Introduction
In the human sperm, the axoneme is composed of nine peripheral microtubule doublets (A, B) and a central pair of microtubules. Doublets are linked by nexin bridges, present dynein arms (outer and inner) and are linked to the central microtubule pair by radial spokes. The central pair is surrounded by a fine fibrillar sheath and a central bridge, the central pair complex (CPC). At the end of the midpiece there is a fibrillar ring of dense material, the annulus, which is the site where the mitochondria helix ends. The axoneme is surrounded by outer dense fibres (ODF) that extend until the end of the proximal principal piece. At the beginning of the principal piece two longitudinal columns connected by semi-circular ribs, the fibrous sheath (FS), appear outside the ODF and prolong up to the end-piece (Holstein & Roosen-Runge, Reference Holstein and Roosen-Runge1981; Inaba, Reference Inaba2003). In sperm motility, dyneins are the motor complexes, and their ATPase activity is regulated by a nexin–dynein regulatory complex that links doublets; and radial spokes, besides providing a radial scaffold between the CPC and doublets, facilitate signal transduction from the CPC to the dyneins, thus governing flagellum beat and waveform (Smith & Yang, Reference Smith and Yang2004; Wirschell et al., Reference Wirschell, Olbrich, Werner, Tritschler, Bower, Sale, Loges, Pennekamp, Lindberg, Stenram, Carlén, Horak, Köhler, Nürnberg, Nürnberg, Porter and Omran2013).
Sperm immotility has been linked to gene mutations (Chemes & Rawe, Reference Chemes and Rawe2003, Reference Chemes and Rawe2010; Turner, Reference Turner2003; Inaba, Reference Inaba2007, Reference Inaba2011), such as those governing dynein arms. The absence of both dynein arms when associated with primary ciliary dyskinesia and situs-inversus is denominated Kartagener syndrome (Zariwala et al., Reference Zariwala, Knowles, Leigh, Pagon, Adam, Ardinger, Bird, Dolan, Fong, Smith and Stephens2014). Other dynein gene mutations were found to cause separate outer (Onoufriadis et al., Reference Onoufriadis, Shoemark, Munye, James and Schmidts2014) or inner (Antony et al., Reference Antony, Becker-Heck, Zariwala, Schmidts, Onoufriadis and Forouhan2012) dynein arm defects. Mutations in genes encoding dynein assembly factors were also observed to cause loss of both dynein arms (Zariwala et al., Reference Zariwala, Gee, Kurkowiak, Al-Mutairi, Leigh and Hurd2013), whereas mutations in genes encoding components of the radial spokes cause loss of the CPC (Castleman et al., Reference Castleman, Romio, Chodhari, Hirst, de Castro, Parker, Ybot-Gonzalez, Emes, Wilson, Wallis, Johnson, Herrera, Rutman, Dixon, Shoemark, Bush, Hogg, Gardiner, Reish, Greene, O’Callaghan, Purton, Chung and Mitchison2009).
