*4.2.1. Mid-piece of the tail*

The outer dense fibers are a morphological extension of the striated columns and capitulum, which are structural elements of the connecting piece of the sperm neck [74]. The outer dense fibers surround the axoneme in the mid-piece of the tail, one fiber overlaying one peripheral microtubule doublet. ODF1 is a major protein of the outer dense fibers (**Figure 5c**, **e**).

The number of mitochondria is reduced as a large portion of the cytoplasm is eliminated with residual bodies from spermatids in the course of spermiogenesis [75]. Up to 75 mitochondria are left in a mature spermatozoon with a minor cytoplasm amount and form a helix around the outer dense fibers and axoneme. The mitochondrial helix has 11–13 turns with two mitochondria per turn. The mitochondrial helix length and the approximate number of turns are constant within a species [76].

The principal piece of the tail harbors glycolytic enzymes, including sperm-specific hexokinase 1, lactate dehydrogenase, and sperm-specific glyceraldehyde 3-phosphate dehydroge-

Ultrastructure of Spermatozoa from Infertility Patients http://dx.doi.org/10.5772/intechopen.71596 85

The complex system of tail elements with their concerted function provides the spermatozoon with the ability to move, that is, to reach and fertilize the oocyte. Any structural alteration of

A functional variant of asthenozoospermia is the most common. Spermatozoa of patients display multiple heterogeneous ultrastructural changes in the axoneme and periaxonemal structures (**Figure 6a**–**e**), such as changes in the number and arrangement of the microtubule doublets, the shape of the outer dense fibers, or the architecture of the fibrous sheath. Quantitative changes in mitochondria and their altered localization were also associated with asthenozoospermia. The percentage of spermatozoa with ultrastructural tail defects is significantly higher in patients with asthenozoospermia. Ultrastructural defects of the tail axoneme were described in drug addicts [85]. Yet smoking and alcohol drinking were not found to affect the ultrastructural parameters of mature spermatozoa, lower sperm counts observed in alcoholics and smokers suggest testicular selection [86]. Functional asthenozoospermia can

**Figure 6.** Longitudinal (a, b) and transverse (c–e) sections through abnormal sperm tails. (a) Lack of annulus (arrow) between the middle and principal piece of the tail; (b)swollen mitochondria (SM) and dislocation of mitochondria (arrow); (c) normal axonema structure and increased quantity of outer dense fibers (OF); (d) disorganization of axonemal microtubules (MT) and outer dense fibers (OF); (e) double tail with (9 + 1) microtubules. The absence of dynein arms in

nase (GAPDHs) [84].

**4.3. Structural abnormalities of the tail**

the system impairs sperm motility.

the right axoneme is revealed. FS, fibrous sheath.

The structure of the mitochondrial helix is stable owing to the so-called mitochondrial capsule, i.e., the outer mitochondrial membrane is coated with keratin-like molecules, which form disulfide bridges between cysteine- and proline-rich selenoprotein regions [77]. Contact zones form at the sites of contacts between mitochondria, indicating that the spermatozoon has a mitochondrial reticulum, similar to the mitochondrial network of the heart muscle rather than individual mitochondria (**Figure 5f**) [78, 79].

Active functional mitochondria were demonstrated to affect the sperm fertilizing potential in many studies. Ultrastructural defects in mitochondria are associated with lower sperm motility. The available data on the role of mtDNA mutations are discrepant. Deletions from mtDNA were considered to be responsible for sperm dysfunction and infertility [80]. However, the difference was not confirmed for several mtRNAs by rtPCR.

Metabolism of sperm mitochondria is still a matter of discussion. It is commonly accepted that ATP produced by mitochondria provides a main source of energy for the dynein motor of the axoneme. In contrast, a compartmentalization hypothesis suggests that glycolysis is a main source of energy for tail movements [81]. Because discrepant experimental data were reported from different studies, the question is still open. It is possible that mitochondria are involved in basic redox processes, which determine the fertilizing potential and lifespan of the spermatozoon, rather than in energy metabolism as a main function.

The mid-piece and principal piece of the tail are separated by a ring structure known as the annulus (**Figure 5e**), which presumably performs a barrier function to prevent molecular diffusion between the two pieces [82].

#### *4.2.2. Principal piece of the tail*

The principal piece of the tail is distal of the mid-piece and is the longest tail segment. The mitochondrial sheath is not found in the principal piece, and a fibrous sheath as another cytoskeletal element of the tail surrounds the axoneme. Two longitudinal columns of the fibrous sheath replace two opposite outer dense fibers and are connected together by numerous circumferential ribs (**Figure 5d**, **e**).

