Immunolocalization of apelin receptor (APJ) in mouse seminiferous epithelium.

The apelin receptor (APJ) belongs to the member of the G protein-coupled receptor family, and expression of APJ has been reported in the different cell types of testis. The seminiferous tubules in the testis can be identified as different stages (I-XII). It has been also suggested that different factors could be expressed in stage and cell-specific manner in the seminiferous tubules. Recently, we also shown that expression of APJ is developmentally regulated in the testis from PND1 to PND42. Therefore, we analyzed the expression of APJ in the testis of adult mice by immunohistochemistry. Immunohistochemistry showed that the APJ was highly specific for the round and elongated spermatids with stage-dependent changes. The seminiferous tubules at stages I-VII showed APJ immunostaining in the spermatid steps 1-8, not steps of 13-16. The seminiferous tubules at stages IX-XII showed APJ immunostaining in the spermatid steps 9-12. These results suggested the possible role of APJ in the spermiogenesis process. The intratesticular administration of APJ antagonist, ML221 showed a few round spermatids in the seminiferous tubules and some of the tubules with complete absence of round spermatid. Overall, we present evidence that APJ expression in spermatid is dependent on the stages of the seminiferous epithelium cycle and APJ could be involved in the differentiation of round spermatid to elongated spermatid.

Three-dimensional analysis and in vivo imaging for sperm release and transport in the murine seminiferous tubule.

Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-µm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid-Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells. In brief: Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells.

Spermatogenesis in the Roborovski hamster (Phodopus roborovskii) and the Chinese hamster (Cricetulus griseus).

BACKGROUND: Spermatogenesis is an elaborately organized and tightly regulated differentiation process. The spermatogenesis duration is stable within a certain species but highly variable between species of the same family. OBJECTIVES: In this study, the spermatogenesis duration of the Roborovski hamster was measured for the first time, and the spermatogenesis duration of the Chinese hamster was re-assessed. MATERIALS AND METHODS: Stage classification and cycle length measurement were carried out by labeling the dividing cells with bromodeoxyuridine and an antibody-based chromogen as well as with the periodic acid-Schiff/hematoxylin stain. Analysis was conducted using reference calculation and linear regression. Morphological measurements completed our set of methods. RESULTS: The mean duration of one seminiferous epithelium cycle was 8.58 ± 0.34 days (mean ± SEM; Phodopus roborovskii) and 16.59 ± 0.47 days (Cricetulus griseus) based on the reference calculation. Slightly higher results were obtained using linear regression analysis: 9.72 ± 0.41 days for P. roborovskii and 17.64 ± 0.61 days for C. griseus. Additionally, a newly developed exemplary flowchart was proposed for the Roborovski hamster to facilitate spermatogenesis stage classification also in other species. The Chinese hamster presented an unexpectedly high paired epididymides weight of 1.701 ± 0.046 g (mean ± SEM) although having a body weight of only 40.5 ± 0.7 g. However, no significant correlation between the relative epididymis weight and spermatogenesis duration in mammals (Spearman rank correlation: r = -0.119, p = 0.607, n = 21) or rodents could be found (r = 0.045, p = 0.903, n = 11). CONCLUSION: Our data emphasize the stability of the spermatogenesis duration within species and its remarkable variability between species. Further research is needed to identify the principal mechanisms and selection drivers that are responsible for such stability within species and the variability between species.

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator: lesson from the testis.

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood-testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell-cell and also Sertoli-spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

Effects of a New Benzimidazole Derivative with Actoprotective Properties on Spermatogenic Epithelium of the Testes and Fertility of Male White Rats.

The effects of a new derivative of benzimidazole (K-134) in doses of 5 and 50 mg/kg on the spermatogenesis and fertilizing ability of spermatozoa were studied on male rats. It was found that 2-month course treatment with the studied substance enhances the producing ability of the spermatogenic epithelium and improves fertilizing ability of spermatozoa.

Duration of the seminiferous epithelium cycle in the frugivorous bat Artibeus lituratus.

