Structural and Ultrastructural Assessment of the Sperm Annulus in Men with Immotile Short-tail Sperm Defects Compared to Normozoospermic Controls: A Case–control Study

ABSTRACT

Background:

The sperm annulus is a fibrous ring structure formed by septin proteins. It connects the mid-piece and principal piece of the sperm flagellum in mammals and plays a crucial role in flagellar stability and motility

Aim:

This study aimed to compare the presence or absence of the sperm annulus in patients with immotile short-tail spermatozoa and a fertile control group

Settings and Design:

This observational case–control study was conducted at a local research institute

Materials and Methods:

Twenty infertile men with short-tail sperm defects (>50% of spermatozoa affected) and 20 fertile men with normal semen parameters were recruited. Immunocytochemistry was performed to assess the presence of the annulus using antibodies against Septin4 and Septin7. The absence of annular structures was confirmed by transmission electron microscopy (TEM)

Statistical Analysis Used:

No statistical analysis was performed in this study

Results:

In the control group, Septin4 and Septin7 were precisely localised at the annulus region between the mid-piece and principal piece of spermatozoa. In contrast, spermatozoa from the patient group exhibited either a complete absence of annular staining or abnormal localisation of the annulus near the sperm head. In one patient, more than 90% of spermatozoa lacked Septin4 and Septin7 expression, as confirmed by TEM

Conclusion:

Absence or structural abnormalities of the sperm annulus in men with short-tail spermatozoa may contribute to impaired motility. Evaluating annulus integrity may serve as a potential diagnostic biomarker in selected cases of male infertility.

INTRODUCTION

Infertility is defined as the failure to achieve pregnancy after 1 year of regular, unprotected intercourse. More than 20 million men worldwide are affected by infertility, which is identified in nearly 85% of infertile couples. Ovulatory dysfunction, tubal disorders and male-factor infertility mainly cause a woman’s infertility. It is estimated that 15% of infertile couples are suffering from unexplained infertility.[1,2] About 50% of infertility cases involve male factors. There are numerous qualitative sperm disorders affecting men worldwide, including teratozoospermia. An abnormal morphology of spermatozoa in semen samples is indicative of this condition. The morphology of spermatozoa is one of the most important and intrinsic characteristics defining the fertilisation capacity of male gametes.[3] Immotile short-tail sperm (ISTS) defect is one of the conditions associated with teratozoospermia. ISTS defects are characterised by a marked shortening of sperm tails, a reduced sperm count and immotile spermatozoa (although in some cases, limited motility is observed in the few remaining spermatozoa). However, the sperm head, nucleus and acrosome are usually normal. Axonemes that are completely absent or severely disorganized (generally lacking central tubules and consisting of doublets with missing or poorly assembled arms, disassembled mitochondria and undetectable dynein) are also common manifestations of this defect. These characteristics usually characterise ISTS defects, but some patients have only displayed some.[4,5,6] The septin family comprises polymerising GTPases that assemble into heteromeric filaments. These filaments are essential for various cellular processes, such as exocytosis, cytokinesis and vesicle trafficking. There are 14 different classes of Septins.[7,8] Septin1, 4, 6, 7 and 12 proteins are components of the mammalian sperm tail annulus. The sperm annulus connects the mid-and principal pieces of the sperm tail in mammals. It acts as a diffusion barrier, and both sperm motility and terminal differentiation depend on its function.[9]

As part of spermatogenesis, Septin4 is essential in maintaining the sperm midpiece and annulus architecture and a ring-like structure between the mid- and the principal pieces of the tail. It also helps maintain the mitochondrial structure in the midpiece of the sperm tail. The absence of Septin4 results in immotile sperm with defective midpieces and annulus, resulting in male sterility in mice.[10]

Septin7 is most often expressed in the annulus of human mature spermatozoa, where it colocalises with Septin4. It is also found in the sperm head and tail, with the tail displaying the maximum expression levels. It was more common for immature sperm and sperm with defects in the tail, head and neck to lack a Septin7 signal. Septin7 regulates the maturation of sperm.[5,11,12] The present study used an immunodetection method with specific antibodies directed at Septin4 and 7 proteins. These proteins were previously described as components of the annulus that can be used as markers of this structure. The method detected annulus in sperm preparations from 20 patients with ISTS. Transmission electron microscopy (TEM) further confirmed the absence of the annulus structure.