The FS is involved in the structural maintenance of the flagellum elasticity but also plays regulatory roles in motility, as the major components of the FS are A-kinase anchor proteins, AKAP4 and AKAP3. They reside in longitudinal columns (AKAP4) and transverse ribs (AKAP3, AKAP4), which serve as a scaffold for glycolytic enzymes and signalling cascades mediated by tyrosine phosphorylations. Also, the longitudinal columns contain the components specific for disulphide bond reduction of cytoskeletal proteins (the sperm thioredoxin family) (Turner et al., Reference Turner, Musse, Mandal, Klotz, Jayes, Herr, Gerton, Moss and Chemes2001; Chemes & Rawe, Reference Chemes and Rawe2003; Eddy et al., Reference Eddy, Toshimori and O’Brien2003; Inaba, Reference Inaba2003, Reference Inaba2011; Moretti et al., Reference Moretti, Scapigliati, Pascarelli, Baccetti and Collodel2007). Albeit of this importance, gene mutations on the FS genes have only been found in one patient (Baccetti et al., Reference Baccetti, Collodel, Estenoz, Manca, Moretti and Piomboni2005a,Reference Baccetti, Collodel, Estenoz, Gambera, Moretti, Serafini and Piombonib). Structural defects causing dysplasia of the fibrous sheath (DFS) are diagnosed by the presence of a marked hypertrophy and hyperplasia of the FS with invasion of the midpiece, in association with a deficient or absent annulus, absent or misaligned mitochondria, and absence of the CPC (50% of the cases) and/or of the dynein arms (Chemes & Rawe, Reference Chemes and Rawe2003, Reference Chemes and Rawe2010). In about 20% of the cases, DFS is associated with primary ciliary dyskinesia (Turner et al., Reference Turner, Musse, Mandal, Klotz, Jayes, Herr, Gerton, Moss and Chemes2001; Chemes & Rawe, Reference Chemes and Rawe2003, Reference Chemes and Rawe2010), and has also been related to consanguinity (Baccetti et al., Reference Baccetti, Capitani, Collodel, Cairano, Gambera, Moretti and Piomboni2001), abnormal segregation of chromosomes (Baccetti et al., Reference Baccetti, Collodel, Estenoz, Manca, Moretti and Piomboni2005a,Reference Baccetti, Collodel, Estenoz, Gambera, Moretti, Serafini and Piombonib; Moretti et al., Reference Moretti, Geminiani, Terzuoli, Renieri, Pascarelli and Collodel2011) and increased sperm ubiquitination (Rawe et al., Reference Rawe, Olmedo, Benmusa, Shiigi, Chemes and Sutovsky2002).
The aim of the present report was to present the ultrastructural aspects of sperm abnormalities found in four patients with DFS and in one patient with situs-inversus totalis, all with total sperm immotility.
Materials and methods
In accordance with the National Law on Medically Assisted Procreation (Law 32/2006) and the National Council on Medically Assisted Procreation guidelines (CNPMA, 2008), semen from five patients were used after patient informed and written consent from cases enrolled in andrology evaluation for infertility.
For transmission electron microscopy processing, liquefied semen was washed with sperm preparation medium (SPM; Medicult Origio, Jyllinge, Denmark), mixed 1:1 with sodium cacodylate buffer, 0.1 M, pH 7.2 (Merck, Darmstadt, Germany) and centrifuged for 10 min at 1500 rpm. The pellet was then fixed with Karnovsky fixative (Sigma-Aldrich, St. Louis, USA) for 2 h at room temperature, washed, post-fixed with 2% osmium tetroxide (Merck) in buffer for 2 h at 4ºC, washed, serially dehydrated in ethanol (50, 75, 90, 95, 2 × 100%, 30 min each) (Panreac, Barcelona, Spain), equilibrated with propylene oxide (2 × 15 min) (Merck), and embedded in Epon (Sigma). Semithin (1 μm) and ultrathin sections (500–700 Å) were prepared with a Diatome diamond knife in an LKB ultramicrotome diamond knife in an ultratome NOVA (Leika Microsystems, Wetzlar, Germany). Semithin sections were stained with aqueous azur II (Merck) and methylene blue (Merck) (1:1) and observed by light microscopy to select the area with a higher concentration of sperm. Ultrathin sections were collected on 300 mesh copper grids (Taab, Berks, England), and sections contrasted with 3% aqueous uranyl acetate (20 min) (BDH, Poole, England) and Reynolds lead citrate (10 min) (Merck). Ultrathin sections were observed in a JEOL 100CXII transmission electron microscope (JEOL, Tokyo, Japan) operated at 60 kV.
Results
Patient 1
This patient was 38 years old, from the littoral north of Portugal and was an economist. At clinical examination he referred mite allergy and sinusitis, with no family consanguinity. The karyotype was normal, there were no Y chromosome microdeletions and the hormonal values were in the normal range. Semen values were 2.8 ml, pH 8.0, 16 × 106 sperm/ml, 100% immotility and 0% normal morphology (95% head; 2% midpiece; 98% flagellum). He performed intracytoplasmic sperm injection (ICSI) treatment cycles but no pregnancy ensued.