The seminiferous epithelium goes through multiple changes which enables the differentiation of a spermatogonia in a fully mature spermatozoon. The timing of these changes is species-specific and influences the duration of the reproductive cycles. Bats are among wild mammals whose coordination between male and female reproductive cycles are imperative, since most females show seasonal preferences, even in the Tropics. This seasonal variation demands constant sperm production ready for spermiation in order to guarantee its genetic dispersion and reproduction success. Despite their abundance, little is known about the duration of reproductive cycles in Neotropical bat species, a relevant information for the species management and for conservational strategies regarding anthropogenic and climate influences on bats reproduction. In this study, we aimed at characterizing the stages of the seminiferous epithelium cycle (SEC) of the fruit bat Artibeus lituratus and to determine its duration based on the immunohistochemical analysis of the bromodeoxyuridine (BrDU) activity. SEC stages were characterized according to the tubular morphology method and the frequency of each stage was estimated. After intratesticular injections of BrDU, the animals were euthanized at different times, and the estimation of SEC duration was performed by observing the most advanced germ cells in the seminiferous epithelium. The most advanced stained cells after 2 days of BrdU injection were the primary spermatocytes in pachytene, transitioning from stages 1-2 of the SEC. Within 2 days, we found a progression of 30.42% of the SEC, and an entire cycle lasted 6.58 days on average. Considering that 4.5 seminiferous epithelium cycles are necessary for the whole spermatogenic processes to be completed, the total length of spermatogenesis in A. lituratus was estimated at 29.61 days. Our findings support a pattern of bimodal seasonal polyestry for this species, with rapid spermatogenic cycles.

Seasonal variation in sperm freezability associated with changes in testicular germinal epithelium in domestic (Ovis aries) and wild (Ovis musimon) sheep.

The aim of this study was to examine ovine sperm cryoresistance during the rutting season (RS) and its association with sperm head area and seminiferous epithelium proliferation. Small ruminants show fluctuating testosterone levels throughout the year, which could interfere with spermatogenesis and sperm cryopreservation. Ejaculates, testicular biopsies and blood were collected during the middle and at the end of the RS (Middle-RS vs End-RS) during periods of high and low testosterone levels in Merino and Mouflon rams. Fresh and frozen-thawed sperm quality, sperm morphometry, seminiferous tubule morphometry and testicular proliferation markers (proliferating cell nuclear antigen, proliferation marker protein Ki-67 and transcription factor GATA-4) were evaluated. Post-thaw sperm viability was higher in the End-RS group in both Merino (69.9±8.2 vs 41.6±7.3%; P=0.020) and Mouflon rams (40.9±3.3 vs 24.2±5.0%; P=0.008). Mouflons had larger sperm head area at the End-RS (38.3±0.2 vs 34.3±0.1µm2; P=0.029), whereas there was no difference between Merino groups (35.7±0.5 vs 34.8±1.0µm2). Seminiferous tubule morphometry and proliferation markers showed higher levels of germinal epithelium proliferation in the Middle-RS of both species. In conclusion, sperm freezability is affected during the RS in domestic and wild rams, which could be correlated with changes that occur during spermatogenesis, since there is an effect of season on cell proliferation in the testis.

Myosin VIIa Supports Spermatid/Organelle Transport and Cell Adhesion During Spermatogenesis in the Rat Testis.

The biology of transport of spermatids and spermatid adhesion across the seminiferous epithelium during the epithelial cycle remains largely unexplored. Nonetheless, studies have implicated the role of motor proteins in these cellular events. In this article, we report findings to unravel the role of myosin VIIa, an F-actin-based barbed (+)-end-directed motor protein, to support cellular transport and adhesion in the testis. Using RNA interference to knock down myosin VIIa in Sertoli cells cultured in vitro as a study model was shown to perturb the Sertoli cell tight junction permeability barrier, mediated through disorganization of actin- or microtubule (MT)-based cytoskeletons owing to disruptive changes on the spatiotemporal expression of F-actin or MT-regulatory proteins. Consistent with these in vitro findings, knockdown of myosin VIIa in the testis in vivo also induced disorganization of the actin- and MT-based cytoskeletons across the seminiferous epithelium, mediated by disruptive changes in the spatiotemporal expression of actin- and MT-based regulatory proteins. More important, the transport of spermatids and organelles across the epithelium, as well as cell adhesion, was grossly disrupted. For instance, step 19 spermatids failed to be transported to the adluminal compartment near the tubule lumen to undergo spermiation; in this manner, step 19 spermatids were persistently detected in stage IX and XII tubules, intermingling with step 9 and 12 spermatids, respectively. Also, phagosomes were detected near the tubule lumen in stage I to III tubules when they should have been degraded near the base of the seminiferous epithelium via the lysosomal pathway. In summary, myosin VIIa motor protein was crucial to support cellular transport and adhesion during spermatogenesis.