MATERIALS AND METHODS

Patients and samples

This observational case–control study was conducted in accordance with the Declaration of Helsinki and was approved by the local Ethics Committee for Human Experimentation (Approval Number: IR.ACECR.ROYAN.REC.1401.058, Date: 14 August 2022). Written informed consent was obtained from all participants. The study included 20 infertile men with short-tail sperm defects (mean age: 40 years) and 20 normozoospermic controls (mean age: 35 years). Patients were required to have more than 50% short-tailed spermatozoa. Short-tailed spermatozoa were classified into five groups based on their percentage in the sample: 50%–60%, 60%–70%, 70%–80%, 80%–90% and 90%–100%, with 5, 5, 4, 2 and 4 patients in each group, respectively.

Semen analysis

Semen samples were collected following a standard abstinence period (2–7 days) and evaluated according to World Health Organization 2010 criteria. Routine semen analysis was performed with computer-assisted sperm analysis software after 30 min of liquefaction at 37°C. Sperm morphology was assessed using Papanicolaou staining, including evaluation of cytoplasmic remnants, acrosomes, necks and tails.

Immunocytochemical analysis

Pellets were resuspended in 1 mL phosphate-buffered saline (PBS) after centrifugation at 2000 rpm for 5 min. Approximately 20 μL of fresh sperm aliquots were placed on slides, air-dried and fixed in 4% paraformaldehyde for 20 min at 4°C. Cells were permeabilised with PBS containing 0.5% Triton X-100. After blocking with 1% bovine serum albumin in PBS, slides were incubated with primary antibodies (rabbit anti-Septin4 or Septin7, Santa Cruz Biotechnology, 1:100) for 2 h at room temperature. Goat anti-rabbit FITC-conjugated antibodies (Santa Cruz Biotechnology, 1:1000) were then applied for 1 h at room temperature. Nuclei were counterstained with DAPI, and slides were analyzed using fluorescence microscopy.

Transmission electron microscopy analysis

Semen samples were washed in M16 medium (Sigma-Aldrich Co. Ltd, Irvine, UK) and centrifuged at 300 × g for 10 min. Samples were fixed in 0.1 M phosphate buffer (pH 7) containing 3% glutaraldehyde for 2 h at room temperature. After two washes in PBS with 1% sucrose, samples were resuspended in 0.2 M sodium cacodylate buffer. Secondary fixation was performed with 1% osmium tetroxide (Agar Scientific Ltd, Essex, UK). Samples were dehydrated in graded alcohols, embedded in Epon resin (Polysciences Inc., Warrington, USA), and sectioned into ultrathin slices (~90 nm) using a diamond knife. Sections were mounted on nickel grids, stained with uranyl acetate and examined using a LEO906 TEM (Zeiss, Germany).

RESULTS

Sperm morphology was evaluated by the Papanicolaou staining method, and samples with morphology of 4% or above were considered normal. The case group also had spermatozoa with amorphous heads, coiled tails, bent necks and bent tails when sperm morphology was examined [Figure 1a and b].

Figure 1.

The arrows highlight the normal sperm in part (a) and the short-tailed sperm in part (b) of the figure.

Immunodetection was performed to assess the expression of Septin4 and Septin7 in groups of short-tailed spermatozoa and in normozoospermic controls. In 20 normal samples, septin expression was detected in the annulus region of spermatozoa [Figure 2].

Figure 2.

