Cloning and developmental analysis of murid spermatid-specific thioredoxin-2 (SPTRX-2), a novel sperm fibrous sheath protein and autoantigen

Thioredoxins compose a growing family of proteins that participate in different cellular processes via redox-mediated reactions. We report here the cloning, developmental expression, and location of murid Sptrx-2. Mouse and rat SPTRX-2 proteins display a high homology to their human ortholog in the thioredoxin and NDP kinase domains, and the coding genes are located at syntenic positions. Northern blotting and in situ hybridization confirmed the testis-specific expression of murine Sptrx-2 mRNA, mostly in round spermatids. Immunohistochemical analysis of the 19 steps of rat spermiogenesis showed that SPTRX-2 expression becomes prominent in the cytoplasmic lobe of step 15-18 spermatids and diminishes in step 19 just before spermiation. However, in the spermatid tail, SPTRX-2 immunoreactivity increased from step 15 to 19 and was confined to the principal piece. By immunogold electron microscopy, SPTRX-2 was first detected scattered throughout the cytoplasm of the axoneme in step 14-15 spermatids, but began to be incorporated by step 16 into the fibrous sheath (FS). During steps 17-18, the labeling increased over the ribs and columns of the assembled FS. It peaked in step 19 and remained in the FS of epididymal spermatozoa. Immunoblots of isolated FS obtained from spermatozoa confirmed that SPTRX-2 is an integral component of the FS and a post-obstruction autoantigen in vasectomized rats. Our data indicate that SPTRX-2 incorporation into the FS lags well behind FS assembly, suggesting it is required during the final stages of sperm tail maturation in the testis and/or epididymis, where extensive disulfide bonding of FS proteins occurs.     

Sak57, an acidic keratin initially present in the spermatid manchette before becoming a component of paraaxonemal structures of the developing tail

We have previously reported that Sak57 (for Spermatogenic cell/Sperm-associated keratin of molecular mass 57 kDa) is an acidic keratin found in rat spermatocytes, spermatids, and sperm. Sak57 displays conserved amino acid sequences found in the 1A and 2A regions of the α-helical rod domain of keratins in human, rat, and mouse. We now report indirect immunofluorescence, confocal laser scanning microscopy and immunogold electron microscopy data showing that Sak57 is associated with the microtubular mantle of the manchette, a transient microtubular structure largely regarded as formed by tubulin and microtubule-associated proteins. The immunocytochemical localization of Sak57 was detected with a polyclonal antiserum to a multiple antigenic peptide (MAP) containing an amino acid sequence known to be present in the 2A region of the α-helical rod domain. During spermiogenic steps 8–12, Sak57 immunoreactive sites were restricted to microtubular mantle of the manchette which encircles the spermatid nucleus during shaping and chromatin condensation. At later stages (spermiogenic steps 12–14), Sak57 immunoreactive sites in the spermatid head region disappeared gradually as specific immunoreactivity appeared along the already assembled axoneme of the developing spermatid tail. Immunogold electron microscopy confirmed the presence of Sak57 immunoreactivity among microtubules of the manchette and on outer dense fibers and the longitudinal columns linking the ribs of the fibrous sheath. Mature spermatids (spermiogenic step 19) displayed tails with an immunofluorescent banding pattern contrasting with the lack of Sak57 immunoreactivity in the head region. Results from this study suggest that, during early spermiogenesis, a microtubular-Sak57 scaffolding is associated with the spermatid nucleus during shaping and chromatin condensation. During late spermiogenesis, the dispersion of the manchette coincides with the progressive visualization of Sak57 in the paraaxonemal outer dense fibers and longitudinal columns of the fibrous sheath in the developing spermatid tail. © 1996 Wiley-Liss, Inc.     

FSCB, a novel protein kinase A-phosphorylated calcium-binding protein, is a CABYR-binding partner involved in late steps of fibrous sheath biogenesis

We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.     

Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa

Previously we reported the cloning of a member of the cysteine-rich secretory protein family, tpx-1, from a testis expression library using an outer dense fiber (ODF)-specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx-1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx-1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx-1 proteins is translationally delayed for 4-5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx-1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx-1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx-1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx-1 may have functional significance in the processes of sperm head development and tail function.     

