**2. Sperm head**

#### **2.1. Chromatin structure in normal spermatozoa**

The nucleus occupies a major part of the sperm head and contains condensed chromatin (**Figure 1a**), which is detected as an electron-dense homogeneous material, with small regions of lower electron density on ultrathin sections. Condensed chromatin is at least 10 times denser than ones in somatic cells [3].

**Figure 1.** (a) Spermatozoon with condensed chromatin (CH) and normal acrosome (A). (b) Postacrosomal segment of perinuclear theca (PS) with characteristic intermittent striation. (c, d) Spermatozoon with immature chromatin (IC). (e) Fragment of sperm acrosome. NE, nuclear envelope; PT, perinuclear theca; IM, inner acrosome membrane; EM, extra acrosome membrane; and PM, plasma membrane.

To achieve this unique extent of compaction, sperm DNA is packaged in a specific manner, which substantially differs from chromatin packaging in somatic cells. In somatic cells, DNA is packaged to produce the so-called nucleosomes. The DNA double helix is wrapped around a specific complex of canonical histones (a histone octamer) [4].

acrosome and nuclear vacuole, and sperm movement. An ultrastructural examination makes it possible to look inside the spermatozoon and to study what is inaccessible by light microscopy, including the extent of chromatin condensation and the structures of the perinuclear theca (PT), its postacrosomal segment, the centriole, the axoneme, and periaxonemal elements of the tail. Every function of the spermatozoon is now possible to attribute to a particular morphological structure owing to the achievements of modern molecular biology, cytology, and genetics. The morphology of spermatozoa reflects how competent they are to fertilize (enter) the

The nucleus occupies a major part of the sperm head and contains condensed chromatin (**Figure 1a**), which is detected as an electron-dense homogeneous material, with small regions of lower electron density on ultrathin sections. Condensed chromatin is at least 10 times denser

**Figure 1.** (a) Spermatozoon with condensed chromatin (CH) and normal acrosome (A). (b) Postacrosomal segment of perinuclear theca (PS) with characteristic intermittent striation. (c, d) Spermatozoon with immature chromatin (IC). (e) Fragment of sperm acrosome. NE, nuclear envelope; PT, perinuclear theca; IM, inner acrosome membrane; EM, extra

oocyte and to provide for embryo development.

**2.1. Chromatin structure in normal spermatozoa**

**2. Sperm head**

72 Spermatozoa - Facts and Perspectives

than ones in somatic cells [3].

acrosome membrane; and PM, plasma membrane.

During sperm maturation, canonical histones are replaced by testis-specific histones and then by protamines, basic proteins with lower molecular weight and high concentration of arginine and cysteine (for a review, see [5, 6]).

As spermatozoa progress through the epididymis, disulfide bridges form between cysteine residues of protamines to further stabilize the DNA-protamine complex and morphologically determine condensation of the dense nucleoprotamine complex in the sperm nucleus [7]. Sperm chromatin is decondensed and acquires a nucleosomal structure after fertilization. The organization of sperm chromatin facilitates the transfer of compacted DNA into the oocyte and ensures its reverse transformation so that genetic information becomes readily available in the developing embryo [8].

Approximately 5–10% of genomic DNA remains free of protamines and preserves a nucleosomal structure in mature human spermatozoa (for a review, see [9]). The role of the residual nucleosomes remained unclear until recently and was explained in three studies, which were published simultaneously in 2010 [10–12]. Residual nucleosomes were found to mark the genes for early embryo development factors and to perform an important function in the epigenetic regulation of embryo development. A gene distribution between protamineassociated and histone-containing (nucleosomal) regions of chromatin follows a certain pattern. Residual nucleosomes occur in the promoters of early developmental genes (e.g., *HOX* gene clusters), imprinted gene loci, and miRNA genes.

Condensation associated with histone-to-protamine replacement metabolically inactivates chromatin and, on the other hand, contributes to its mechanical and chemical stability, thus protecting the paternal genome from nucleases while spermatozoa travel through the male and female reproductive tracts and interact with the oocyte. Residual nucleosomes mark early developmental genes. Normal chromatin condensation is indicative of the sperm potential to produce a normally developing embryo.

#### **2.2. Abnormal chromatin condensation in spermatozoa**

Spermatozoa with incomplete chromatin condensation in the nucleus are almost always detectable in ejaculate samples from fertile donors. Granular and fibrillary structures of approximately 40 nm in diameter are seen in these cells. The chromatin structure observed in the spermatozoa is similar to that of elongated spermatids, and their chromatin is consequently known as immature chromatin (**Figure 1c**, **d**) [13].

What is a possible role of distorted chromatin compaction? The disturbance of chromatin condensation is a consequence of a reduced protamine content [14]. Hammoud et al. [15] have recently found that defects in histone-to-protamine exchange lead to a random distribution of nucleosomal (histone-associated and potentially active) chromatin in infertile patients, in contrast to a programmed nucleosomal chromatin distribution in fertile men. Distorted chromatin compaction in spermatozoa seems to lead to substantial post-fertilization defects. Abnormal (insufficient) chromatin condensation was shown to delay the first cell division cycle and to subsequently cause damage to the embryo [16]. Such defects can be responsible for ART failures [17] and early pregnancy losses [18].