A total of 18 polypeptides were identified in the fibrous sheath. The polypeptides form a scaffold for glycolytic enzymes and act as signaling molecules upon induction of sperm motility (for a review, see [83]). A-kinase anchoring proteins 3 and 4 (AKAP3 and AKAP4) are major components of the fibrous sheath and probably form its integral cytoskeletal structure. AKAP3 and AKAP4 are associated with each other and bind to cAMP-dependent protein kinase A through its regulatory subunit. The AKAP3 and AKAP4 genes were sequenced, and the binding sites identified.

The principal piece of the tail harbors glycolytic enzymes, including sperm-specific hexokinase 1, lactate dehydrogenase, and sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDHs) [84].

#### **4.3. Structural abnormalities of the tail**

The number of mitochondria is reduced as a large portion of the cytoplasm is eliminated with residual bodies from spermatids in the course of spermiogenesis [75]. Up to 75 mitochondria are left in a mature spermatozoon with a minor cytoplasm amount and form a helix around the outer dense fibers and axoneme. The mitochondrial helix has 11–13 turns with two mitochondria per turn. The mitochondrial helix length and the approximate number of turns are

The structure of the mitochondrial helix is stable owing to the so-called mitochondrial capsule, i.e., the outer mitochondrial membrane is coated with keratin-like molecules, which form disulfide bridges between cysteine- and proline-rich selenoprotein regions [77]. Contact zones form at the sites of contacts between mitochondria, indicating that the spermatozoon has a mitochondrial reticulum, similar to the mitochondrial network of the heart muscle

Active functional mitochondria were demonstrated to affect the sperm fertilizing potential in many studies. Ultrastructural defects in mitochondria are associated with lower sperm motility. The available data on the role of mtDNA mutations are discrepant. Deletions from mtDNA were considered to be responsible for sperm dysfunction and infertility [80]. However, the

Metabolism of sperm mitochondria is still a matter of discussion. It is commonly accepted that ATP produced by mitochondria provides a main source of energy for the dynein motor of the axoneme. In contrast, a compartmentalization hypothesis suggests that glycolysis is a main source of energy for tail movements [81]. Because discrepant experimental data were reported from different studies, the question is still open. It is possible that mitochondria are involved in basic redox processes, which determine the fertilizing potential and lifespan of

The mid-piece and principal piece of the tail are separated by a ring structure known as the annulus (**Figure 5e**), which presumably performs a barrier function to prevent molecular dif-

The principal piece of the tail is distal of the mid-piece and is the longest tail segment. The mitochondrial sheath is not found in the principal piece, and a fibrous sheath as another cytoskeletal element of the tail surrounds the axoneme. Two longitudinal columns of the fibrous sheath replace two opposite outer dense fibers and are connected together by numerous cir-

A total of 18 polypeptides were identified in the fibrous sheath. The polypeptides form a scaffold for glycolytic enzymes and act as signaling molecules upon induction of sperm motility (for a review, see [83]). A-kinase anchoring proteins 3 and 4 (AKAP3 and AKAP4) are major components of the fibrous sheath and probably form its integral cytoskeletal structure. AKAP3 and AKAP4 are associated with each other and bind to cAMP-dependent protein kinase A through its regulatory subunit. The AKAP3 and AKAP4 genes were sequenced, and

constant within a species [76].

84 Spermatozoa - Facts and Perspectives

fusion between the two pieces [82].

*4.2.2. Principal piece of the tail*

cumferential ribs (**Figure 5d**, **e**).

the binding sites identified.

rather than individual mitochondria (**Figure 5f**) [78, 79].

difference was not confirmed for several mtRNAs by rtPCR.

the spermatozoon, rather than in energy metabolism as a main function.

The complex system of tail elements with their concerted function provides the spermatozoon with the ability to move, that is, to reach and fertilize the oocyte. Any structural alteration of the system impairs sperm motility.