Ultrastructure of spermiogenesis in a freshwater stingray, Himantura signifer

Ultrastructural changes of spermatids during spermiogenesis in a freshwater stingray, Himantura signifer, are described. Differentiation of spermatids begins with modification of the nuclear envelope adjacent to the Golgi apparatus, before the attachment of the acrosomal vesicle. A fibrous nuclear sheath extends over the nuclear surface from the site of acrosomal adherence. The conical apical acrosome is formed during nuclear elongation. At the same time, chromatin fibers shift from an initially random arrangement, assume a longitudinal orientation, and become helical before final nuclear condensation. An axial midpiece rod is formed at the posterior end of nucleus and connects to the base of the sperm tail. Numerous spherical mitochondria surround the midpiece axis. The tail originating from the posterior end of the midpiece is composed of the usual 9 + 2 axoneme accompanied by two longitudinal columns, which are equal in size and round in cross section. The two longitudinal columns are absent at the end piece. A distinctive feature of freshwater stingray sperm is its spiral configuration.     

Translation and assembly of CABYR coding region B in fibrous sheath and restriction of calcium binding to coding region A

CABYR is a highly polymorphic, sperm flagellar calcium-binding protein that is tyrosine as well as serine/threonine phosphorylated during capacitation. Six alternative splice variants of human CABYR (I-VI) have previously been identified, involving two coding regions, CR-A and CR-B, separated by an intervening stop codon. It is presently unknown if proteins encoded by the predicted coding region B of CABYR are translated during spermiogenesis, where they localize, or which CABYR isoforms bind calcium. Immunofluorescent and electron microscopic studies using polyclonal antibodies generated to the recombinant c-terminal 198 aa CABYR-B localized the isoforms containing CABYR-B to the ribs and longitudinal columns of the fibrous sheath in the principal piece of the flagellum. Antisera to recombinant CABYR-A and CABYR-B proteins recognized distinct populations of CABYR isoforms encoded by either CR-A alone and/or CR-B as well as a common population of CABYR isoforms. Only the recombinant CABYR-A and not the CABYR-B bound calcium in vitro, which is consistent with the hypothesis that CABYR-A is the only form that binds calcium in sperm. These observations confirmed that, despite the presence of the stop codon in CR-A, splice variants containing CR-B are expressed during spermiogenesis and assemble into the fibrous sheath of the principal piece; however, calcium binding occurs only to those CABYR isoforms containing CABYR-A.     

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis

During mammalian spermatogenesis, the diploid spermatogonia mature into haploid spermatozoa through a highly controlled process of mitosis, meiosis and post-meiotic morphological remodeling (spermiogenesis). Despite important progress made in this area, the molecular mechanisms underpinning this transformation are poorly understood. Our analysis of the expression and function of the putative serine–threonine kinase Fused (Fu) provides critical insight into key steps in spermatogenesis. In this report, we demonstrate that conditional inactivation of Fu in male germ cells results in infertility due to diminished sperm count, abnormal head shaping, decapitation and motility defects of the sperm. Interestingly, mutant flagellar axonemes are intact but exhibit altered periaxonemal structures that affect motility. These data suggest that Fu plays a central role in shaping the sperm head and controlling the organization of the periaxonemal structures in the flagellum. We show that Fu localizes to multiple tubulin-containing or microtubule-organizing structures, including the manchette and the acrosome–acroplaxome complex that are involved in spermatid head shaping. In addition, Fu interacts with the outer dense fiber protein Odf1, a major component of the periaxonemal structures in the sperm flagellum, and Kif27, which is detected in the manchette. We propose that disrupted Fu function in these structures underlies the head and flagellar defects in Fu-deficient sperm. Since a majority of human male infertility syndromes stem from reduced sperm motility and structural defects, uncovering Fu?s role in spermiogenesis provides new insight into the causes of sterility and the biology of reproduction.     

Developmental expression and characterization of FS39, a testis complementary DNA encoding an intermediate filament-related protein of the sperm fibrous sheath.

