.3 Morphological and biochemical changes of spermatozoa during epididymal transit

Table 20.4 Spermatozoal characteristics in different parts of the epididymis in bulls

Table 20.5 Role of epididymal proteins in sperm maturation

selected proteins into the spermatozoa in an apocrine manner without fusing the sperm plasma membrane. Different proteins present in the epididymosomes and their roles are summarized in Table 20.5.

20.1.5 SpermatogenicCycle

Upon examining the cross-sections of a seminiferous tubule, it may find definite cellular associations among the sperm forming cells as the seminiferous germinal epithelium cells (type A spermatogonia, the stem cells) are continuously regenerated by mitotic division to replenish the parental cell in a cyclic process. Each such spermatogenic association is designated as a stage of the seminiferous epithelial cycle. The time required to reappear in the same stage within a given seminiferous tubular segment is called the spermatogenic cycle (Fig. 20.4).

The cycle has two key features: firstly, the new spermatogonia start their divisions at a constant time interval

Fig. 20.4 Spermatogenic cycle of bull. [Figure showing the germ cell differentiation and progression from type A spermatogonia to spermatozoa in the 12 stages (written horizontally in roman I to XII) of the seminiferous epithelium cycle (presented vertically in roman I to IV) based on the development of the acrosome during spermiogenesis. Each stage can be found within the same transverse section of the seminiferous tubule. The duration of each cycle is about 13.5 days. Around 4.5 cycles are required to develop a spermatozoon from a spermatogonium. So the total duration of spermatogenesis is about

4.5 x 13.5 days = 61 days. The cell progression is continued from the right side in each row (cycle). The specific cell type in each cycle for a particular stage (column) is presented by alphabet(s) like, A = type A spermatogonia, In = intermediate spermatogonia, B1 = type B1 sper­matogonium, B2 = type B2spermatogonium, PL = preleptotene sper­matocyte, L = leptotene spermatocyte, Z = zygotene spermatocyte, P = pachytene spermatocyte, S-II = secondary spermatocyte, R-1 to R-7 = round spermatids and E-8 to E-14 = elongating/elongated spermatids, and Sp = Spermatozoon. Duration of mitotic proliferation continued for 21 days (all the stages of A to P), meiosis (S-II) continued for 23 days, and spermiogenesis (R-1 to E-14) for 17 days.] [Source: Segatelli et al. 2013; Staub and Johnson 2018]

Fig. 20.5 Effect of heat stress or warm climate in spermatogenesis and semen production

at one point of the tubule. Secondly, the rate of germinal cell differentiation is always the same with a fixed duration. Therefore, the duration of a cycle is fixed and species-specific (Table 20.5). In a cycle, diverse developmental stages of spermatozoa are found in different regions of the seminifer­ous tubule. In a particular segment of the tubule, the recur­rence of the same stage of the stem cell occurs. Various stages of spermatozoa are progressed cyclically. This process is continued longitudinally from the base of the seminiferous tubule towards the lumen.

20.1.6 SpermatogenicWave

The spermatogenic stages are highly coordinated not only with time but also in space. The serial transverse section of the seminiferous tubule exhibits that stage I is followed by II and stage III is by stage IV, and so on. The spermatogenic wave is the distance between the same stages within the seminiferous tubule, and one tubule may have several com­plete waves.

20.1.7 Duration of Spermatogenesis

The time to complete a spermatogenic cycle varies between species and depends upon the number of spermatogenic stages in a cycle. In bull and mouse, one cycle consists of 12 different stages of cells; a rat has 14 different stages of cells in a cycle. To complete the entire spermatogenesis process for generating a single spermatozoon, a 4¹∕₂ cycle

(3.9-4.7) is usually required in almost all domesticated mammals. The duration of spermatogenesis varies within 30-75 days in all domestic mammals (Table 20.6).

20.1.8 GermCellDegeneration

All the cells that are generated during various stages of spermatogenesis are not differentiated into spermatozoa. Few cells degenerate at different stages of spermatogenesis, and the numbers of degenerated cells are species-specific. In bull, about of 30% cells degenerate between spermatogonia A to intermediate spermatogonia. Additional 30% degenera­tion occurs during the formation of type B spermatogonia. In stallion, mostly degeneration occurs during the formation of type B spermatogonia. Some cells also degenerate at the end of meiosis. In humans, 30-40% of cells degenerate at the end of meiosis.

20.1.9 SpermatogenicEfficiency

Spermatogenic efficiency denotes daily sperm production (DSP) per gram of testes. It can be measured qualitatively by studying histological sections of seminiferous tubules and quantitatively by cell counting. The spermatogenic efficiency is effective for species comparison (Table 20.5). The sper- matogenic efficiency depends upon the number of SSCs and Sertoli cells. Domestic animals can categorize into three groups based on their spermatogenic efficiency. Animals like boar, stallion, buck, ram, rabbit, and mice have high

Table 20.6 Duration of a Spermatogenic cycle and spermatogenesis and daily production of spermatozoa with testicular weight in different mammals

Data compiled from various sources

spermatogenic efficiency and can produce 20-30 million sperm per gram of testis. The bull, buffalo bull, and cats have average spermatogenic efficiency and can produce 10— 20 million sperm per gram of testis. Humans have low spermatogenic efficiency and can produce less than ten mil­lion sperm per gram of testis. The lower spermatogenic efficiency in humans can be explained by their fewer germ cells, longer duration of spermatogenesis, and longer cycle length. In addition, the proportion of seminiferous tubules and the seminiferous epithelium is lower in humans com­pared to stallions, bulls, and rats. The spermatogenic effi­ciency can also explain puberty. As a thumb rule, 50 million spermatozoa with 10% motility denote puberty in the bull at about 42 weeks (38-46 weeks), depending on breed.

20.1.10 Factors Affecting Spermatogenesis

Several factors alter spermatogenic efficiency, and they can classify into physical factors, chemical factors, nutritional factors, endocrine factors, genetic factors, age, pathological factors, and miscellaneous factors. The detailed mechanisms of these factors affecting spermatogenesis are summarized in Table 20.7.

cell pool in avian testes. Hence, more spermatozoa produce within a short time. The epididymal transit time is also less in avian spermatozoa. Spermatogenic efficiency is around four times more than mammals (four times more spermatozoa per gram of testis). The spermatozoa of chicken have spindle­shaped heads and are challenging to differentiate from mid­piece. The spermatozoa are stored in extragonadal ducts, and their survivability is poor. Therefore, more mating is required to ensure more viable spermatozoa fertilize the ovum when the hens are in a clutch.

Know More....

The efficiency of the Sertoli cells for spermatozoa pro­duction is considered the major factor in assessing the spermatogenic efficiency of any animal. The wild boar (Sus scrofa scrofa) is one of the species that contain the highest number of Sertoli cells (42 million per gram of testis) and Leydig cells (157 million per gram of testis) among the mammals. But, the efficiency of sperm pro­duction by the Sertoli cells is less in wild boar, nearly 50% than the domestic pig; the size of the Leydig cells (400 μm³) is about fivefold smaller than the domestic pig. Thus, the spermatogenic efficiency of wild boar is less.

20.1.11 Special Characteristics in Avian Spermatogenesis

Avian spermatogenesis divides into spermatocytogenesis and spermiogenesis. But, the duration of spermatogenesis is gen­erally four times faster than mammals. Spermatogenic cycle time is also less with less mitotic division. There is no stem

20.2