Morphofunctional evaluation of the testis, duration of spermatogenesis and spermatogenic efficiency in the Japanese fancy mouse (Mus musculus molossinus).

Japanese fancy mouse, mini mouse or pet mouse are common names used to refer to strains of mice that present with different colour varieties and coat types. Although many genetic studies that involve spotting phenotype based on the coat have been performed in these mice, there are no reports of quantitative data in the literature regarding testis structure and spermatogenic efficiency. Hence, in this study we researched testis function and spermatogenesis in the adult Japanese fancy mouse. The following values of 68 ± 6 mg and 0.94 ± 0.1% were obtained as mean testis weight and gonadosomatic index, respectively. In comparison with other investigated mice strains, the fancy mouse Leydig cell individual size was much smaller, resulting in higher numbers of these cells per gram of testis. As found for laboratory mice strains, as a result of the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle have been described in this study. The combined frequencies of pre-meiotic and post-meiotic stages were respectively 24% and 64% and very similar to the laboratory mice. The more differentiated germ cell types marked at 1 h or 9 days after tritiated thymidine administration were preleptotene/leptotene and pachytene spermatocytes at the same stage (VIII). The mean duration of one spermatogenic cycle was 8.8 ± 0.01 days and the total length of spermatogenesis lasted 37.8 ± 0.01 days (4.5 cycles). A high number of germ cell apoptosis was evident during meiosis, resulting in lower Sertoli cell and spermatogenic efficiencies, when compared with laboratory mice strains.

Spermatogenic dynamics of the spiny rat Kannabateomys amblyonyx (Wagner, 1845) (Rodentia, Echimyidae).

Rodents are distributed worldwide, playing important ecological roles regarding preservation of forest areas. Thus, the study of their reproductive biology is a key for conservation initiatives that prevent extinction and/or improve species management. The present study aimed to describe the spermatogenic dynamics of the spiny rat Kannabateomys amblyonyx, an endemic species from Atlantic Rainforest areas, in Brazil. The average body weight was 418.43g with a gonadosomatic index of 0.41%. The testicular parenchyma organization followed the pattern described for other rodent species, with a large amount of seminiferous tubules occupying 93.57% of the organ, in a total of 26.04m per testicle. Stage I of the seminiferous tubule cycle was the most frequent in K. amblyonyx, while stage IV the most scarce. Each tubular section in stage I showed 0.47 type-A spermatogonia, 11.78 primary spermatocytes in pre-leptotene, 3.81 in zygotene and 14.31 in pachytene, whereas 32.19 cells were round spermatids and 6.23 were Sertoli cells. From these results it was possible to determine the sperm reserve of 274.49×106 cells per gram of testis. The mitotic and meiotic indexes were 25.06 and 2.25 cells, respectively, whereas the spermatogenic yield was 69.73 cells. Those findings are significant since this is the first study regarding the reproductive aspects of the only Echimyidae species in Brazil, which shows a monogamous mating system.

Spermatogenesis in a neotropical marsupial species, Philander frenatus (Olfers, 1818).

Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.

Transformation, migration and outcome of residual bodies in the seminiferous tubules of the rat testis.

