Annual changes in the testicular activity of the river sculpin, Cottus hangiongensis Mori, with emphasis on the occurrence of aberrant spermatids during spermatogenesis

Annual changes in the spermatogenetic activity of the testis were studied histologically in the river sculpin. Coitus hangiongensis , sampled monthly from a river in southern Hokkaido, Japan. A pair of sperm reservoirs, consisting of many anastomosing lacunae, was present along the dorsomedian edge of the paired testes, and functioned also as a sperm-transporting system instead of the typical sperm duct. Spermatogenesis occurred actively in August, yielding an increasing number of mature spermatozoa in October. This process advanced, but slowly during the succeeding winter months, until March. The testis became functionally mature during the spawning period in April and May. In July, small numbers of spermatocytes were found to have appeared already, which indicated a relatively short period of post-spawning testicular regression. In November, germinal cysts containing aberrant binuclear spermatids began to appear within the seminal lobules. The paired nuclei of aberrant spermatids gradually enlarged, and the cells were released into the lumina of the seminal lobules simultaneously with the release of mature spermatozoa from the germinal cysts. During the functional maturity stage, lumina of seminal lobules which had expelled mature spermatozoa to sperm reservoirs became filled with these abnormal bodies. Discussion includes the occurrence of aberrant spermatids which resulted in the formation of 'spermatid masses' as has been described in other cottids.     

Differential basic nucleoprotein kinetics in the two kinds of lepidoptera spermatids: Nucleate (eupyrene) and anucleate (apyrene)

Normal lepidopteran males produce two kinds of spermatozoa: nucleate (eupyrene) and anucleate (apyrene). Eupyrene spermatozoa have the usual type of elongate nuclei. But in apyrene spermatids, the nuclei never elongate and the chromatin remains in a telophase-like condition until enucleation occurs. The study of the differential nucleoprotein kinetics of the two types of spermatids, using the fluorescent dye sulfoflavine, shows that: (1) In the elongate eupyrene nuclei, lysine-rich nucleoproteins are replaced by arginine-rich ones, while in the non-elongating apyrene nuclei only lysine-rich nucleoproteins are detected. However, nuclear elongation is not causally related to nucleoprotein transitions as transitions occur in the eupyrene spermatids after nuclear elongation. (2) The replacement of the nucleoproteins occurs in the eupyrene nuclei in a polarized manner. This may be correlated with the heterogeneous ultrastructural configuration of the chromatin fibers in elongating spermatid nuclei, as shown in other insect species. (3) Concomitantly with the eupyrene spermatid nucleoprotein transition, the cytoplasm of the head cyst cell shows an increasing amount of cytoplasmic lysine-rich proteins, while no such a phenomenon occurs in apyrene cysts. This differential pattern distribution may reflact functional differences among the two types of cysts and is probably related to the regulation of the dichotomy in lepidopteran spermatogenesis.     

Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.     

Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This “elementary” fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30–40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called “immature chromatin,” which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.     

The blood-testis barrier and its breakdown following immunization to testis material in the Nile tilapia,Oreochromis niloticus

Summary The blood-testis barrier and its changes following immunization to testis material, were investigated by light- and electron microscopy in a teleost fish, the Nile tilapia Oreochromis niloticus, using horseradish peroxidase and bovine serum albumin as tracers. In the normal testis, histochemistry using horseradish peroxidase revealed that a barrier composed of junctional complexes connecting adjacent Sertoli cells existed around the central lumina of the seminal lobules, and also around the germ-cell cysts containing spermatids at the middle or late phase of chromatin condensation. By contrast, bovine serum albumin was prevented from passing through the basement membrane and could not penetrate any of the spermatogenetic cysts, indicating that the basement membrane may be an ion-selective barrier. In tilapia immunized with allogeneic testis homogenate emulsified in Freund's complete adjuvant, bovine serum albumin could penetrate the spermatogenetic cysts, and horseradish peroxidase was able to pass through the intercellular spaces between Sertoli cells to the region nearer the seminal lobule lumen, due to the junctional complexes becoming loosened. The results suggest that the blood-testis barrier, both junctional complexes and the basement membrane, are broken down during immune responses.     