Immunofluorescence assay of Septin4, 7 in human spermatozoa. Septin4, 7 was expressed in the annulus of spermatozoa. (A1, B1) DAPI (blue), (A2, B2) Septin4, 7 (green) label the annulus region of spermatozoa, (A3, B3) bright field Arrows indicate the annulus region labeled by DAPI (A1, B1), Septin 4 and 7 antibody (A2, B2), and Bright Field (A3, B3)

It has been found that most of the short-tailed sperm in the 50%–60% and 60%–70% groups had annulus. Spermatozoa with normal or relatively longer tails displayed an annulus between the midpiece and principal piece, whereas short-tailed spermatozoa exhibited a defective annulus positioned near the sperm head. A significant proportion of men with more than 70% short-tailed spermatozoa showed absence of the annulus [Figure 3]. There was a different position of the annulus in these patients, which was near the sperm head [Figure 4]. Analysis of immotile sperm with elongated tails revealed an intact and normally structured annulus. This finding confirms that the structural defects, including absence or mislocalisation of the annulus observed in short-tailed sperm, are not present in these cells, indicating that annulus disruption is specifically associated with short-tailed sperm defects. In a representative case, immunofluorescence analysis revealed that ~90% of spermatozoa lacked Septin4 and Septin7 staining at the annulus region. This observation was corroborated by TEM, which confirmed the structural absence of the annulus. These findings indicate a widespread disruption of annulus organisation in this patient. The individual, a 33-year-old male, exhibited the following semen parameters [Figure 5].

Figure 3.

Proportion of sperm lacking annulus increases with higher percentages of short-tailed sperm, highlighting their strong association

Figure 4.

Expression of Septin4 and 7 proteins in the annulus in a patient with short-tail spermatozoa. A1 and B1 represent the nucleus of the cells. A2 and B2 are fluorescent stains showing the expression of these proteins in the annulus region of spermatozoa using the Septin4 and Septin7 antibodies. A3 and B3 are bright-field images. The location of the annulus could be seen near the sperm head, The arrows in each panel indicate the annulus region where Septin4 and Septin7 are localized.

Figure 5.

Immunodetection of Septin4 and Septin7 in semen smears from one (case) of the 20 patients studied. The annulus structure was completely absent. DAPI (Blue), (A2, B2) Septin4, 7 (Green), (A3, B3) bright field, Arrows indicate the annulus, showing no Septin4 or Septin7 signal

Concentration: 40 million/mL, short tail: 75%, abnormal morphology: 100%, Total DNA fragmentation index: 34.3%, total motility: 0%.

TEM revealed no abnormalities in sperm heads or acrosomes of patients with predominantly short-tailed spermatozoa. However, axonemal microtubules were severely defective, showing irregular and disorganized structures. The fibrous and mitochondrial sheaths were also irregular, and the annulus was completely absent [Figure 6].

Figure 6.

(a,b) Transmission electron microscopy analysis of a patient with 75% short-tailed spermatozoa. Almost all sperm are absent the annulus ring. The sperm head was normal in shape, but there was disorganisation of the axonemal microtubules, hyperplasia of the fibrous sheath, extra cytoplasmic droplets and mitochondrial defects. Arrows indicate the annulus and other structures, showing abnormalities in fibrous sheath, microtubules, mitochondria, and cytoplasmic droplets. An: Annulus, Ax: Axoneme, ISTS: Immotile short tail sperm, FS: Fibrous sheath, M: Mitochondrion, N: Nucleus

