Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida,Cotylea)

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + “1” micro‐tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron‐dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread‐like chromatin mixed with electron‐dense intranuclear spindle‐shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron‐dense bodies may provide characters useful for phylogenetic analysis.     

Ultrastructure of Spermatogenesis in the Testis of Paragonimus heterotremus

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.     

Generation of flagella by cultured mouse spermatids

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.     

Differentiation of binuclear spermatozoa in mice

In an electron microscopy study of abnormal spermatogenesis in mice, we have found that two discrete haploid nuclei may be located in a single spermatid cytoplasm after the second meiotic division. The spermatid continues to differentiate and forms a binucleate spermatozoon with both nuclei separately packaged within the sperm head. The Golgi apparatus of the double spermatid forms a single proacrosome that attaches to both nuclei. Apparently, one acrosomal structure differentiates to cover and compartmentalize the two haploid nuclei within the sperm head. Chromatin condensation appears normal. The head morphology and number of flagella vary in mature spermatozoa produced by this process. This work demonstrates one pathway by which polyploid spermatids continue to differentiate to spermatozoa after failure of cytoplasmic division or possibly cellular fusion.     

The restructuring of the flagellar base and the flagellar necklace during spermatogenesis of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Transmission electron microscopy was used to study the development of the flagellar base and the flagellar necklace during spermatogenesis in a moth (Ephestia kuehniella Z.). Until mid-pachytene, two basal body pairs without flagella occur per cell. The basal bodies, which contain a cartwheel complex, give rise to four flagella in late prophase I. The cartwheel complex appears to be involved in the nucleation of the central pair of axonemal microtubules. In spermatids, there is one basal body; this is attached to a flagellum. At this stage, the nine microtubular triplets of the basal body do not terminate at the same proximal level. The juxtanuclear triplets are shifted distally relative to the triplets distant from the nuclear envelope. Transition fibrils and a flagellar necklace are formed at the onset of axoneme elongation. The flagellar necklace includes Y-shaped elements that connect the flagellar membrane and the axonemal doublets. In spindle-containing spermatocytes, the flagellar necklace is no longer detectable. During spermatid differentiation, the transition fibrils move distally along the axoneme and a prominent middle piece appears. Our observations and those in the literature indicate certain trends in sperm structure. In sperms with a short middle piece, we expect the presence of a flagellar necklace. The distal movement of the transition fibrils or equivalent structures is prevented by the presence of radial linkers between the flagellar membrane and the axonemal doublets. On the other hand, the absence of a flagellar necklace at the initiation of spermiogenesis enables the formation of a long middle piece. Thus, in spermatozoa possessing an extended middle piece, a flagellar necklace may be missing.     

Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision

Reunov, A.A., Yurchenko, O.V., Alexandrova, Y.N. and Radashevsky, V.I. 2009. Spermatogenesis in Boccardiella hamata (Polychaeta: Spionidae) from the Sea of Japan: sperm formation mechanisms as characteristics for future taxonomic revision. —Acta Zoologica (Stockholm) 91 : 477–456. To characterize novel features that will be useful in the discussion and validation of the spionid polychaete Boccardiella hamata from the Sea of Japan, the successive stages of spermatogenesis were described and illustrated. Spermatogonia, spermatocytes and early spermatids are aflagellar cells that develop synchronously in clusters united by a cytophore. At the middle spermatid stage, the clusters undergo disintegration and spermatids produce flagella and float separately in coelomic fluid as they transform into sperm. Spermatozoa are filiform. The ring‐shaped storage platelets are located along the anterior nuclear area. The nucleus is cupped by a conical acrosome. A nuclear plate is present between the acrosome and nucleus. The nucleus is a cylinder with the implantation fossa throughout its length and with the anterior part of the flagellum inside the fossa. There is only one centriole, serving as a basal body of the flagellum, situated in close vicinity of the acrosomal area. A collar of four mitochondria is located under the nuclear base. The ultrastructure of B. hamata spermatozoa from the Sea of Japan appears to be close to that of B. hamata from Florida described by Rice (Microscopic Anatomy of Invertebrates, Wiley‐Liss, Inc., New York, 1992), suggesting species identity of the samples from the two regions. However, more detailed study of Florida’s B. hamata sperm is required for a reliable conclusion concerning the similarity of these two polychaetes. In addition to sperm structure, features such as the cytophore‐assigned pattern of spermatogenic cell development, the synchronous pattern of cell divisions, the non‐flagellate early spermatogenic stages, and the vesicle amalgamation that drives meiotic cell cytokinesis and spermatid diorthosis will likely be useful in future testing of the validity of B. hamata and sibling species throughout the world.     