Observations at the ultrastructural level revealed that most of the sperm heads were abnormal (Fig. 1 A), with a conserved structure of the basal plate, centriole, striated columns and ODF (Fig. 1 B). The midpiece was short, with a few dispersed mitochondria that presented a light appearance. There was no visible annulus material and the midpiece was continuous with the principal piece. The principal piece was enlarged by the presence of a hyperplastic and hypertrophic FS (Fig. 1 A) that invaded the midpiece (Fig. 1 A, B). Regarding the axoneme, the proximal principal piece was characterized by the loss of the CPC and of the radial spokes (Fig. 1 C), whereas in the distal principal piece in addition microtubule doublet disorganization was observed (Fig. 1D).
Ultrastructure of sperm from four patients with dysplasia of the fibrous sheath. (A–D) Patient 1. (A) Longitudinal section of the sperm; abnormal head with excess of cytoplasm (cy), a regular nucleus (N) and a displaced acrosomal vesicle (AV); in the midpiece (MP) mitochondria (m) are scarce; the dysplastic fibrous sheath (FS) invades the MP region; there is no visible annulus material (arrows). (B) The neck region appears conserved, with the basal plate (BP), centriole (Ce), striated columns (SC) and outer dense fibres (ODF); mitochondria (m) display pale areas. There is no visible annulus material (arrows). (C) Transverse section at the proximal principal piece; dynein arms (white arrowheads) are present; absence of the central pair complex and radial spokes (*). (D) Transverse section at the distal principal piece; absence of the central pair complex and radial spokes (*) and microtubule doublet disorganization (arrow). (E–G) Patient 2. (E) Longitudinal section of the sperm; the apical region of the MP is enlarged with aggregates of pale mitochondria (m), followed by a mitochondrial helix; the annulus (arrows) is present. (F) The apical region of the principal piece presents a row of FS followed by a dysplastic FS. The annulus (arrows) is present. There is no visible annulus material (arrows). (G) Transverse section at the principal piece; the axoneme presents a reduced number of microtubule doublets (arrow); the central pair complex is present (black arrowheads). (H) Patient 3. Transverse section at the principal piece; the axoneme shows absence of the central pair complex (*), a reduced number of radial spokes (arrow), and absence of dynein arms (white arrowheads) and nexin bridges (dotted line). (I, J) Patient 4. (I) Transverse section at the principal piece; microtubule doublets (small arrow) and ODF (large arrow) are conserved; the central pair complex and radial spokes are missing (*). (J) Transverse section at the principal piece; disorganization and increased number of microtubule doublets (small arrow) and ODF (large arrow). Bars: (A, B, F), 0.5 μm; (C, D, G–J), 0.1 μm; (E), 1 μm.
Patient 2
This patient was 45 years old, from the littoral centre of Portugal and was a sales representative, performing several hours of driving daily. At clinical examination he had no upper respiratory complaints or family consanguinity. The karyotype was normal, there were no Y chromosome microdeletions and the hormonal values were in the normal range. Semen values were 3.3 ml, pH 7.5, 39.6 × 106 sperm/ml, 100% immotility and 0% normal morphology (100% head; 48% midpiece; 80% flagellum). He performed ICSI treatment cycles, first with ejaculated sperm and then with testicular sperm obtained by testicular sperm extraction (TESE) without achieving a successful pregnancy.