Ultrastructural observations of eu- and paraspermiogenesis in the cottid fish Hemilepidotus gilberti (Teleostei: Scorpaeniformes: Cottidae)

The developmental process of eu- and paraspermatozoa in the cottid fish, Hemilepidotus gilberti, was observed by electron microscopy. Euspermatozoa of H. gilberti consist of a thin disk-like sperm head (about 3 microm in length), a short middle piece, and a long flagellum, but lack an acrosome. On the other hand, during spermiogenesis, aberrant spermatids, rich in cytoplasm and possessing binuclei, develop into cysts containing spermatids. The developing aberrant spermatids connect with normal spermatids and euspermatozoa by intercellular bridges. The early phase of chromatin condensation in aberrant spermatids is almost identical to that in normal spermatids, but the nuclei in the later phase develop into a mass of highly electron-dense globules. Since the aberrant spermatids complete karyokinesis but not cytokinesis at telophase of the second meiotic division, they are considered to develop into hyperpyrenic cells due to incomplete cytokinesis of the second meiotic division. These spermatids are oval in shape (5-7 microm in diameter) and lack a flagellum. The aberrant spermatids of H. gilberti are shed along with euspermatozoa and amount to about 50% of semen in volume. Judging from their form and developmental process, aberrant spermatids produced in H. gilberti are considered hyperpyrenic paraspermatozoa.     

Ultrastructural observations of euspermatozoa and paraspermatozoa in a copulatory cottoid fish Blepsias cirrhosus

Euspermatozoa and paraspermatozoa of a copulatory (internal insemination with external sperm transfer) cottoid fish Blepsias cirrhosus were observed ultrastructurally. Euspermatozoa of B. cirrhosus consisted of an acrosome‐less, thin, disk‐like sperm head (1·6-2·0 μm in length and 1·3-1·6 μm in width), a long middle piece, and a long flagellum ( c . 30 μm). Aberrant spermatids, which were rich in cytoplasm and possessed two nuclei, occurred in testicular lobules. They were also observed in semen and were round (5·0-5·3 μm in diameter) and biflagellate, suggesting that they are released along with euspermatozoa at ejaculation. The nuclei of aberrant spermatids developed into masses of highly electron‐dense globules. Judging from their form, nuclear condition, and connection with normal spermatids by intercellular bridges during spermiogenesis, aberrant spermatids of B. cirrhosus are considered hyperpyrenic paraspermatozoa formed by incomplete cytokinesis at the second meiotic division.     

Temporal pattern of the double sperm line production in Tubifex tubifex (Annelida,Oligochaeta)

Laboratory cohort cultures of the tubificid Tubifex tubifexwere carried out at 21 °C in homogeneous conditions. Our aim was to study the temporal pattern of spermatogenesis of the two sperm lines which characterise this species. Starting from the second week after cocoon laying, we analysed the seminal vesicle content. We counted the cysts in the vesicles and arranged them into three classes: premeiotic, paraspermatids and euspermatids. Our results show that spermatogenesis begins early, at week 3, but cysts of spermatids are not found until week 5 for the parasperm line and week 6 for the eusperm line. These data are derived from a preliminary study of complex population dynamics. Nonetheless, they allow us to formulate some hypothesis regarding the mechanism which regulate the production of the two types of sperm.     

Histological structure of the male reproductive organs and spermatogenesis in a copulating sculpin, <Emphasis Type="Italic">Radulinopsis taranetzi</Emphasis> (Scorpaeniformes: Cottidae)

Characteristics of the structure and function of male reproductive organs in the copulating sculpin Radulinopsis taranetzi were investigated based on histological observations. The male reproductive organs comprised three parts: a pair of testes, a seminal vesicle, and a penis. Germinal cells matured in cysts located in the small seminal lobules. Asynchronous spermatogenesis advanced rapidly from the posterior to the anterior region of the testes. After sperm matured in the posterior part of the testes, the seminiferous epithelium of the seminal lobules synthesized and secreted eosinophilic fluid that showed a positive periodic acid–Schiff (PAS) reaction into the seminal lobules. Spermatozoa excreted from the posterior part of the testes were stored together with the secretion in the seminal vesicle and showed no activity in the seminal fluid. Histological observations throughout the year suggest that the fluid is secreted and spermatozoa are stored in the seminal vesicles during February to July, which is presumably when mating occurs. The importance of testicular maturation and the secretion of eosinophilic fluid during this long reproductive period is also discussed.     