Spermiogenesis in three species of cicadas (Hemiptera: Cicadidae)

Spermiogenesis in three species of cicadas representing one cicadettine (Monomatapa matoposa Boulard) and two cicadines (Diceroprocta biconica [Walker] and Kongota punctigera [Walker]) was investigated by light and electron microscopy. Although spermiogenesis was occurring in the testis of adult males of all species, earlier spermiogenic stages were observed in D. biconica only. While spermiogenesis was similar to that described for other insects, some differences were noted. For example granular material did not assemble around the centriole to form a centriolar adjunct but did accumulate in the cytoplasm of early spermatids adjacent to a region of the nuclear membrane where nuclear pores were aggregated. In late spermatids this material accumulated anterior to the mitochondrial derivatives in a developing postero‐lateral nuclear groove. While this material has been named the ‘centriolar adjunct’ by previous authors, its formation away from the centriole raises questions about its true identity. Second, during acrosome maturation an ante‐acrosomal region of cytoplasm develops. Although present in later spermatids, this region is lost in spermatozoa. Interspecific variations in chromatin condensation patterns and the number of microtubule layers encircling the spermatid nucleus during spermiogenesis were noted.     

Spermiogenesis and spermatozoon ultrastructure of the davaineid cestode Raillietina micracantha (Fuhrmann, 1909)

Miquel, J., Torres, J., Foronda, P. and Feliu, C. 2010. Spermiogenesis and spermatozoon ultrastructure of the davaineid cestode Raillietina micracantha. — Acta Zoologica (Stockholm) 91 : 212–221 The spermiogenesis and the ultrastructural organization of the spermatozoon of the davaineid cestode Raillietina micracantha are described by means of transmission electron microscopy. Spermiogenesis begins with the formation of a zone of differentiation containing two centrioles. One of the centrioles develops a free flagellum that later fuses with a cytoplasmic extension. The nucleus migrates along the spermatid body after the proximodistal fusion of the flagellum and the cytoplasmic extension. During advanced stages of spermiogenesis a periaxonemal sheath and intracytoplasmic walls appear in the spermatids. Spermiogenesis finishes with the appearance of two helicoidal crested bodies at the base of spermatids and, finally, the narrowing of the ring of arched membranes detaches the fully formed spermatozoon. The mature spermatozoon of R. micracantha is a long and filiform cell, tapered at both ends, which lacks mitochondria. It exhibits two crested bodies of different lengths, one axoneme of the 9 + ‘1’ pattern of trepaxonematan Platyhelminthes, twisted cortical microtubules, a periaxonemal sheath, intracytoplasmic walls, granules of glycogen and a spiralled nucleus. The anterior extremity of the spermatozoon is characterized by the presence of an electron‐dense apical cone and two spiralled crested bodies while the posterior extremity of the male gamete exhibits only the axoneme and an electron‐dense posterior tip.     

Spermatogenesis of the spider crab Maja brachydactyla (Decapoda: Brachyura)