Experiments were performed to study the transformation, migration and outcome of residual bodies (RBs) in the seminiferous tubules of the rat testes. One part of the testes from adult Sprague-Dawley rats was used to generate paraffin sections to observe RBs and RB precursors through specific staining, and the other part of the testes was used to generate ultrathin sections to observe RBs under a transmission electron microscope. Deep blue particles of different sizes were observed in some seminiferous tubules through specific staining for RBs and RB precursors. These particles first appeared in the seminiferous tubules at stage I of the spermatogenic cycle, and after spermiation, the particles travelled rapidly towards the deeper region of the seminiferous epithelium and soon appeared close to the basement membrane of the seminiferous tubule. All of the particles in the tubules disappeared at stage IX. Using transmission electron microscopy, components of different electron densities were observed in the RBs on the surface of the seminiferous epithelium, all of which gradually formed in the cytoplasm of spermatozoon in later stages of spermiogenesis. After the spermatozoa were released, the RBs in the epithelium travelled quickly to the edge of the tube and were gradually transformed into lipid inclusions. These lipid inclusions ultimately became lipidlike particles. The lipidlike particles were discharged into the interstitial tissue. RBs initiate their own digestive process before their formation during spermiation in the rat testes. After spermiation, the RBs transform into lipid inclusions and finally into lipidlike particles. These lipidlike particles can be eliminated from the seminiferous tubules.

Duration of spermatogenesis and daily sperm production in the rodent Proechimys guyannensis.

The spiny rat (Proechimys guyannensis) is a neotropical rodent that is used in biomedical research, particularly research related to chronic resistance to epilepsy and infectious diseases. To our knowledge, there are few reports concerning the reproductive biology of this species. Therefore, besides providing basic biometric and morphometric data, in the present study we investigated testis function and spermatogenesis in adult spiny rats. The mean testis weight and gonadosomatic index obtained were 1.63 ± 0.2 g and 1.15 ± 0.1% respectively. Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized. Stages VI and VII presented the highest frequencies (~17-19%), whilst stages II to V showed the lowest frequencies (~2-4%). The most advanced germ cell types labelled at 1 h or 20 days after BrdU injections were respectively preleptotene/leptotene spermatocytes at stage VII and elongated spermatids at stage III. The mean duration of one cycle was 7.5 ± 0.01 days and the entire spermatogenic process lasted 33.7 ± 0.06 days (~4.5 cycles). The seminiferous tubules (ST) occupied ~96 ± 1% of the testis parenchyma, whereas Leydig cells comprised only 1.5 ± 0.4%. The number of Sertoli cells (SC) per testis gram and the SC efficiency (spermatids/SC) were respectively 78 × 106 ± 11 × 106 and 7.9 ± 1. The daily sperm production per testis gram (spermatogenic efficiency; daily sperm production (DSP)/g/testis) was 78 × 106 ± 8 × 106. To our knowledge, this spermatogenic efficiency is among the highest found for mammals investigated to date and is probably related to the very short duration of spermatogenesis and the very high ST percentage and SC number obtained for this species.

Germ cell transport across the seminiferous epithelium during spermatogenesis.

Transport of germ cells across the seminiferous epithelium is crucial to spermatogenesis. Its disruption causes infertility. Signaling molecules, such as focal adhesion kinase, c-Yes, c-Src, and intercellular adhesion molecules 1 and 2, are involved in these events by regulating actin-based cytoskeleton via their action on actin-regulating proteins, endocytic vesicle-mediated protein trafficking, and adhesion protein complexes. We critically evaluate these findings and provide a hypothetical framework that regulates these events.

Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis.

A-single spermatogonia heterogeneity and cell cycles synchronize with rat seminiferous epithelium stages VIII-IX.

In mammalian testes, "A-single" spermatogonia function as stem cells that sustain sperm production for fertilizing eggs. Yet, it is not understood how cellular niches regulate the developmental fate of A-single spermatogonia. Here, immunolabeling studies in rat testes define a novel population of ERBB3(+) germ cells as approximately 5% of total SNAP91(+) A-single spermatogonia along a spermatogenic wave. As a function of time, ERBB3(+) A-single spermatogonia are detected during a 1- to 2-day period each 12.9-day sperm cycle, representing 35%-40% of SNAP91(+) A-single spermatogonia in stages VIII-IX of the seminiferous epithelium. Local concentrations of ERBB3(+) A-single spermatogonia are maintained under the mean density measured for neighboring SNAP91(+) A-single spermatogonia, potentially indicative of niche saturation. ERBB3(+) spermatogonia also synchronize their cell cycles with epithelium stages VIII-IX, where they form physical associations with preleptotene spermatocytes transiting the blood-testis barrier and Sertoli cells undergoing sperm release. Thus, A-single spermatogonia heterogeneity within this short-lived and reoccurring microenvironment invokes novel theories on how cellular niches integrate with testicular physiology to orchestrate sperm development in mammals.

