Axonemal doublet microtubules can split into two complete singlets in human sperm flagellum tips

Abstract

Motile flagella are crucial for human fertility and embryonic development. The distal tip of the flagellum is where growth and intra-flagellar transport are coordinated. In most model organisms, but not all, the distal tip includes a 'singlet region', where axonemal doublet microtubules (dMT) terminate and only complete A-tubules extend as singlet microtubules (sMT) to the tip. How a human flagellar tip is structured is unknown. Here, the flagellar tip structure of human spermatozoa was investigated by cryo-electron tomography, revealing the formation of a complete sMT from both the A-tubule and B-tubule of dMTs. This different tip arrangement in human spermatozoa shows the need to investigate human flagella directly in order to understand their role in health and disease.

Keywords: axoneme; cilia; cryo-electron microscopy; cryo-electron tomography; fibrous sheath; singlet region.

Figure 1

Ultrastructure of mammalian spermatozoa. (A) Cartoon cross sections through the sperm flagellum illustrate its internal ultrastructure. The sperm tail distal tip contains spatially disorganized sMTs. More proximally, MTs are organized in an axoneme with nine dMTs and two CP MTs (9 + 2 arrangement). Even more proximal parts of the sperm tail are surrounded and protected by the FS 31, 38 and by the outer dense fibers, that enhance structural integrity 34, 35 and modulate the flagellar beat 36, 37. The mitochondrial sheath that wraps around the tail's mid‐piece provides energy for cell motility. The arrows indicate which flagellar regions the cross sections represent. (B) The flagellum is divided into three structural regions. The most distal segment of the sperm flagellum is the end piece, which is defined by the absence of the FS. The principal piece includes most of the tail length and is protected by the FS. The mid‐piece is defined by the presence of the mitochondrial sheath wrapped around it. The connecting piece (also called neck) links the spermatozoon head to its flagellum. The head is the most anterior part of the cell and it carries the genetic material in the nucleus.

Figure 2

The human sperm tail thickness correlates to the MT number in the end piece and to the presence of FS in the principal piece. (A–E) Montaged or single frame cryo‐EM of human spermatozoon end pieces highlight morphological heterogeneity in sperm tails. The PM and the MTs of the flagellum are clearly visible. (F) The thickness of the flagellum increases with the distance from the tip. The striped background indicates the measured average extent of the singlet region and the gray background indicates the measured average extent of the end piece. Each data point represents the mean thickness of at least five individual flagella measured from montages of cryo‐EMs (up to 20 flagella per data point in the end piece). The high standard deviation (error bars) is due to the intrinsic heterogeneity of the samples. (G) Cryo‐ET revealed that the MT number correlates with the flagellum thickness in the singlet region (R ² = 0.808). dMTs were counted as two MTs. (H–J) 23 nm‐thick tomographic slices through the principal pieces of three different human sperm cells. (H) Transition point between the end piece and the principal piece. Longitudinal (H–I) and tangential (J) views of the principal piece reveal the rib‐like morphology of the FS. Arrowheads indicate interconnections between adjacent ribs. All scale bars are 100 nm.

Figure 3

dMTs can split into two complete sMTs in human flagella. (A) The distance between the MT termination point and the flagellum tip was measured in cryo‐ETs for each MT in the singlet region of three different sperm cells. Tomographic views of the three imaged tips are shown in Fig. S2. The tips contained 10, 11, and 14 sMTs, respectively. The vertical bars represent sMTs (in random order), the top of the graph represents the flagellum tip and the spacing between them represents the distance of the MT termination points from the tip. (B–C) 23 nm‐thick tomographic cross‐sectional slices of the singlet region in cell3 from panel A, containing 14 sMTs. The two panels show the same image, but sMTs are circled and counted in panel C. (D–F) dMTs can transition into singlets by three modes. (1) The A‐ and B‐tubules split into two complete sMT (D); (2) The B‐tubule terminates while the A‐tubule continues into the singlet region (E); (3) The B‐tubule splits from the A‐tubule and continues into the singlet region as an incomplete MT (F). A‐tubules are drawn and marked in light blue and B‐tubules in dark blue. Panels show one example for each transition mode in longitudinal tomographic views (Tomo) and models. Cross‐view MT models (Cross) are shown for each observed event. Black arrows connect different cross‐view models of the same dMT from most proximal to most distal. Dark blue arrowheads point at the B‐tubule termination point. Panel F does not contain a longitudinal model and includes a tomographic view of the incomplete extension of the B‐tubule. Additional tomographic views of each transition mode are shown in Fig. S3. Longitudinal views are 8 nm‐thick tomographic slices. Scale bars are 100 nm in panels B–C and 20 nm in panels D–F.

Figure 4

Splitting of dMTs in bovine sperm flagella. Thin sections through the singlet region of bovine sperm cells show at least 18 (A–B) and 15 (C–D) sMTs. Panels A–B and C–D, respectively, show the same images, but sMTs are counted in panels B and D. (E) Cryo‐EM of a flattened bovine sperm flagellum tip. Five events of dMTs splitting into two sMTs are shown (arrowheads) and at least 16 sMT ends are visible (numbered). One event of B‐tubule termination is shown by the white arrow. Scale bars are 100 nm in panels A–D and 200 nm in panel E.