A functional variant of asthenozoospermia is the most common. Spermatozoa of patients display multiple heterogeneous ultrastructural changes in the axoneme and periaxonemal structures (**Figure 6a**–**e**), such as changes in the number and arrangement of the microtubule doublets, the shape of the outer dense fibers, or the architecture of the fibrous sheath. Quantitative changes in mitochondria and their altered localization were also associated with asthenozoospermia. The percentage of spermatozoa with ultrastructural tail defects is significantly higher in patients with asthenozoospermia. Ultrastructural defects of the tail axoneme were described in drug addicts [85]. Yet smoking and alcohol drinking were not found to affect the ultrastructural parameters of mature spermatozoa, lower sperm counts observed in alcoholics and smokers suggest testicular selection [86]. Functional asthenozoospermia can

**Figure 6.** Longitudinal (a, b) and transverse (c–e) sections through abnormal sperm tails. (a) Lack of annulus (arrow) between the middle and principal piece of the tail; (b)swollen mitochondria (SM) and dislocation of mitochondria (arrow); (c) normal axonema structure and increased quantity of outer dense fibers (OF); (d) disorganization of axonemal microtubules (MT) and outer dense fibers (OF); (e) double tail with (9 + 1) microtubules. The absence of dynein arms in the right axoneme is revealed. FS, fibrous sheath.

be secondary to a varicocele, infections of reproductive organs, and exogenous exposures [58]. Spermiogram parameters are possible to correct with medications in men diagnosed with functional asthenozoospermia. When the treatment is ineffective, ICSI is likely to help.

within human *GAPDS* gene were assayed. In all five studied semen DFS samples, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of *sGAPD* [91]. Pereira et al. [92] found heterozygous deletion in the *DNAH5* gene,

Ultrastructure of Spermatozoa from Infertility Patients http://dx.doi.org/10.5772/intechopen.71596 87

DSF has an autosomal recessive inheritance. The genetic risk is now impossible to estimate. A few cases of live births after ICSI with spermatozoa of DSF patients were reported in the

PCD is an autosomal recessive disorder and is highly heterogeneous genetically. PCD affects the axonemal structures (microtubules and dynein arms) of cilia and flagella (**Figure 7d**–**f**). Bronchial and pulmonary diseases are the main pathology in PCD because infections and

Headaches are common in PCD patients because lack of ciliary motility in the brain ventricles impairs circulation of the cerebrospinal fluid. Situs inversus is additionally observed in half of the PCD patients, possibly resulting from lack of ciliary motility in embryonic Hensen's node, which is responsible for the unidirectional fluid flow and thereby establishes left-right

Fertility is impaired in male patients because their spermatozoa are absolutely immotile or defects occur in efferent seminiferous ducts lined by ciliated epithelia. In a semen analysis, gross ejaculate parameters (volume, pH, viscosity, and color) and the concentration and count

Transmission electron microscopy (TEM) is commonly used to detect PCD. TEM reports lack of outer and/or inner dynein arms, the two central microtubules, or radial spokes and changes

Molecular methods to diagnose PCD have intensely been developed in the past years. Unicellular algae of the genus Chlamydomonas, which have two flagella, provide a convenient model to study the molecular composition of the axoneme. Axoneme protein genes identified in Chlamydomonas are candidate genes for PCD. A total of 16 mutations of PCD candidate genes were identified from 1999 to 2011 by genetic methods (analysis of linkage groups identified by homozygosity mapping), proteome analysis, and sequencing (mostly Sanger sequencing). Since 2011, mutations of 18 other genes have been described via whole-

PCD is genetically heterogeneous. Mutations of two genes, *DNAI1* and *DNAH5*, are the most common in PCD. *DNAI1* and *DNAH5* code for proteins of outer dynein arms of the axoneme. *DNAI1* mutations were found in 14% of PCD patients [97]. The *DNAH5* product is a major motor protein of outer dynein arms. Its mutations were observed in more than 25% of PCD patients [98]. Many proteins are involved in building the axoneme. Several proteins are common for epithelial cilia and sperm tails. Patients homozygous for mutations of their genes develop the total set of PCD signs, including bronchial and pulmonary diseases, changes in asymmetry of visceral organs, and immotile sperm. Other axonemal proteins are tissue specific, and mutations

bronchiectasis develop when respiratory cilia have motility defects or are immotile.

asymmetry [94]. The prevalence of PCD at birth is 1/10,000 to 1/20,000 [95].

of morphologically normal spermatozoa are within the normal ranges.

exome and whole-genome sequencing (for the review see [73, 96]).

but not mutations in *AKAP3* and *AKAP4* in four patients with DFS.

medical literature [93].

*4.4.2. Primary ciliary dyskinesia (PCD)*

in microtubule arrangement.