DISCUSSION

The sperm tail and its defects are associated with asthenoteratozoospermia and male infertility.[13,14,15] Septins, including Septin1, 2, 4, 6, 7 and 12, are components of the sperm annulus, and reduced septin expression has been observed in sterile men with abnormal sperm motility and morphology. Septin4 plays a crucial role in the localisation of septin proteins. In Septin4-null mice, sperm exhibit reduced motility, infertility and bent tails, with the annulus undetectable by electron microscopy.[9] Septin7 signals are frequently absent in immature or morphologically abnormal spermatozoa, a feature that is significantly more common in asthenospermic patients.[13] Septin12 forms filaments with Septin4, 6 and 7; however, in the absence of Septin4, filament formation is disrupted, resulting in defective sperm tails.[16] In one patient with short-tail spermatozoa, 90% of spermatozoa lacked Septin4 and/or Septin7, which was confirmed by TEM. Sugino et al. examined Japanese asthenozoospermic cases and reported defective Septin4 or Septin7 labeling in 10 cases (13%–15%), suggesting that septins could serve as diagnostic markers.[17] In contrast, Lhuillier et al. analyzed 75 French asthenozoospermic cases and observed only one missing annulus, with differences possibly attributable to ancestry, cohort size or other factors.[18] Hosseinifar et al. analyzed sperm from 100 Iranian men with asthenozoospermia, detecting Septin4 and Septin7 immunocytochemically. Electron microscopy revealed a missing annulus in only one case, suggesting that septin antibodies may serve as potential diagnostic markers for annulus defects.[19] A previous study on 254 men identified SLC26A8, a transmembrane protein localised to the annulus, as associated with asthenozoospermia (mean progressive motility, 24%). However, annulus defects were observed in only 1.2% of cases, indicating that annulus integrity is not a primary endpoint for screening or classifying asthenozoospermic patients.[20] Rafaee et al. analyzed 90 Iranian patients with teratozoospermia, assessing genetic variations in exons 5-7 of Septin12 and evaluating annulus status. One patient carrying the c. 474G>A variant exhibited short-tailed sperm, with 99% of sperm lacking an annulus; however, no correlation was found between Septin12 polymorphisms and the defect.[21] Wang et al. reported two unrelated Chinese patients with biallelic loss-of-function variants in Septin4. Absence of Septin4 disrupted Septin12 filament formation, leading to defective sperm tails and midpiece abnormalities, including mitochondrial sheath disorganization and annulus loss.[16] In the present study, electron microscopy revealed similar midpiece defects; however, Septin4 mutations were not investigated. Despite annulus defects, sperm heads were morphologically normal, while axonemal microtubules exhibited complete disorganization. Additional abnormalities included fibrous sheath hyperplasia, extra cytoplasmic droplets, and mitochondrial irregularities in annulus-less spermatozoa. Hwang et al. investigated heterozygous and homozygous Dnah2 mutations in patients with multiple morphological abnormalities of the sperm flagella. In wild-type sperm, Septin4 and Septin12 were localised between the midpiece and principal piece, whereas in Dnah2 Δ/Δ sperm, these septins were expressed near the connection piece and TEM confirmed dysregulation of the centriole and/or annulus.[22] In our study, most short-tailed sperm expressed Septin4 and Septin7 near the sperm head, consistent with previous reports, although no genetic analyses were performed. In groups 1 and 2, Septin4 and Septin7 were present in the annulus at the normal position between the midpiece and principal piece, with average annulus presence of 60% and 50%, respectively. In groups 3 and 4, annulus signals were displaced toward the sperm head, with averages of 32% and 29%. In group 5, Septin4 and Septin7 were mostly absent, with only 13% of sperm exhibiting an annulus. Septins, especially SEPT4 and SEPT12, are essential for organising the sperm annulus, preserving tail integrity and supporting progressive motility. Beyond structural roles, septins influence oocyte activation and early embryonic cytoplasmic dynamics, emphasising their relevance in fertilisation and early development. Dysfunction of septins compromise sperm function and may reduce ART success.[23] In our study, among six patients with severe short-tail sperm defects, only one received medical treatment and five underwent seven ART cycles, all unsuccessful. In contrast, Hosseini et al. reported successful fertilisation and live births in 117 similar patients using ICSI.[24] This difference is probably due to our limited sample size, highlighting the need for further studies on annulus status as a potential prognostic marker for ART outcomes.

CONCLUSIONS

The presence of ISTS defects in infertile men is frequently associated with the absence or mislocalisation of the annulus, a critical cytoskeletal component essential for flagellar structure and function. Disruption of the annulus strongly correlates with impaired sperm motility and abnormal tail morphology. These findings highlight the importance of assessing the annulus as a diagnostic marker in male infertility cases predominantly characterised by short-tail sperm defects. Further studies are warranted to elucidate the underlying mechanisms and to explore potential therapeutic strategies targeting annulus-related abnormalities.

Authors’ contributions

AM: Manuscript preparation, data acquisition and analysis, editing and review. RA: Technical assistance, manuscript editing and supervision. SGMA: Study design and data acquisition. SM: Study design, methodology, results interpretation, review and editing and overall supervision.

Conflicts of interest

There are no conflicts of interest.

Data availability statement

Data are available on reasonable request.

Funding Statement

Financial support for this study was provided by Royan Institute.