This study describes spermatogenesis in a majid crab (Maja brachydactyla) using electron microscopy and reports the origin of the different organelles present in the spermatozoa. Spermatogenesis in M. brachydactyla follows the general pattern observed in other brachyuran species but with several peculiarities. Annulate lamellae have been reported in brachyuran spermatogenesis during the diplotene stage of first spermatocytes, the early and mid‐spermatids. Unlike previous observations, a Golgi complex has been found in mid‐spermatids and is involved in the development of the acrosome. The Golgi complex produces two types of vesicles: light vesicles and electron‐dense vesicles. The light vesicles merge into the cytoplasm, giving rise to the proacrosomal vesicle. The electron‐dense vesicles are implicated in the formation of an electron‐dense granule, which later merges with the proacrosomal vesicle. In the late spermatid, the endoplasmic reticulum and the Golgi complex degenerate and form the structures–organelles complex found in the spermatozoa. At the end of spermatogenesis, the materials in the proacrosomal vesicle aggregate in a two‐step process, forming the characteristic concentric three‐layered structure of the spermatozoon acrosome. The newly formed spermatozoa from testis show the typical brachyuran morphology. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Ultrastructure of spermiogenesis and spermatozoa in Prorhynchus sp. (Platyhelminthes,Lecithoepitheliata, Prorhynchidae)

Summary

Mature sperm of Prorhynchus sp. have an elongated nucleus, multiple mitochondria and dense bodies, and two free axonemes which are located in grooves of the main shaft for much of their length. The axonemes are subterminally inserted and have the typical 9+ ‘1’ arrangement unique to Platyhelminthes and synapomorphic for taxa of Trepaxonemata. The testis follicles examined had small numbers of developing spermatids and very few mature sperm were present. During spermiogenesis, spermatids remain joined in clusters by distinctive bridges. In each spermatid two centrioles (with an intercentriolar body between them) give rise to free axonemes which grow out in opposite directions from each other. Indistinct ciliary rootlets are present. The axonemes are carried distally from the main spermatid mass on an elongating process and turn back towards the main spermatid mass. Nucleus, mitochondria and dense bodies move into the shaft, and the spermatid elongates before detaching from others in the cluster. This is the first detailed study of sperm and spermiogenesis in Lecithoepitheliata. Mature sperm are distinctly different from those of prolecithophorans, to which they are reputedly related, the latter having aflagellate sperm without dense bodies.     

Ultrastructure of sperm development in the free-living marine nematode Metachromadora itoi (Chromadoria, Desmodorida)

The spermatogenesis of the free‐living marine nematode Metachromadora itoi was studied with electron microscopy. Spermatocytes and early spermatids have no cytoplasmic components specific for nematodes, i.e. membranous organelles (MO) and fibrous bodies (FB). The late spermatids are subdivided into the residual body and the main cell body with a centrally located nucleus devoid of a nuclear envelope. A pair of 9 × 2 centrioles is associated with the nuclei of spermatids and spermatozoa. The nucleus of the mature spermatid is surrounded by a thick mass of radially arranged FB delimited externally by a discontinuous layer of mitochondria, which underlie a thin ectoplasm. Sperm development is accompanied by transfer of FB matter through the mitochondrion layer into the ectoplasm. The immature spermatozoa from the testis have the centrally located nucleus surrounded by a transparent halo with remnants of FB. The halo is delimited by a sphere of mitochondria that underlie the thick fibrous ectoplasm, a derivative of the FB. In the mature spermatozoa the ectoplasm is transformed into the prominent unpolarized pseudopod. The central nucleus is surrounded by a transparent halo and a sphere of mitochondria, which underlie the pseudopod. MO were not found throughout spermatogenesis. In general, spermatogenesis in M. itoi differs from that observed in many nematodes but resembles in some details the sperm development in some chromadorid and tylenchomorph nematodes. The phylogenetic importance of this sperm development is discussed.     

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.     

Fine structure of spermiogenesis with special reference to the spermatid morulae of the freshwater mussel Prisodon alatus (Bivalvia,Unionoidea)