#### **4.4. Genetically determined forms of asthenozoospermia**

#### *4.4.1. Dysplasia of the fibrous sheath of the tail*

Chemes et al. [87] proposed the term dysplasia of the fibrous sheath (DFS) for the disorder, which is also known as stump tail syndrome and short-tail spermatozoa. Spermatozoa have substantially reduced, if any, motility due to fibrous for DSF [88]. In spermatozoa, the location of longitudinal columns and transverse ribs of the fibrous layer has been disturbed. There are changes in the structure of the mitochondrial helix—a significant shortening and disruption of localization. Anomalies in the structure of the fibrous sheath often put together with the absence of a central pair of the axoneme microtubules (**Figure 7a**–**c**).

A mouse model of DFS was obtained by targeted disruption of the *Akap4* gene, and spermatozoa of mutant mice had short thick tails, which were morphologically identical to those in DFS patients [89]. However, consistent human data were reported from only one study. Baccetti et al. [90] used PCR and observed intragenic deletions of *AKAP4* and *AKAP3*, which code for major structural components of the fibrous sheath, in one DFS patient. No abnormality was detected in other samples.

We found a decrease in the activity of the glycolytic sperm-specific enzyme glyceraldehyde-3-phosphate dehydrogenase (sGAPD) and atypical localization of the enzyme. Mutations

**Figure 7.** (a) Sperm with dysplasia of the fibrous sheath (DFS) of the tail. The lack of mitochondria is revealed (arrow). Transverse (b) and longitudinal (c) sections through the tail with DFS. The lack of the central pair of microtubules (asterisk). (d) Sperm with primary ciliary dyskinesia (PCD). Transverse (e) and longitudinal (f) sections through the tail with PCD. The absence of dynein arms is revealed on the transverse section. OF, outer dense fibers; FS, fibrous sheath; and M, mitochondria.

within human *GAPDS* gene were assayed. In all five studied semen DFS samples, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of *sGAPD* [91]. Pereira et al. [92] found heterozygous deletion in the *DNAH5* gene, but not mutations in *AKAP3* and *AKAP4* in four patients with DFS.

DSF has an autosomal recessive inheritance. The genetic risk is now impossible to estimate. A few cases of live births after ICSI with spermatozoa of DSF patients were reported in the medical literature [93].

### *4.4.2. Primary ciliary dyskinesia (PCD)*

be secondary to a varicocele, infections of reproductive organs, and exogenous exposures [58]. Spermiogram parameters are possible to correct with medications in men diagnosed with functional asthenozoospermia. When the treatment is ineffective, ICSI is likely to help.

Chemes et al. [87] proposed the term dysplasia of the fibrous sheath (DFS) for the disorder, which is also known as stump tail syndrome and short-tail spermatozoa. Spermatozoa have substantially reduced, if any, motility due to fibrous for DSF [88]. In spermatozoa, the location of longitudinal columns and transverse ribs of the fibrous layer has been disturbed. There are changes in the structure of the mitochondrial helix—a significant shortening and disruption of localization. Anomalies in the structure of the fibrous sheath often put together with the

A mouse model of DFS was obtained by targeted disruption of the *Akap4* gene, and spermatozoa of mutant mice had short thick tails, which were morphologically identical to those in DFS patients [89]. However, consistent human data were reported from only one study. Baccetti et al. [90] used PCR and observed intragenic deletions of *AKAP4* and *AKAP3*, which code for major structural components of the fibrous sheath, in one DFS patient. No abnormal-

We found a decrease in the activity of the glycolytic sperm-specific enzyme glyceraldehyde-3-phosphate dehydrogenase (sGAPD) and atypical localization of the enzyme. Mutations

**Figure 7.** (a) Sperm with dysplasia of the fibrous sheath (DFS) of the tail. The lack of mitochondria is revealed (arrow). Transverse (b) and longitudinal (c) sections through the tail with DFS. The lack of the central pair of microtubules (asterisk). (d) Sperm with primary ciliary dyskinesia (PCD). Transverse (e) and longitudinal (f) sections through the tail with PCD. The absence of dynein arms is revealed on the transverse section. OF, outer dense fibers; FS, fibrous sheath;

**4.4. Genetically determined forms of asthenozoospermia**

absence of a central pair of the axoneme microtubules (**Figure 7a**–**c**).

*4.4.1. Dysplasia of the fibrous sheath of the tail*

86 Spermatozoa - Facts and Perspectives

ity was detected in other samples.

and M, mitochondria.

PCD is an autosomal recessive disorder and is highly heterogeneous genetically. PCD affects the axonemal structures (microtubules and dynein arms) of cilia and flagella (**Figure 7d**–**f**). Bronchial and pulmonary diseases are the main pathology in PCD because infections and bronchiectasis develop when respiratory cilia have motility defects or are immotile.