Higher percentage of spermatozoa with immature chromatin was observed in semen of the patients with arrest of embryonic development compared with fertile men, and the difference was statistically significant. Semen samples with increased percentage of spermatozoa with immature chromatin in the men with embryo development arrest in reproductive history were 2.2 times more frequent than in the control group (44 vs. 20%) [19].

The question arises as to whether defects in chromatin condensation are associated with DNA fragmentation in spermatozoa. An early hypothesis suggested that defects in histone-to-protamine exchange and, therefore, in chromatin condensation inevitably lead to higher sperm DNA fragmentation [20].

A higher count of spermatozoa with immature, insufficiently condensed chromatin in semen provides an independent diagnostic sign and shows no association with a higher count of spermatozoa with DNA fragmentation [19, 21]. Clinically, fertility disorders are associated with both higher percentage of spermatozoa with immature chromatin and higher percentage of spermatozoa with DNA fragmentation in the ejaculate, but the disorders differ in nature between the two cases. Diagnosing the nature of damage to sperm nuclear material makes it possible to choose a treatment adequate to the observed defect [22].

#### **2.3. Vacuoles in the sperm nucleus**

Hollows, which were initially described as vacuoles varying in size and location, can be detected in chromatin of sperm nuclei [23]. Vacuoles are actually indentations in the nucleus, as is seen on ultrathin sections. Chemes and Alvarez Sedò [24] have proposed the term lacunae or lacunar defects for nuclear vacuoles. Lacunae vary in location and texture. **Figure 2a**, **b** shows a lacuna surrounded by a membrane with membrane whorls (MWs), which consist of double membranes with septal complexes [25]; the lacuna is interconnected with nuclear pockets at the base of the head (**Figure 1a**).

On the other hand, no correlation of the presence of large vacuoles in spermatozoa has been observed for spermiogram parameters, DNA damage, and live birth rate [29]. IMSI does not

**Figure 2.** Vacuoles in the sperm nucleus. The lacuna surrounded by membrane with membrane whorls (MWs) (a, b). The lacuna is interconnected with nuclear pockets at the base of the head (a, arrow). Invaginations of the nucleus (I) not surrounded by membrane (d, e), and two large lacunae (L) without visible contact with the subacrosomal space (c). A,

Ultrastructure of Spermatozoa from Infertility Patients http://dx.doi.org/10.5772/intechopen.71596 75

Haraguchi et al. [31] have used immunochemistry with electron microscopy and detected proteasomes in nuclear vacuoles and clear spots of condensed chromatin. Nuclear vacuoles and nuclear pockets at the base of the nucleus were assumed to function as proteolytic centers to resorb the molecules (somatic and sperm-specific histones and transit proteins) that are released during chromatin reprogramming. A positive correlation between the presence of vacuoles and the acrosomal reaction [32], vacuoles and capacitation [33] similarly indicates that vacuoles are related to physiological properties of spermatozoa and has no effect on their

The acrosome is a secretory vesicle derived from the Golgi apparatus. The acrosome forms a cap on the anterior pole of the nucleus and consists of an outer membrane, inner membrane, and matrix. The outer acrosomal membrane is adjacent to the plasma membrane covering the head of the spermatozoon. A layer sandwiched between the inner acrosomal membrane and the nuclear envelope is known as the perinuclear theca (PT), which has a medium electron

improve the outcome of ART after two successive IVF-ICSI failures [30].

acrosome and PS, postacrosomal segment of perinuclear theca.

fertilizing potential.

**2.4. Acrosome and perinuclear theca**

density and is approximately 200 nm thick (**Figure 1e**).

Invaginations of another type can also be detected in sperm nuclei. The invaginations may occur in both basal and apical parts of the nucleus, are not surrounded by a membrane, and contain granular material (**Figure 2d**, **e**). A connection (contact) between a lacuna and the subacrosomal space cannot always be seen in ultrathin sections, and the lacuna consequently appears to be a vacuole in nuclear chromatin. DNA is absent from lacunae (**Figure 2c**) [26].

Moving sperm organelle morphology examination (MSOME) using high-resolution microscopy at magnifications exceeding 5000× makes it possible to select in vivo the vacuole-free spermatozoa and to perform intracytoplasmic morphologically selected sperm injection (IMSI) [27].

There are data that IMSI of spermatozoa without vacuoles or with one small vacuole substantially increases the yield of blastocysts as compared with spermatozoa containing large vacuoles or spermatozoa with more than two small vacuoles [28].

**Figure 2.** Vacuoles in the sperm nucleus. The lacuna surrounded by membrane with membrane whorls (MWs) (a, b). The lacuna is interconnected with nuclear pockets at the base of the head (a, arrow). Invaginations of the nucleus (I) not surrounded by membrane (d, e), and two large lacunae (L) without visible contact with the subacrosomal space (c). A, acrosome and PS, postacrosomal segment of perinuclear theca.