The ultrastructural study revealed that most of the sperm nuclei were abnormal, with a conserved structure of the basal plate, centriole, striated columns, ODF and annulus (Fig. 1 E). The apical region of the midpiece was enlarged by the presence of extra cytoplasm and contained aggregated and misaligned mitochondria, whereas the basal region of the midpiece acquired a regular mitochondrial helix appearance. All mitochondria had a light appearance. The midpiece was separated from the principal piece by the annulus. Bellow this point, the principal piece was enlarged by the presence of an excess of cytoplasm, with the FS material forming a unilateral longitudinal row. This was followed by a disorganized FS with mild hypertrophy and hyperplasia and without a great enlargement of the principal piece (Fig. 1 E, F). We did not find any normal axoneme, most presenting a variable reduced number of microtubule doublets, radial spokes and ODF, with presence of the CPC (Fig. 1 G).
Patient 3
This patient was 40 years old, from the littoral centre of Portugal and was an accountant. At clinical examination he referred chronic bronchitis and no familiar consanguinity. The karyotype was normal, there were no Y chromosome microdeletions and the hormonal values were in the normal range. Semen values were 4 ml, pH 7.8, 58.5 × 106 sperm/ml, 100% immotility and 8% normal morphology (86% head; 62% midpiece; 20% flagellum). As the patient presented a voluminous left testicle varicocele he was submitted to surgery. After that he performed ICSI treatment cycles without success.
Under the electron microscope, most of the sperm heads were abnormal, with a conserved structure of the basal plate, centriole, striated columns and ODF. The midpiece was short, with a few pale and disorganized mitochondria. There was no visible annulus material and the midpiece was continuous with the principal piece. The principal piece was enlarged by the presence of hyperplastic and hypertrophic FS that invaded the midpiece. At the axoneme, the large majority of the sections showed absence of dynein arms, nexin bridges and CPC, with presence of a variable number of radial spokes (Fig. 1 H).
Patient 4
This patient was 43 years old, from the littoral centre of Portugal and was a long distance driver. At clinical examination he referred no respiratory complaints or family consanguinity. The karyotype was normal, there were no Y chromosome microdeletions and the hormonal values were in the normal range. Semen values showed 2.2 ml, pH 7.6 and very rare sperm with total immotility. He performed ICSI treatment cycles without achieving a successful pregnancy.
At the electron microscope, most of the sperm heads presented an abnormal morphology, with a conserved structure of the basal plate, centriole, striated columns and ODF. The midpiece was short, with a few pale mitochondria. There was no visible annulus material and the midpiece was continuous with the principal piece. The principal piece was enlarged by a hyperplastic and hypertrophic FS that invaded the midpiece. Relatively to the axoneme, most of the sperm presented absence of the CPC and radial spokes (Fig. 1 I). However, some sperm evidenced total microtubule disorganization with an increased number of microtubule doublets and ODF (Fig. 1 J).
Patient 5
This patient was 42 years old, from northern Portugal and was a Judicial officer. At clinical examination he referred no family consanguinity but reported chronic respiratory complaints, with nasal polyps, chronic sinusitis, rhinitis and bronchitis. He also presented a situs-inversus totalis. As he did not perform nasal cilia analysis, primary ciliary dyskinesia could not be comproved. The karyotype was normal, there were no Y chromosome microdeletions, cystic fibrosis transmembrane receptor (CFTR) mutations or adverse CFTR polymorphisms, and the hormonal values were in the normal range. Semen values showed 5.6 ml, pH 7.8 with very rare sperm with total immotility. He undertook TESE-ICSI treatment cycles and after a successful pregnancy the couple had male twins, who now are 5 years old and are perfectly healthy.
Ultrastructural examination revealed that all sperm had an abnormal head, with a conserved structure of the basal plate, centriole, striated columns, ODF, annulus and FS. The midpiece was short and enlarged due to the presence of aggregated and misaligned pale mitochondria (Fig. 2 A). At the axoneme, all sections showed absence of dynein arms and nexin links, with presence of radial spokes and CPC (Fig. 2 B).