Within the testicular cysts of the mussel Prisodon alatus are numerous somatic host cells described as Sertoli cells (SC), each containing a variable number of young spermatid morulae. Among them, several free spermatid morulae, spermatids, and spermatozoa were observed. Each free spermatid morula is surrounded by an external membrane. The early spermatids enclosed within the morulae have dense and homogeneous chromatin, and the cytoplasm occupies little space around the nucleus. Later, during spermiogenesis, the SC show lysis and disrupt to liberate the spermatid morulae. The membrane of the free morula is then disrupted, releasing the young spermatids. The SC disappear just after the appearance in the testis of a large number of free young spermatids. The nucleus of each free spermatid becomes gradually smaller and denser by the appearance of a granular pattern of condensed chromatin. During the maturation phase of the spermatids, the cytoplasm becomes more voluminous, and mitochondria and centrioles are more evident. Then, flagellogenesis occurs, and the nucleus gradually condenses into thicker strands. In the mature sperm, the apical zone has a disc-shaped acrosomal vesicle and the midpiece contains five mitochondria and two centrioles located at the same level. The flagellum has the common 9+2 microtubular pattern. The results are discussed with particular reference to Sertoli cells and clusters of spermatid morulae with those of species of closely related taxa in the bivalves. J. Morphol. 238:63–70, 1998. © 1998 Wiley-Liss, Inc.     

Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda)

Levron, C., Yoneva, A. and Kalbe, M. 2011. Spermatological characters in the diphyllobothriidean Schistocephalus solidus (Cestoda). —Acta Zoologica (Stockholm) 00 : 1–8. The spermiogenesis and the mature spermatozoon of Schistocephalus solidus (Cestoda: Diphyllobothriidea) are described using transmission electron microscopy. Spermiogenesis in S. solidus begins with the formation in the spermatid of a differentiation zone surrounded by cortical microtubules and delimited by arching membranes. This conical area presents two centrioles associated with striated rootlets and a median cytoplasmic extension between them. The centrioles are separated by an intercentriolar body composed of three electron‐dense plates dividing four electron‐lucent plates. The centrioles give rise to two flagella that undergo a rotation and later fuse proximodistally with the median cytoplasmic expansion. The presence of an electron‐dense material in the distal part of the differentiation zone is observed in the early stage of spermiogenesis. This pattern corresponds to Type I spermiogenesis according to the classification proposed by Bâ and Marchand (Mémoires du Muséum National d’Histoire Naturelle 1995; 166 : 87). The mature spermatozoon of S. solidus presents the Type I pattern defined by Levron et al. (Biological Reviews 2010; 85 : 523). It consists of five regions that exhibit two axonemes, parallel cortical microtubules, nucleus and electron‐dense zones. The anterior tip of the spermatozoon possesses only a few singlets. The axonemes are of a 9 + ’1’ trepaxonematan pattern and do not reach the posterior extremity of the mature spermatozoon.     

Autophagy regulates spermatid differentiation via degradation of PDLIM1

Spermiogenesis is a complex and highly ordered spermatid differentiation process that requires reorganization of cellular structures. We have previously found that Atg7 is required for acrosome biogenesis. Here, we show that autophagy regulates the round and elongating spermatids. Specifically, we found that Atg7 is required for spermatozoa flagella biogenesis and cytoplasm removal during spermiogenesis. Spermatozoa motility of atg7-null mice dropped significantly with some extra-cytoplasm retained on the mature sperm head. These defects are associated with an impairment of the cytoskeleton organization. Functional screening revealed that the negative cytoskeleton organization regulator, PDLIM1 (PDZ and LIM domain 1 [elfin]), needs to be degraded by the autophagy-lysosome-dependent pathway to facilitate the proper organization of the cytoskeleton. Our results thus provide a novel mechanism showing that autophagy regulates cytoskeleton organization mainly via degradation of PDLIM1 to facilitate the differentiation of spermatids.     

FINE STRUCTURAL CHANGES IN THE FLAGELLUM OF THE SPERMATID IN EXPERIMENTAL CRYPTORCHIDISM OF THE RAT

Rat testes were confined to the abdominal cavity by operation. After 1 to 26 days they were excised, fixed with osmium tetroxide, sectioned, and examined with the electron microscope. Changes in the axial filament complex of the spermatid flagellum appeared 2 days after operation, and the arrangement of filaments in the middle- and main pieces of some spermatid tails was disordered as compared to the 9 + 2 filament arrangement in the tails of the control spermatids and in other flagella and cilia. In cross-sections, the filaments in the experimental material were nine or less in number, and each of them was single and dense. Occasionally some were double, and in those instances one filament was dense and the other was light and tubular. The central filaments were obscure. In longitudinal sections,the filaments were not parallel to the main axis of the flagella or to each other. It was assumed that the central filaments were more sensitive to the experimental conditions than the peripheral pairs of filaments. Furthermore, the light filaments of the peripheral pairs were more sensitive than the dense filaments. Besides the axial filament complex, the fibrous sheath which surrounds it in the main piece was also changed. The plasma membrane of the changed flagella disappeared or became fragmented.     