On the other hand, no correlation of the presence of large vacuoles in spermatozoa has been observed for spermiogram parameters, DNA damage, and live birth rate [29]. IMSI does not improve the outcome of ART after two successive IVF-ICSI failures [30].

Haraguchi et al. [31] have used immunochemistry with electron microscopy and detected proteasomes in nuclear vacuoles and clear spots of condensed chromatin. Nuclear vacuoles and nuclear pockets at the base of the nucleus were assumed to function as proteolytic centers to resorb the molecules (somatic and sperm-specific histones and transit proteins) that are released during chromatin reprogramming. A positive correlation between the presence of vacuoles and the acrosomal reaction [32], vacuoles and capacitation [33] similarly indicates that vacuoles are related to physiological properties of spermatozoa and has no effect on their fertilizing potential.

#### **2.4. Acrosome and perinuclear theca**

chromatin compaction in spermatozoa seems to lead to substantial post-fertilization defects. Abnormal (insufficient) chromatin condensation was shown to delay the first cell division cycle and to subsequently cause damage to the embryo [16]. Such defects can be responsible

Higher percentage of spermatozoa with immature chromatin was observed in semen of the patients with arrest of embryonic development compared with fertile men, and the difference was statistically significant. Semen samples with increased percentage of spermatozoa with immature chromatin in the men with embryo development arrest in reproductive history

The question arises as to whether defects in chromatin condensation are associated with DNA fragmentation in spermatozoa. An early hypothesis suggested that defects in histone-to-protamine exchange and, therefore, in chromatin condensation inevitably lead to higher sperm

A higher count of spermatozoa with immature, insufficiently condensed chromatin in semen provides an independent diagnostic sign and shows no association with a higher count of spermatozoa with DNA fragmentation [19, 21]. Clinically, fertility disorders are associated with both higher percentage of spermatozoa with immature chromatin and higher percentage of spermatozoa with DNA fragmentation in the ejaculate, but the disorders differ in nature between the two cases. Diagnosing the nature of damage to sperm nuclear material makes it

Hollows, which were initially described as vacuoles varying in size and location, can be detected in chromatin of sperm nuclei [23]. Vacuoles are actually indentations in the nucleus, as is seen on ultrathin sections. Chemes and Alvarez Sedò [24] have proposed the term lacunae or lacunar defects for nuclear vacuoles. Lacunae vary in location and texture. **Figure 2a**, **b** shows a lacuna surrounded by a membrane with membrane whorls (MWs), which consist of double membranes with septal complexes [25]; the lacuna is interconnected with nuclear

Invaginations of another type can also be detected in sperm nuclei. The invaginations may occur in both basal and apical parts of the nucleus, are not surrounded by a membrane, and contain granular material (**Figure 2d**, **e**). A connection (contact) between a lacuna and the subacrosomal space cannot always be seen in ultrathin sections, and the lacuna consequently appears to be a vacuole in nuclear chromatin. DNA is absent from lacunae (**Figure 2c**) [26].

Moving sperm organelle morphology examination (MSOME) using high-resolution microscopy at magnifications exceeding 5000× makes it possible to select in vivo the vacuole-free spermatozoa and to perform intracytoplasmic morphologically selected sperm injection (IMSI) [27].

There are data that IMSI of spermatozoa without vacuoles or with one small vacuole substantially increases the yield of blastocysts as compared with spermatozoa containing large

for ART failures [17] and early pregnancy losses [18].

DNA fragmentation [20].

74 Spermatozoa - Facts and Perspectives

**2.3. Vacuoles in the sperm nucleus**

pockets at the base of the head (**Figure 1a**).

were 2.2 times more frequent than in the control group (44 vs. 20%) [19].

possible to choose a treatment adequate to the observed defect [22].

vacuoles or spermatozoa with more than two small vacuoles [28].

The acrosome is a secretory vesicle derived from the Golgi apparatus. The acrosome forms a cap on the anterior pole of the nucleus and consists of an outer membrane, inner membrane, and matrix. The outer acrosomal membrane is adjacent to the plasma membrane covering the head of the spermatozoon. A layer sandwiched between the inner acrosomal membrane and the nuclear envelope is known as the perinuclear theca (PT), which has a medium electron density and is approximately 200 nm thick (**Figure 1e**).

The acrosome covers the anterior two-thirds of the sperm head. Relative to the acrosome, the head can be divided into three regions: acrosomal, equatorial, and postacrosomal. Only the PT with its characteristic intermittent striation occurs between the nucleus and the plasma membrane in the postacrosomal region of the spermatozoon (**Figure 1b**).

and enter the oocyte upon fertilization. In contrast to the acrosome, which rapidly responds to exogenous factors, the PT is resistant to extraction with denaturing agents and high-salt buffers. The putative oocyte-activating factor MN13 was found in the PT [40]. MN13 is located in

Ultrastructure of Spermatozoa from Infertility Patients http://dx.doi.org/10.5772/intechopen.71596 77

Phospholipase C zeta (PLCζ) is another protein found in the postacrosomal segment of the PT