Proteins immunologically related to intermediate filaments have been identified in the sperm fibrous sheath but remain uncharacterized. We isolated and characterized a novel intermediate filament-related protein (FS39) localized to the fibrous sheath of the sperm tail. We used Northern blot analysis to establish that FS39 is transcribed predominantly in the testis of mice >18-20 days old. At this age, spermatogenesis has proceeded to the development of the first round haploid spermatids. In situ hybridization revealed that FS39 mRNA is first detectable in late step 3 spermatids, is at its highest level during steps 9 and 10, and diminishes in steps 13 and 14. Western blot analysis identified a single protein of 39 kDa in mouse and rat testis and epididymis, suggesting the protein is conserved in rodents. Indirect immunofluorescence localized FS39 to the fibrous sheath of the sperm tail, and in testis sections expression was detected from step 13 and step 14 spermatids onward, indicating FS39 is under translational control. Southern blot analysis showed FS39 to be a single copy gene, and hybridization to human genomic DNA suggested that a human equivalent gene is present. These results demonstrate that FS39 is transcribed in testis tissue during the haploid phase of spermatogenesis, is present in mature sperm, and codes for a novel 39-kDa intermediate filament-related protein of the fibrous sheath.     

Protein domains govern the intracellular distribution of mouse sperm AKAP4

A-kinase anchor proteins (AKAPs) spatially restrict cAMP-dependent protein kinase by tethering it to various cellular structures. In the polarized sperm cell, various compartmentalized functions, such as motility generated by the flagellum, are modulated by cAMP-dependent protein kinase. This important regulatory enzyme is associated with AKAP4, the principal component of the fibrous sheath; AKAP4 is synthesized as a precursor, pro-AKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. To define the domains responsible for the intracellular distribution and assembly of AKAP4 into a macromolecular complex, various AKAP4-green fluorescent protein (GFP) constructs were introduced into somatic cell lines. The presence of the pro domain, either alone or as part of pro-AKAP4, resulted in a diffuse cytoplasmic localization of the GFP fusion protein, suggesting that, the pro domain keeps the AKAP4 precursor unassembled in vivo until it is transported to the developing tail structure and incorporated into the fibrous sheath. When the mature AKAP4-GFP fusion protein was expressed, it localized in a punctate cytoplasmic pattern. Two domains critical for this punctate localization, T2a and T2b, are homologous to the T2-tethering domain of rat AKAP5 that is important for binding to the actin cytoskeleton in transfected HEK293 cells. In contrast to AKAP5, the distribution of AKAP4 was dependent on the microtubular cytoskeleton. The interaction of AKAP4 with the microtubular network provides evidence that the longitudinal columns of the fibrous sheath, which contain AKAP4, may interact directly with the outer microtubular doublets of the sperm axoneme.     

New insights into the assembly of the periaxonemal structures in mammalian spermatozoa

Disruption of Ube2b in the mouse has revealed that the regular and symmetric organization of the fibrous sheath of the sperm flagella is dependent on expression of the ubiquitin-conjugating enzyme UBE2B. These data could cast light on how a component of the ubiquitin-proteasome pathway participates in the assembly of flagellar periaxonemal structures. Data in the literature support the notion of involvement of ubiquitin-proteasome pathways in the assembly of cytoskeletal components in somatic cells. This review attempts to integrate recent knowledge regarding flagellar components that could be related to proteasome components and, therefore, could be targets of UBE2B in the spermatid. An attempt is made to characterize the human flagellar anomalies of infertile patients, which are the closest to those of Ube2b-deficient mice. These new insights regarding the assembly of mammalian sperm flagella provide a basis for studying the ontogenesis of flagellar accessory structures and suggest leads for medical and genetic investigations.     