Headaches are common in PCD patients because lack of ciliary motility in the brain ventricles impairs circulation of the cerebrospinal fluid. Situs inversus is additionally observed in half of the PCD patients, possibly resulting from lack of ciliary motility in embryonic Hensen's node, which is responsible for the unidirectional fluid flow and thereby establishes left-right asymmetry [94]. The prevalence of PCD at birth is 1/10,000 to 1/20,000 [95].

Fertility is impaired in male patients because their spermatozoa are absolutely immotile or defects occur in efferent seminiferous ducts lined by ciliated epithelia. In a semen analysis, gross ejaculate parameters (volume, pH, viscosity, and color) and the concentration and count of morphologically normal spermatozoa are within the normal ranges.

Transmission electron microscopy (TEM) is commonly used to detect PCD. TEM reports lack of outer and/or inner dynein arms, the two central microtubules, or radial spokes and changes in microtubule arrangement.

Molecular methods to diagnose PCD have intensely been developed in the past years. Unicellular algae of the genus Chlamydomonas, which have two flagella, provide a convenient model to study the molecular composition of the axoneme. Axoneme protein genes identified in Chlamydomonas are candidate genes for PCD. A total of 16 mutations of PCD candidate genes were identified from 1999 to 2011 by genetic methods (analysis of linkage groups identified by homozygosity mapping), proteome analysis, and sequencing (mostly Sanger sequencing). Since 2011, mutations of 18 other genes have been described via wholeexome and whole-genome sequencing (for the review see [73, 96]).

PCD is genetically heterogeneous. Mutations of two genes, *DNAI1* and *DNAH5*, are the most common in PCD. *DNAI1* and *DNAH5* code for proteins of outer dynein arms of the axoneme. *DNAI1* mutations were found in 14% of PCD patients [97]. The *DNAH5* product is a major motor protein of outer dynein arms. Its mutations were observed in more than 25% of PCD patients [98].

Many proteins are involved in building the axoneme. Several proteins are common for epithelial cilia and sperm tails. Patients homozygous for mutations of their genes develop the total set of PCD signs, including bronchial and pulmonary diseases, changes in asymmetry of visceral organs, and immotile sperm. Other axonemal proteins are tissue specific, and mutations of their genes cause mosaic ciliopathy, such as asthenozoospermia and anosmia, or asthenozoospermia and swelling of the nasopharyngeal mucosa, which we identified in our patients.

(HSV), we proved this by immunofluorescence (IF) using the monoclonal antibodies to HSV1 and HSV2 (**Figure 8c**, **d**) and *in situ* hybridization (ISH) with biotinylated probes for HSV (**Figure 8e**). Infection was observed in both the total sperm fraction and the isolated fraction of motile spermatozoa. The persistence of the HSV genome in motile, morphologically normal sperms indicates that the virus may be transmitted vertically to the offspring via natural

Ultrastructure of Spermatozoa from Infertility Patients http://dx.doi.org/10.5772/intechopen.71596 89

Herpetic infection of spermatozoa was significantly more common in infertile men and men whose spouses had a history of spontaneous miscarriage or ART failure as compared with fertile men. Specific antiherpetic treatment of men diagnosed with HSV infection of spermatozoa results in a substantial, almost fivefold increase in the rates of blastocyst formation after

Bacterial colonies were detected in ejaculate samples from patients with fertility disorders. In the colonies, heteromorphic microorganisms were held together in a diffusive substance, probably of a polysaccharide nature, or covered with membranes as bacterial biofilms. The

**Figure 9.** (a) Bacterial microcolony (B) attached to sperm head (H). (c) Bacterial microcolony (B) attached to sperm tail (T). (b, d) Bacterial microcolonies attached to the epithelial cells (EC). A diffuse substance (a–c) or membranes (Me) (d)

fertilization or various ARTs, including IVF or ICSI.

ICSI and clinical pregnancy after ART [100].

**6. Bacterial infection of the ejaculate**

are detected between bacterial cells.

The development of ICSI allowed men with pronounced asthenozoospermia, including forms with genetic causes, to have children. The consequences of using ICSI in PCD and DSF are poorly understood because the disorders are rare and only few live births after ICSI have been reported (20 cases according to PubMed). PCD patients with andrological symptoms naturally had no offspring before the advent of ICSI. PCD is an autosomal recessive disorder and is expressed only in homozygotes and compound heterozygotes, when both alleles of one gene are affected. This circumstance reduces PCD risk in ICSI offspring, but makes it more likely for the mutations to accumulate in the population and to occur in homozygote at a higher rate in the long term.