Thus, PLCζ and probably other proteins of the postacrosomal sheath of the PT act as oocyteactivating factors. The postacrosomal sheath is the first to contact the oocyte, and its dissolution (disassembly) is sufficient for triggering early events of oocyte activation. The oocyte-activating factors are transmitted from the sperm PT into the oocyte cytoplasm after the incorporation and rapid dissolution of the PT. In the normal fertilization cycle, the PT dissolves in the oocyte cytoplasm simultaneously with decondensation of the sperm nucleus and initiates division of the maternal pronucleus by hydrolyzing a membrane-bound phospholipid substrate, triggering cytoplasmic Ca2+ oscillations [42]. In the case of ICSI, activation occurs only in the oocytes that contain a partly or completely dissolved PT. When the PT dissolves only partly, the residual PT postacrosomal sheath may persist at the apical side of the paternal pronucleus and may delay or arrest zygote development [43]. Dissolution of the subacrosomal part of the PT is essential for complete DNA decondensation in the paternal

Electron microscopic examination of the acrosome provides an experimentally grounded alternative to sperm penetration assays. The method reliably reports the integrity of the acrosome and the status of its enzymatic system and the postacrosomal segment, which is involved in sperm attachment to the oocyte. A higher percentage of spermatozoa with abnormal acrosomes in an ejaculate sample can be responsible for idiopathic infertility when the

Lack of an acrosome is identified as primary when resulting from spermiogenesis defects. Globozoospermia of a presumably genetic nature provides a classical example of the primary

Globozoospermia is an uncommon male fertility disorder. Round-headed cells may account for up to 6% of the total sperm count in the ejaculate in fertile men [44], while 100% of spermatozoa have round heads in total globozoospermia. The sperm count and motility are not affected in globozoospermia. An ultrastructural examination shows that acrosomes are completely absent from round heads or that a rudimentary acrosome occurs at the nuclear pole

Defects of chromatin condensation in the nucleus are additionally seen in the majority of ejaculate samples. Heterogeneity is also possible; i.e., spermatozoa with normal condensed chromatin and those with decondensed chromatin may be detected in one ejaculate sample. Both

periodic striations, which form the postacrosomal sheath of the PT.

and is thought to act as an oocyte-activating factor [41].

pronucleus and the start of DNA synthesis in both pronuclei.

spermiogram parameters are within the normal ranges.

**2.6. Acrosomal abnormalities**

*2.6.1. Primary lack of an acrosome*

opposite to the tail (**Figure 3a**).

lack of an acrosome.

Material contained in the lumen of the acrosome has a medium electron density and is known as the acrosomal matrix [34]. Zona pellucida (ZP)-binding proteins are found in the acrosomal matrix. Proacrosin is the most important of all ZP-binding proteins of the acrosome. Proacrosin was long believed to be a main lytic protein essential for sperm penetration through the ZP. However, proacrosin knockout mice were found to be fertile [35], although their spermatozoa penetrate through the ZP slower than spermatozoa of wild-type mice. Acrosin probably plays a role in maturation and packaging of other acrosomal matrix proteins. The acrosomal matrix contains several other ZP-binding proteins.

The acrosome of a capacitated spermatozoon interacts with ZP glycoprotein 1 (ZP1) of the oocyte to trigger fusion of the plasma and outer acrosomal membranes, the membrane ends fuse, and vesicles form. Then proteases are released from the acrosome and digest the ZP. The process is known as the acrosomal reaction, which consists in exocytosis and allows the spermatozoon to pass through the ZP. Acrosome-reacted spermatozoa subsequently bind with ZP2, another glycoprotein, which is responsible for sperm adhesion to the oocyte [36]. The inner acrosomal membrane remains intact.

The plasma membrane and the outer acrosomal membrane of the equatorial segment are not involved in forming vesicles during the acrosomal reaction. The equatorial segment is a region where fusion of the spermatozoon and oocyte plasma membrane is triggered. The sperm plasma membrane of the equatorial segment fuses with microvilli of the oolemma, the membranes fuse, and sperm components are thus delivered into the ooplasm. The equatorial segment protein (ESP) is found in the equatorial segment of the acrosome in human spermatozoa [37]. ESP is detectable throughout the acrosome biogenesis. It is thought that ESP plays a role in adhesion of the spermatozoon to the oocyte and their fusion at the oolemma level. Fujihara et al. [38] identified sperm equatorial segment protein 1 (SPESP1), which is specific to the equatorial segment. Spermatozoa of transgenic mice devoid of *SPESP1* (*Spesp1−/−)* fuse with eggs at a far lower rate. SPESP1 seems to be responsible for maintaining the integrity of the equatorial segment after the acrosomal reaction. Membranes of the equatorial segment are disrupted after the acrosomal reaction in Spesp1−/− mice, whereas the equatorial segment is preserved in wild-type mice.

An important role is ascribed to Izumo. The Izumo family includes four proteins, Izumo1-4. Izumo1 is a membrane immunoglobulin protein with an extracellular immunoglobulin domain of 145 residues and an N-terminal domain. The sperm protein Izumo1 on the equatorial segment of the acrosome-reacted spermatozoon recognizes its receptor, JUNO, on the oocyte surface. Human Izumo1 forms a high-affinity complex with the Juno receptor of the oocyte and changes its conformation [39].