Identification of a protein in the fibrous sheath of the sperm flagellum

The fibrous sheath is a unique cytoskeletal component in the principal-piece segment of the mammalian sperm flagellum. Monoclonal antibody ATC was shown by indirect immunofluorescence (IIF) to bind to the principal piece of the flagellum of permeabilized mouse, rat, and hamster sperm, but not to that region of guinea pig, rabbit, or human sperm. IIF on isolated fibrous sheaths confirmed that the antigen was present in the fibrous sheath of mouse, rat, and hamster sperm. On Western blots of mouse spermatozoa, ATC identified a relatively insoluble major antigen with an apparent molecular weight of 67,000 (Mr 67,000). Hamster sperm fibrous sheaths contain an antigen of Mr 66,000, while rat sperm fibrous sheaths contain an antigen of Mr 65,500. The antigen was first detected in late spermatids, as determined by immunohistochemical procedures on sections of mouse, rat, and hamster testis. The antigen was not detected on Western blots of mouse brain, kidney, liver, or thymus. These results indicate that ATC recognizes a protein integral to the fibrous sheath of the principal piece of sperm detected by immunohistochemistry late in spermiogenesis that is probably restricted to the male germ cell line.     

Spermiogenesis and spermatozoon ultrastructure of the dilepidid cestode Molluscotaenia crassiscolex (von Linstow, 1890), an intestinal parasite of the common shrew Sorex araneus

Marigo, A.M., Bâ, C.T. and Miquel, J. 2011. Spermiogenesis and spermatozoon ultrastructure of the dilepidid cestode Molluscotaenia crassiscolex (von Linstow, 1890), an intestinal parasite of the common shrew Sorex araneus. —Acta Zoologica (Stockholm) 92 : 116–125. Spermiogenesis in Molluscotaenia crassiscolex begins with the formation of a differentiation zone containing two centrioles. One of the centrioles develops a flagellum directly into the cytoplasmic extension. The nucleus elongates and later migrates along the spermatid body. During advanced stages of spermiogenesis, a periaxonemal sheath appears in the spermatid. Spermiogenesis finishes with the appearance of a single helicoidal crested body at the base of the spermatid and, finally, the narrowing of the ring of arched membranes causes the detachment of the fully formed spermatozoon. The mature spermatozoon of M. crassiscolex exhibits a partially detached crested body in the anterior region of the spermatozoon, one axoneme, twisted cortical microtubules, a periaxonemal sheath, and a spiralled nucleus. The anterior spermatozoon extremity is characterized by the presence of an electron‐dense apical cone and a single spiralled crested body, which is attached to the sperm cell in the anterior and posterior areas of region I, whereas in the middle area it is partially detached from the cell. This crested body is described for the first time in cestodes. The posterior extremity of the male gamete exhibits only the disorganizing axoneme. Results are discussed and compared particularly with the available ultrastructural data on dilepidids sensu lato.     

AN ULTRASTRUCTURAL STUDY OF PLANT SPERMATOGENESIS : Spermatogenesis in Nitella

Spermatogenesis in the charophyte Nitella has been followed in antheridia prepared for light and electron microscopy. The antheridial filament cells contain paired centrioles which are similar in structure and behavior to the centrioles of animal cells. In the early spermatid, the centrioles undergo an initial elongation at their distal ends and become joined by a spindle-shaped fibrous connection. At the same time, their proximal ends are closely associated with the development of a layer of juxtaposed microtubules which will form the microtubular sheath. The architectural arrangement of these microtubules suggests that they constitute a cytoskeletal system, forming a framework along which the mitochondria and plastids become aligned and along which the nucleus undergoes extensive elongation and differentiation. The microtubular sheath persists in the mature sperm. During mid-spermatid stages, the centrioles give rise to the flagella and concomitantly undergo differentiation to become the basal bodies. The Golgi apparatus goes through a period of intensive activity during mid-spermatid stages, then decreases in organization until it can no longer be detected in the late spermatid. An attempt is made to compare similarities between plant and animal spermiogenesis.     