Ultrastructure of sperm from a patient with situs-inversus totalis. (A, B) Patient 5. (A) Longitudinal section of the sperm; the midpiece (MP) is enlarged with aggregates of misaligned pale mitochondria (m). The annulus is present (arrows), and at the principal piece the outer dense fibres (ODF) and the fibrous sheath (FS) have a normal appearance. (B) Transverse section at the principal piece; the axoneme shows a conserved central pair complex (black arrowhead) and radial spokes (arrow); dynein arms (white arrowheads) and nexin bridges (dotted line) are missing. Bars: (A), 0.5 μm; (B), 0.1 μm.
Discussion
Several studies presented the ultrastructural features of sperm with DFS and asthenozoospermia. In a series of five patients, several degrees of hyperplasia and disorganization of the FS were observed, associated with variable axoneme disruption. Two of these patients had chronic respiratory complains, and none presented situs-inversus (Chemes et al., Reference Chemes, Brugo, Zanchetti, Carrere and Lavieri1987; Reference Chemes, Morero and Lavieri1990). In a study of 42 patients, authors observed marked hypertrophy and hyperplasia of the FS, with the annulus placed beneath the striated columns, absent midpiece and mitochondria poorly assembled or absent. Of these patients, 24% had respiratory symptoms without situs-inversus (with absence of dynein arms), whereas in the remainder variable axoneme disruption was observed (Chemes et al., Reference Chemes, Olmedo, Carrere, Oses, Carizza, Leisner and Blaquier1998). In another study of six patients, the authors also found a marked hypertrophy and hyperplasia of the FS. One patient had severe respiratory symptoms. The annulus was placed beneath the striated columns, the midpiece was not formed and mitochondria were poorly assembled or absent. In these cases, variable axoneme disruption associated with partial inner dynein arm deficiency was also observed (Rawe et al., Reference Rawe, Galaverna, Acosta, Olmedo and Chemes2001).
Our results showed the same pattern of hyperplastic and hypertrophic FS in three cases, but sperm had absence of the annulus and total immotility. Additionally, the axoneme presented specific changes, such as absence of the CPC and radial spokes, with one case additionally presenting absence of the dynein arms and nexin bridges (case with chronic bronchitis). We also present a case with a different pattern of DFS, where sperm was totally immotile, but there was the presence of the annulus without invasion of the MP by the dysplastic FS. In this case, sperm displayed a variable axoneme disruption. In all these four cases, mitochondria exhibited pale spaces, a finding that has been related to unexplained asthenozoospermia (Pelliccione et al., Reference Pelliccione, Micillo, Cordeschi, D’Angeli, Necozione, Gandini, Lenzi, Francavilla and Francavilla2011).
In a case report of tail stump spermatozoa, associated with total sperm immotility, authors observed a variety of anomalies, including presence or absence of the annulus, 65% with absent CPC and in the remaining a decrease or an increase in the number of doublets. About 48% of sperm presented an accumulation of cytoplasm with dispersed mitochondria and dysplastic fibres. In other sperm, the midpiece was enlarged, containing misaligned mitochondria. An annulus separated the midpiece from the principal piece, where a dysplastic FS was evident (Barthelemy et al., Reference Barthelemy, Tharane, Lebos, Lecomte and Lansac1990). This report is more close to our findings, as it included only patients with totally immotile sperm, the annulus could be present (as in our patient 2) or absent (as in our patients 1, 3 and 4), the CPC could be absent (as in our patients 1, 3 and 4), with some sperm presenting an increase in the number of microtubule doublets associated with axoneme disorganization (as in our patients 2 and 4).
Some studies included the analysis of FS genes and sperm chromosome constitution. In 13 patients with DFS and asthenozoospermia, an intragenic deletion of the AKAP3 and AKAP4 genes was found, together with an increase of sex chromosome disomy and diploidy. Sperm showed marked hypertrophy and hyperplasia of the FS that invaded the midpiece, which was short and presented poorly assembled mitochondria. There was no reference to the annulus. The lack of one or two dynein arms was frequently observed and in some cases the absence of the CPC. (Baccetti et al., Reference Baccetti, Collodel, Estenoz, Manca, Moretti and Piomboni2005a,Reference Baccetti, Collodel, Estenoz, Gambera, Moretti, Serafini and Piombonib). This increase in the frequencies of sex chromosome disomy and diploidy was also observed in a study of two patients with DFS associated with sperm motility (one case) and total immotility (one case) (Moretti et al., Reference Moretti, Geminiani, Terzuoli, Renieri, Pascarelli and Collodel2011). According to these authors these patients should thus be genetically counselled about the risks on the offspring.