#### **2.5. Perinuclear theca**

The PT is a cytoskeletal structure that harbors a specific oocyte-activating factor (for a review, see [40]). The PT and its postacrosomal segment remain associated with the sperm nucleus and enter the oocyte upon fertilization. In contrast to the acrosome, which rapidly responds to exogenous factors, the PT is resistant to extraction with denaturing agents and high-salt buffers.

The putative oocyte-activating factor MN13 was found in the PT [40]. MN13 is located in periodic striations, which form the postacrosomal sheath of the PT.

Phospholipase C zeta (PLCζ) is another protein found in the postacrosomal segment of the PT and is thought to act as an oocyte-activating factor [41].

Thus, PLCζ and probably other proteins of the postacrosomal sheath of the PT act as oocyteactivating factors. The postacrosomal sheath is the first to contact the oocyte, and its dissolution (disassembly) is sufficient for triggering early events of oocyte activation. The oocyte-activating factors are transmitted from the sperm PT into the oocyte cytoplasm after the incorporation and rapid dissolution of the PT. In the normal fertilization cycle, the PT dissolves in the oocyte cytoplasm simultaneously with decondensation of the sperm nucleus and initiates division of the maternal pronucleus by hydrolyzing a membrane-bound phospholipid substrate, triggering cytoplasmic Ca2+ oscillations [42]. In the case of ICSI, activation occurs only in the oocytes that contain a partly or completely dissolved PT. When the PT dissolves only partly, the residual PT postacrosomal sheath may persist at the apical side of the paternal pronucleus and may delay or arrest zygote development [43]. Dissolution of the subacrosomal part of the PT is essential for complete DNA decondensation in the paternal pronucleus and the start of DNA synthesis in both pronuclei.

#### **2.6. Acrosomal abnormalities**

The acrosome covers the anterior two-thirds of the sperm head. Relative to the acrosome, the head can be divided into three regions: acrosomal, equatorial, and postacrosomal. Only the PT with its characteristic intermittent striation occurs between the nucleus and the plasma

Material contained in the lumen of the acrosome has a medium electron density and is known as the acrosomal matrix [34]. Zona pellucida (ZP)-binding proteins are found in the acrosomal matrix. Proacrosin is the most important of all ZP-binding proteins of the acrosome. Proacrosin was long believed to be a main lytic protein essential for sperm penetration through the ZP. However, proacrosin knockout mice were found to be fertile [35], although their spermatozoa penetrate through the ZP slower than spermatozoa of wild-type mice. Acrosin probably plays a role in maturation and packaging of other acrosomal matrix pro-

The acrosome of a capacitated spermatozoon interacts with ZP glycoprotein 1 (ZP1) of the oocyte to trigger fusion of the plasma and outer acrosomal membranes, the membrane ends fuse, and vesicles form. Then proteases are released from the acrosome and digest the ZP. The process is known as the acrosomal reaction, which consists in exocytosis and allows the spermatozoon to pass through the ZP. Acrosome-reacted spermatozoa subsequently bind with ZP2, another glycoprotein, which is responsible for sperm adhesion to the oocyte [36]. The

The plasma membrane and the outer acrosomal membrane of the equatorial segment are not involved in forming vesicles during the acrosomal reaction. The equatorial segment is a region where fusion of the spermatozoon and oocyte plasma membrane is triggered. The sperm plasma membrane of the equatorial segment fuses with microvilli of the oolemma, the membranes fuse, and sperm components are thus delivered into the ooplasm. The equatorial segment protein (ESP) is found in the equatorial segment of the acrosome in human spermatozoa [37]. ESP is detectable throughout the acrosome biogenesis. It is thought that ESP plays a role in adhesion of the spermatozoon to the oocyte and their fusion at the oolemma level. Fujihara et al. [38] identified sperm equatorial segment protein 1 (SPESP1), which is specific to the equatorial segment. Spermatozoa of transgenic mice devoid of *SPESP1* (*Spesp1−/−)* fuse with eggs at a far lower rate. SPESP1 seems to be responsible for maintaining the integrity of the equatorial segment after the acrosomal reaction. Membranes of the equatorial segment are disrupted after the acrosomal reaction in Spesp1−/− mice, whereas the equatorial segment is preserved in wild-type mice.

An important role is ascribed to Izumo. The Izumo family includes four proteins, Izumo1-4. Izumo1 is a membrane immunoglobulin protein with an extracellular immunoglobulin domain of 145 residues and an N-terminal domain. The sperm protein Izumo1 on the equatorial segment of the acrosome-reacted spermatozoon recognizes its receptor, JUNO, on the oocyte surface. Human Izumo1 forms a high-affinity complex with the Juno receptor of the oocyte and

The PT is a cytoskeletal structure that harbors a specific oocyte-activating factor (for a review, see [40]). The PT and its postacrosomal segment remain associated with the sperm nucleus

membrane in the postacrosomal region of the spermatozoon (**Figure 1b**).

teins. The acrosomal matrix contains several other ZP-binding proteins.

inner acrosomal membrane remains intact.