A comparison of the annual changes in testicular activity and serum androgen levels between the early and delayed maturing groups of male Cottus hangiongensis

Synopsis Annual changes in testicular activity and concentration of two serum androgens were monitored in two groups of the river-sculpin Cottus hangiongensis collected from the upper and lower reaches of a river at southern Hokkaido, Japan. One of them (early maturing group) underwent testicular maturation with aberrant spermatids and spermatid masses produced during the reproductive cycle. Moreover, regular seasonal changes in serum testosterone and 11-ketotestosterone concentrations were observed. On the other hand, in the other group (delayed maturing group), although body size of the fish was large enough to undergo reproduction, annual changes in gonadosomatic index and testicular activity did not vary much. During the months of active testicular development in the early maturing group, spermatogenesis was observed to begin in some regions of the testes of delayed maturing fish, but always resulted in the formation of aberrant spermatids and spermatid masses. Moreover, concentration of serum androgens did not significantly vary throughout the year. Results suggest that low androgen production is a proximal factor for delayed sexual maturity in the delayed maturing group, and that the occurrence of aberrant spermatids and spermatid masses during spermatogenesis is not linked to the delayed maturity.     

Ultrastructural study of spermatogenesis in<Emphasis Type="Italic"> Phoronopsis harmeri</Emphasis> (Lophophorata,Phoronida)

The process of sperm development in Phoronopsis harmeri was studied by electron microscopy. Developing spermatogenical cells are aggregated around the capillaries of the haemal plexus. The spermatogonia, which are situated around the capillary walls of the caeca, are remarkable for the presence of germ-line vesicles and contain their centrioles near the cell membrane. The spermatocytes and spermatids are flagellated cells arranged in clusters. During spermiogenesis the basal body/flagellum complex migrates to the apical pole of the spermatid. The acrosome-like structure arises from material produced by the Golgi complex. It lacks a surrounding membrane and has a fibrillar content. The nucleus elongates and the condensation of chromatin is caused by an activation of 'initiation centres'. The late spermatid and the spermatozoon appear as two-armed 'V'-shaped cells in which one arm contains the nucleus and posteriorly located mitochondria, and the other one is the axoneme. Spermatogenesis of P. harmeri is an interesting example of gamete differentiation where advanced sperm structure is combined with a plesiomorphic pattern of sperm development characterized as 'flagellate spermatogenesis'. Communicated by H.-D. Franke     

Les éléments cytoplasmiques au cours de la spermiogenèse du TritonPleurodeles waltlii Michah

Summary The development of the different components of thePleurodeles spermatozoon neck region have been investigated and described from the early beginning spermiogenesis process. In some cases their origine is not well defined. Some dictyosomes remain in a constant relationship with the pericentriolar granule and the two different rings, even after they had left the acrosomal region.The development of the neck in a deep postnuclear niche, does not seem related with the centrioles. The nuclear origine of some basic proteins of the neck is discussed in relation to the close correspondence between their appearance and the substitution of the lysine rich histone by the arginine rich histone in the nuclear. The various morphologic signs of transit at the nuclear envelope level have been investigated. The ways along which some materials is eventually transferred out of the nucleus remained an open question. ThePleurodeles spermatid neck region is compared with the same region of the insect and mammalian sperm.Some components of thePleurodeles young spermatids display common characters with the mammalian chromatoid body. Their development are fairly similar. Thus the Urodele spermatid seems to posses a chromatoîd body like most other vertebrate spermatids.

Equipe de Recherche Associée au C.N.R.S., E.R.A. n^o 129.