黄毛鼠精子尾部主段超微结构

黄毛鼠精子尾主段具有纤维鞘。纤维鞘具背腹两条纵柱和环肋。纤维鞘之内分布着从中段延伸下来的外周致密纤维。在主段的近中段端,有９条外周致密纤维。随后,外周致密纤维逐渐变细,且逐一终止,在９条外周致密纤维中,最早终止的是Ｆ８,随后的终止顺序是Ｆ３、Ｆ４、Ｆ７、Ｆ２、Ｆ９、Ｆ５、Ｆ６、Ｆ１。在主段的近末段端,没有外周致密纤维。根据外周致密纤维的数量,可以将主段分为１０个区域。从近中段端至近末段端,这１０个     

The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis

Fertilization in mammals occurs via a series of well-defined events in the secluded environment of the female reproductive tract. The mode of selection of the fertilizing spermatozoon nevertheless remains unknown. As has become evident during in vitro fertilization by sperm microinjection into the oocyte, abnormal spermatozoa can successfully fertilize oocytes. Under these extreme conditions, post-fertilization events, early embryonic development and implantation are significantly compromised indicating that the contribution of spermatozoa extends beyond sperm penetration. Microscopic identification of normal spermatozoa is a well-standardized procedure but insights into the mechanisms that lead to aberrant sperm differentiation and into the subcellular nature of sperm abnormalities have only recently begun to be obtained. The spermatozoon is the result of a complex development in which spermatid organelles give rise to various structural components with characteristic functions. Similar to other differentiated cells, the spermatozoon has a specific pathology that is most clearly identified by ultrastructural evaluation coupled with immunocytochemistry and molecular techniques. This multidisciplinary approach allows the precise characterization of sperm abnormalities, including structural, molecular and functional aspects. We summarize here studies of the physiopathology of spermiogenesis in two abnormal sperm phenotypes of infertile men: dysplasia of the fibrous sheath and acephalic spermatozoa/abnormal head-tail attachment. The characterization of the abnormalities of the tail cytoskeleton and centrioles has uncovered aspects of the subcellular basis of pathological spermiogenesis, has suggested experimental approaches to explore the nature of these anomalies and has opened the way for genetic studies that will ultimately lead to the design of the therapeutic tools of the future.     

优雅蝈螽精子形成过程中F-肌动蛋白的动态变化

为阐明F-肌动蛋白在优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl精子形成过程中的动态变化, 本研究利用微分干涉相衬技术和免疫荧光技术首次对优雅蝈螽精子形成过程中的F-肌动蛋白进行了细胞定位, 利用透射电镜技术从超微水平观察了优雅蝈螽精子顶体复合体的结构。结果显示： 精子形成早期, F-肌动蛋白富集于亚顶体区域, 形态由“球状”转变为“棒锥状”; 精子形成中期, F-肌动蛋白呈“倒Y型”分布于亚顶体区域和细胞核前端两侧; 精子形成后期, 亚顶体区域的F 肌动蛋白解聚消失, F-肌动蛋白呈“箭头状”, 仅分布于顶体复合体扩张的两翼中。F-肌动蛋白动态变化伴随着细胞核和精子头部的形态改变, F-肌动蛋白的动态装配在精子顶体复合体形态构建和细胞核的形变中起着重要的作用。本研究还发现未成熟的精子尾部有一些富含F-肌动蛋白的细胞质微滴, 与精子形成过程中多余细胞质和细胞器的外排有关。F-肌动蛋白的动态变化研究为进一步阐明细胞骨架蛋白在昆虫精子形成过程中的功能和作用机制奠定了基础。     

Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed

The completion of spermiogenesis requires condensation of the haploid spermatid genome. This task is accomplished in a gradual and relentless manner by first erasing the nucleosomal organization of chromatin while the DNA is protected by transient nuclear proteins TP1 and TP2. Then, the more permanent protamines come into play to stabilize the spermatid genome until fertilization occurs. Mice lacking TPI manage to produce relatively structurally normal sperm, although fertility is reduced and chromatin condensation is abnormal despite the compensatory expression of TP2. TP1 and TP2 appear to have the house-keeping function of reestablishing continuity when chromatin breaks take place during the remodeling process. DNA single-strand breaks are frequently observed when spermiogenesis is half completed. There is a temporal relationship between TP1 and DNA breaks: TP1 nuclear levels increase and the frequency of DNA breaks become less prominent as spermiogenesis is reaching completion. TP1 seems to hold the broken ends together until an as-yet-unidentified ligase bridges the gap.     

Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re‐evaluated.