76 Spermatozoa - Facts and Perspectives

changes its conformation [39].

**2.5. Perinuclear theca**

Electron microscopic examination of the acrosome provides an experimentally grounded alternative to sperm penetration assays. The method reliably reports the integrity of the acrosome and the status of its enzymatic system and the postacrosomal segment, which is involved in sperm attachment to the oocyte. A higher percentage of spermatozoa with abnormal acrosomes in an ejaculate sample can be responsible for idiopathic infertility when the spermiogram parameters are within the normal ranges.

#### *2.6.1. Primary lack of an acrosome*

Lack of an acrosome is identified as primary when resulting from spermiogenesis defects. Globozoospermia of a presumably genetic nature provides a classical example of the primary lack of an acrosome.

Globozoospermia is an uncommon male fertility disorder. Round-headed cells may account for up to 6% of the total sperm count in the ejaculate in fertile men [44], while 100% of spermatozoa have round heads in total globozoospermia. The sperm count and motility are not affected in globozoospermia. An ultrastructural examination shows that acrosomes are completely absent from round heads or that a rudimentary acrosome occurs at the nuclear pole opposite to the tail (**Figure 3a**).

Defects of chromatin condensation in the nucleus are additionally seen in the majority of ejaculate samples. Heterogeneity is also possible; i.e., spermatozoa with normal condensed chromatin and those with decondensed chromatin may be detected in one ejaculate sample. Both

oocyte-activating activity, ICSI is insufficient in globozoospermia. The development of oocyte

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Secondary lack of an acrosome results from a premature acrosomal reaction, i.e., the acrosome is lost in acrosome-reacted spermatozoa (**Figure 3b**). Disruption of the plasma membrane is observed in this case, and the inner acrosomal membrane adjacent to the nuclear envelope is seen on the sperm surface in the acrosomal region. The outer acrosomal membrane and the plasma membrane form bubbles during the acrosomal reaction. In the case of a physiological acrosomal reaction, the postacrosomal segment and its plasma membrane are preserved in

The percentage of spermatozoa with a secondary loss of the acrosome (i.e., acrosome-reacted spermatozoa) in ejaculate samples are 18.22 ± 8.27% in fertile men and 26.37 ± 12.81% in infertile patients with normal spermiogram parameters (p < 0.05) [52]. A higher percentage of acrosomereacted spermatozoa (with acrosome degradation) in the ejaculate may impair its fertilization potential. Leukocytospermia with an enhanced production of reactive oxygen species by leukocytes is one of the possible causes of an early acrosomal reaction. Our findings indicate that bacterial microcolonies present in the ejaculate may also cause a premature acrosomal reaction, and their presence is not always accompanied by an inflammatory response. We analyzed the results of electron microscopic examinations of 746 semen samples from patients with fertility disorders. Bacterial microcolonies were detected in 186 of the 746 samples (25%), and a higher (more than 20%) content of spermatozoa with a secondary loss of the acrosome was observed in 112 of the 186 samples (60%). In the absence of bacterial infection, a higher content of acro-

A higher leukocyte count in the ejaculate was detected in 36 of the 186 samples with bacterial

Electron microscopy is a gold-standard test for acrosomal reaction, although a number of

Irregular acrosome (**Figure 3c**) and lack of acrosomal contents (**Figure 3d**) (enzymatic insufficiency of the acrosome) are found in both pronounced teratozoospermia and normospermia. Proteolytic enzymes of the acrosome dissolve the zona pellucida to allow fusion of the spermatozoon and the oolemma. When the process is disturbed as a result of acrosome loss or dysfunction, spermatozoa lose their fertilizing potential. Irregularly T-shaped acrosomes

Spermatozoa that have an enlarged perinuclear space and lack the postacrosomal sheath of the PT account for 2–5% of the total sperm count in semen from fertile men (**Figure 4b**–**d**). The abnormality is often combined with the presence of excess residual cytoplasm on the

activation methods made it possible to achieve a better success rate [54].

some-reacted spermatozoa was found in 117 of the 560 samples (20%) [55].

other tests are now available to assess the penetrating potential of spermatozoa.

*2.6.4. Enlarged subacrosomal space and lack of a PT and postacrosomal segment*

can be detected in binuclear spermatozoa (**Figure 4a**).

*2.6.2. Secondary lack of an acrosome*

the live spermatozoon.

microcolonies (19%).

*2.6.3. Irregular acrosome*

**Figure 3.** (a) Round acrosomeless sperm heads from globozoospermia. Some nuclei are with condensed chromatin (CH) and one nucleus with immature chromatin (IC). (b) Secondary lack of acrosome. The intact internal acrosomal membrane (IM) and postacrosomal segment (PS) are visible. The outer acrosomal membrane and the plasma membrane form bubbles (B). (c) Acrosome with irregular contours (RA); (d) "empty" acrosome (EA).

within- and between-sample heterogeneity are observed. Higher contents of spermatozoa with immature chromatin [45] were observed in globozoospermia in the majority of studies.