Spermatogenic cycle length and sperm production in the freshwater turtle Kinosternon scorpioides.

Kinosternon scorpioides is a Brazilian freshwater turtle that belongs to the class Reptilia, encompassing almost 10,000 species. Nevertheless, very little is known about the testicular quantitative parameters, particularly those related to spermatogenesis, in this vertebrate class. Our main objectives were to investigate in detail the structure and function of the testis in K. scorpioides, particularly the aspects related to spermatogenic cycle length and Sertoli cell (SC) and spermatogenic efficiencies. Nine sexually mature turtles were examined, and intraperitoneal bromodeoxyuridine injections were administered to estimate duration of spermatogenesis. Based on the acrosome development in spermatids and the overall germ cell associations, 10 stages of the seminiferous epithelium cycle were characterized. Similar to birds, humans, and some primate species, several stages were observed per seminiferous tubule cross-sections. One spermatogenic cycle and the entire spermatogenic process lasted, respectively, 12 and 53 days. The SC efficiency (number of round spermatids per SC) and daily sperm production per gram of testis were, respectively, 20 and 40 million spermatids. As established for mammals, our findings suggest that SC efficiency is also a critical determinant of sperm production in reptiles. To our knowledge, this is the first study to investigate the kinetics of spermatogenesis and testis function in any reptilian species. Besides allowing a better understanding of reproductive biology in reptiles, these data will be useful in comparative studies. Moreover, these results could provide the basis for investigations related to the evaluation of spermatogonial stem cell physiology niche in Kinosternon scorpioides.

Stereology as a valuable tool in the toolbox of testicular research.

The morphology of the testis may be altered in various developmental, physiological and pathological conditions and these changes are reflected by the alterations in the reproductive capacity. Studying testicular morphology under these conditions or following therapeutic interventions relies on quantitative data. Design-based stereology provides quantitative morphological data on the most important characteristics of the testis. The total volume of the testis, seminiferous tubules, interstitial tissues and germinal epithelium, length, diameter and cross sectional area of the tubules as well as the number of Sertoli, Leydig, myoid cells, spermatogonia, spermatocytes and spermatids can be estimated. The present paper explains and demonstrates accurate and efficient stereological methods of sampling and analysis of testicular specimens according to the basics made by the pioneer stereologists. Second-order stereology can provide additional information on the spatial arrangement of the content of the tissues, cells or organelles in testis. These methods are valuable enough to be included in the toolbox of testicular research and are essential whenever quantitative data on morphological characteristics of the testis are required including testicular biopsy of human or in experimental studies.

Ultrastructural study of spermiogenesis in a rare desert amphisbaenian Diplometopon zarudnyi.

Spermiogenesis, in particular the head differentiation of Diplometopon zarudnyi, was studied at the ultrastructural level by Transmission Electron Microscope (TEM). The process includes acrosomal vesicle development, nuclear elongation, chromatin condensation and exclusion of excess cytoplasm. In stage I, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. This step commences with an acrosome vesicle forming from Golgi transport vesicles; simultaneously, the nucleus begins to move eccentrically. In stage II, the round proacrosomal vesicle is flattened by projection of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle as the fibrous layer forms the subacrosomal cone. Circular manchettes surrounded by mitochondria develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. The nucleus has uniform diffuse chromatin with small indices of heterochromatin. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. In stage III, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; coarse granules replace the small ones within the nucleus. One endonuclear canal is present where the perforatorium resides. In stage IV, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the residues of the cytoplasmic lobes are removed. In stage V, the sperm head matures.