Septins (small GTPases associated with membranes, actin and microtubules) and the testis anion transporter TAT1 are the structural components of the sperm annulus. The annulus is an electron dense ring-shaped structure located at the junction of the midpiece with the principal piece, establishing a compartmentalization of the sperm flagellum. Although its absence has been related to partial sperm immotility, no mutations or deficient mRNA expression have been observed in these cases, which suggests that these proteins could not reach the annulus (Lhuillier et al., Reference Lhuillier, Rode, Escalier, Lorès, Dirami, Bienvenu, Gacon, Dulioust and Touré2009). In our report, all cases (patients 1, 3 and 4) with severe dysplastic FS (with invasion of the midpiece) had no visible annulus material. As patient 2 had an annulus and a different kind of DFS presentation, results suggest that pathology also affects the annulus only when the FS is severely dysplastic.
Some cases with primary ciliary dyskinesia (PCD) and dynein arm gene mutations also present total sperm immotility and absent dynein arms, with the prototype being Kartagener syndrome (Afzelius & Eliasson, Reference Afzelius and Rune Eliasson1979; Baccetti et al., Reference Baccetti, Burrini, Capitani, Collodel, Moretti, Piomboni and Renieri1993; Inaba, Reference Inaba2011; Lee, Reference Lee2011). However, not all cases with PCD have absence of dynein arms, as in a case report of a patient with PCD, authors found absence of the CPC and a variable number of doublets in some sperm, but dynein arms were intact. In this case report, the annulus was poorly developed or absent, there were few mitochondria, and the FS was thin and disorganized (Mclachlan et al., Reference Mclachlan, Ishikawa, Osianlis, Robinson, Merriner, Healy, de Kretser and O’Bryan2012), which suggest a less severe form of DFS. In our present report of four patients with DFS, all with total sperm immotility, two presented mild respiratory symptoms. Patient 1 had a 9+0 pattern and absent radial spokes, with conserved dynein arms, whereas patient 3 (chronic bronchitis), in addition to the 9+0 pattern and partial absence of the radial spokes, had a total absence of dynein arms and nexin bridges. A less severe form of DFS was observed in patient 2, who had no respiratory complaints, and there was a disorganization of doublets at the axoneme. In contrast, patient 5 had severe respiratory symptoms, situs-inversus, total sperm immotility, and absence of dynein arms and nexin bridges. In these cases, the ciliary mucosa was investigated to rule out PCD.
In conclusion, we present three cases with DFS associated with total sperm immotility, abnormal mitochondria, and absence of the annulus, CPC and radial spokes, of which one had, in addition, absence of dynein arms and nexin bridges. We also describe a patient with total sperm immotility and a different presentation of DFS, as the annulus was present and the dysplastic FS was restricted to the principal piece. These findings thus confirm the heterogeneity of the DFS condition. The fifth patient had severe respiratory symptoms associated with situs-inversus totalis, total sperm immotility, and absence of dynein arms and nexin bridges, thus also further supporting previous observations.
Acknowledgements
We would like to acknowledge Ilda Pires, Biologist, MSc, ESHRE senior clinical embryologist and Madalena Cabral, BSc, biologist, embryologist for semen processing at CHVNG; and Ana Gonçalves, BSc, biochemist and Cláudia Osório, BSc, biologist for semen processing at CGR.
Funding
UMIB is funded by National Funds through the Foundation for Science and Technology (FCT), under the Pest-OE/SAU/UI0215/2014.
Disclosure statement
The authors have nothing to declare.