Kullander and Rausing [46] were the first to assume a genetic nature for globozoospermia. Cases with a family history of the disorder supported the assumption. Mutations or deletions of three genes—*SPATA16*, *PICK1*, and *DPY19L2*—were detected in globozoospermia in molecular genetic studies. A homozygous mutation of *SPATA16* (3q26.32) was found in three brothers with globozoospermia [47]. A mutation of *PICK1* (22q12.3-q13.2) was identified in a globozoospermia patient [48]. The protein products of the two genes occur in the Golgi apparatus and are involved in vesicular trafficking, which is necessary for acrosome biogenesis in spermatids during spermiogenesis [49]. A deletion of *DPY19L2* was observed in the majority of total globozoospermia cases; Dpy19l3 protein is essential for a nuclear flattening and the formation of the acrosome [50].

The identification of the missense mutation L967Q of the gene *VPS54* [51], gene *GM130* inactivation [52], and some others lead to phenotypic globozoospermia in mouse model. These factors are related to the function of the Golgi apparatus vesicles, and these mutations are not identified in men.

The postacrosomal sheath of the PT is absent in patients with globozoospermia. PLCζ is found in extremely small, if any, amounts in spermatozoa of mice and human patients with a *Dpy19l2* mutation and the globozoospermia phenotype [53]. Because these proteins possess oocyte-activating activity, ICSI is insufficient in globozoospermia. The development of oocyte activation methods made it possible to achieve a better success rate [54].

#### *2.6.2. Secondary lack of an acrosome*

Secondary lack of an acrosome results from a premature acrosomal reaction, i.e., the acrosome is lost in acrosome-reacted spermatozoa (**Figure 3b**). Disruption of the plasma membrane is observed in this case, and the inner acrosomal membrane adjacent to the nuclear envelope is seen on the sperm surface in the acrosomal region. The outer acrosomal membrane and the plasma membrane form bubbles during the acrosomal reaction. In the case of a physiological acrosomal reaction, the postacrosomal segment and its plasma membrane are preserved in the live spermatozoon.

The percentage of spermatozoa with a secondary loss of the acrosome (i.e., acrosome-reacted spermatozoa) in ejaculate samples are 18.22 ± 8.27% in fertile men and 26.37 ± 12.81% in infertile patients with normal spermiogram parameters (p < 0.05) [52]. A higher percentage of acrosomereacted spermatozoa (with acrosome degradation) in the ejaculate may impair its fertilization potential. Leukocytospermia with an enhanced production of reactive oxygen species by leukocytes is one of the possible causes of an early acrosomal reaction. Our findings indicate that bacterial microcolonies present in the ejaculate may also cause a premature acrosomal reaction, and their presence is not always accompanied by an inflammatory response. We analyzed the results of electron microscopic examinations of 746 semen samples from patients with fertility disorders. Bacterial microcolonies were detected in 186 of the 746 samples (25%), and a higher (more than 20%) content of spermatozoa with a secondary loss of the acrosome was observed in 112 of the 186 samples (60%). In the absence of bacterial infection, a higher content of acrosome-reacted spermatozoa was found in 117 of the 560 samples (20%) [55].

A higher leukocyte count in the ejaculate was detected in 36 of the 186 samples with bacterial microcolonies (19%).

Electron microscopy is a gold-standard test for acrosomal reaction, although a number of other tests are now available to assess the penetrating potential of spermatozoa.

#### *2.6.3. Irregular acrosome*

within- and between-sample heterogeneity are observed. Higher contents of spermatozoa with immature chromatin [45] were observed in globozoospermia in the majority of studies. Kullander and Rausing [46] were the first to assume a genetic nature for globozoospermia. Cases with a family history of the disorder supported the assumption. Mutations or deletions of three genes—*SPATA16*, *PICK1*, and *DPY19L2*—were detected in globozoospermia in molecular genetic studies. A homozygous mutation of *SPATA16* (3q26.32) was found in three brothers with globozoospermia [47]. A mutation of *PICK1* (22q12.3-q13.2) was identified in a globozoospermia patient [48]. The protein products of the two genes occur in the Golgi apparatus and are involved in vesicular trafficking, which is necessary for acrosome biogenesis in spermatids during spermiogenesis [49]. A deletion of *DPY19L2* was observed in the majority of total globozoospermia cases; Dpy19l3 protein is essential for a nuclear flattening and the formation of the acrosome [50]. The identification of the missense mutation L967Q of the gene *VPS54* [51], gene *GM130* inactivation [52], and some others lead to phenotypic globozoospermia in mouse model. These factors are related to the function of the Golgi apparatus vesicles, and these mutations are not

form bubbles (B). (c) Acrosome with irregular contours (RA); (d) "empty" acrosome (EA).

**Figure 3.** (a) Round acrosomeless sperm heads from globozoospermia. Some nuclei are with condensed chromatin (CH) and one nucleus with immature chromatin (IC). (b) Secondary lack of acrosome. The intact internal acrosomal membrane (IM) and postacrosomal segment (PS) are visible. The outer acrosomal membrane and the plasma membrane

The postacrosomal sheath of the PT is absent in patients with globozoospermia. PLCζ is found in extremely small, if any, amounts in spermatozoa of mice and human patients with a *Dpy19l2* mutation and the globozoospermia phenotype [53]. Because these proteins possess

identified in men.

78 Spermatozoa - Facts and Perspectives

Irregular acrosome (**Figure 3c**) and lack of acrosomal contents (**Figure 3d**) (enzymatic insufficiency of the acrosome) are found in both pronounced teratozoospermia and normospermia. Proteolytic enzymes of the acrosome dissolve the zona pellucida to allow fusion of the spermatozoon and the oolemma. When the process is disturbed as a result of acrosome loss or dysfunction, spermatozoa lose their fertilizing potential. Irregularly T-shaped acrosomes can be detected in binuclear spermatozoa (**Figure 4a**).

#### *2.6.4. Enlarged subacrosomal space and lack of a PT and postacrosomal segment*

Spermatozoa that have an enlarged perinuclear space and lack the postacrosomal sheath of the PT account for 2–5% of the total sperm count in semen from fertile men (**Figure 4b**–**d**). The abnormality is often combined with the presence of excess residual cytoplasm on the

fertility disorders. Acrosomal hypoplasia is a common component of pronounced teratozoospermia, is well detectable by electron microscopy, and is essential to diagnose because acro-

Ultrastructure of Spermatozoa from Infertility Patients http://dx.doi.org/10.5772/intechopen.71596 81

The connecting piece connects the head with the tail (**Figure 5a**). A thin basal plate occurs at the base of the head, it has a concave shape, forming an implantation fossa. The region beneath the basal plate harbors nine striated columns, which continue caudally as outer dense fibers. Striated columns are the part of the connecting structures of the neck. The basal plate is at the base of the head nucleus. A centriole is enclosed in an electron-dense capitulum. The centriole is a universal element of animal eukaryotic cells and plays a role in the formation of the mitotic spindle.

A typical centrosome (cell center) of immature germline cells consists of two cylindrical centrioles, each consists of nine symmetrically oriented microtubule triplets, of 0.5 μm in length and 0.2 μm in diameter. Two centrioles are positioned in an orthogonal orientation, the axis of the daughter centriole being perpendicular to that of the mother centriole. A typical centriole has a 9 + 0 organization of microtubule triplets. In a mature spermatozoon, the distal centriole gives

**Figure 5.** (a) The connecting piece of normal spermatozoon. (b) Decapitated spermatozoon. B, basal plate; C, centriole; Ca, capitulum; SC, striated column, OF, outer dense fibers; and M, mitochondria. (c) Transverse section through the midpiece of spermatozoon tail; (d) transverse section through the principal piece of spermatozoon; (e) longitudinal section through the middle and principal piece of the tail; (f) the site of contact between mitochondria (arrow). Ax, axoneme, dynein arms of peripheral microtubule doublets are visible. M, mitochondria; FS, fibrous sheath; and An, annulus.

somal insufficiency is possible to correct using ICSI (for a review, see [58]).

**3. Connecting piece (neck) of the spermatozoon**

**Figure 4.** (a) Binuclear spermatozoa with T-shaped acrosome (TA). (b) Sperm with small cytoplasmic droplet on the head (CD) lacking the PT and its postacrosomal segment and with enlarged subacrosomal space (SS). A, irregular acrosome. (c, d) Spermatozoa with excess residual cytoplasm on the head (RH) and on the neck (RN), irregular acrosomes (A), and enlarged subacrosomal space (SS).

head (**Figure 4c**, **d**). The disorder is sometimes referred to as type II globozoospermia. Sperm heads appear to be spherical under a light microscope, but an ultrastructural study shows that spermatozoa have normal elongate nuclei, whereas their heads look round because of excess residual cytoplasm. This form of pathology also impairs fertility, but differs from globozoospermia [56] because lack of the PT and its postacrosomal segment suggests lack of the oocyte-activating factor.

In some cases, a small cytoplasmic droplet on the head is found in spermatozoa lacking the PT and its postacrosomal segment, so that the spermatozoa appear to be normal by light microscopy (**Figure 4b**). The pathology is detectable only by electron microscopy and may cause idiopathic infertility while the conventional spermiogram parameters are within the normal ranges. ICSI with the oocyte activation methods developed for patients with globozoospermia could solve the problem for these patients. A promising method was tested in a mouse model; i.e., recombinant PLCζ was injected to allow fertilization with spermatozoa of *PLCζ-/-I489F* mutant mice [57].

The acrosome is the most labile component of the spermatozoon. According to our data, the percentage of spermatozoa with abnormal acrosome shapes is 50.12 ± 8.70% in fertile men. Alterations of the acrosome shape or lack of the acrosomal contents are greater in men with fertility disorders. Acrosomal hypoplasia is a common component of pronounced teratozoospermia, is well detectable by electron microscopy, and is essential to diagnose because acrosomal insufficiency is possible to correct using ICSI (for a review, see [58]).