Histology of salmonid testes during maturation

The commonly applied classification systems of fish gonad maturity divide the maturation process into certain stages. However, the scales do not entirely reflect the continuity of the maturation process. Based on light microscope observations, the paper describes a comprehensive pattern of testicular transformations during maturation. The study was carried out on precocious underyearling and 1-year-old males of sea trout (Salmo trutta m. trutta L.), 1-year-old males of salmon (Salmo salar L.), and males of brown trout (Salmo trutta m. fario L.) aged from 7 months to 4 years. A total of 821 gonads collected during all seasons of the year were examined. The fish were fixed in Bouin's fluid. Histological slides of the mid-part of the gonad were made using the standard paraffin technique. The 3-6 microm sections were stained with Heidenhain haematoxylin. Histological changes of testes during maturation were similar in the three species studied. Immature and resting gonads contained type A spermatogonia in lobules only. The appearance of cystic structures containing type B spermatogonia in the lobules signalled the beginning of the sexual cycle in male gonads. Type B spermatogonia underwent synchronous mitotic divisions resulting in an increase in the total number of spermatogonia. As the spermatogenesis continued, the gonads showed a gradual increase in the number of cysts containing cells at all the spermatogenetic stages: type B spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa. The well-formed spermatozoa were released to the lobule lumen once the Sertoli cells and spermatozoa connections broke up and the cyst disappeared. This was a continuous process observed throughout the spawning season. The spermatozoa were moved to the efferent duct. While some of the germ cells were completing spermatogenesis, the lobules contained less and less cysts with type B spermatogonia, primary and secondary spermatocytes, and spermatids; eventually all the cells completed spermatogenesis. At the end of maturation, vacuoles, up to 18.9 microm in final diameter (brown trout), appeared in the Sertoli cells. The vacuoles were visible in the lobule wall epithelium for a prolonged period of time. In most salmonid individuals examined, the reproductive cycles were observed to overlap. In some fish, the preparation for another cycle began very early, i.e., at the and of preceding spermatogenesis, which had not been observed before. Gonad maturation in some males was incomplete.     

Testis structure and symplastic spermatid formation during spermatogenesis of pipefishes

The pipefishes Syngnathus abaster and S. acus have paired testes of atypical organization. Each testis is a hollow tube consisting of a single germinal compartment of the tubular type. During the reproductive period, the germinal epithelium consists of small spermatocysts containing spermatogonia or primary spermatocytes. Cysts of older germ cells, such as secondary spermatocytes and spermatids were never observed. Developing symplastic spermatids were found in the lumen of the tubule together with mature sperm and large droplet-containing cells. Most of the spermatids were giant cells with four nuclei at the same developmental stage. Symplastic spermatids, which presumably form by nuclear division not followed by cytokinesis, are a stage of spermatogenesis in pipefishes.     

Control of spermatogenesis resumption in post-diapausing larvae of the codling moth

The lepidopteran primary spermatocytes produce first eupyrene (nucleated) and later apyrene (anucleated) spermatozoa. The shift to apyrene commitment of the spermatocytes is related to an apyrene-spermatogenesis-inducing factor (ASIF) becoming active towards pupation. During diapause, the primary spermatocytes lyse and spermatogenesis ceases. The renewal of the dichotomous spermatogenesis in the testes of post-diapausing, last-instar larvae of the codling moth was studied in vivo and in vitro. In vivo, the post-diapausing larvae resume the two types of spermatogenesis. Since ASIF activity is related to pupation, the earliest apyrene spermatids appear one day before pupation, as in non-diapausing larvae. In vitro, renewal of spermatogenesis occurs if 20-hydroxy-ecdysone is added to the medium, but only eupyrene spermatids occur since the testes are explanted before ASIF activity has started. These spermatids are unreduced and develop directly from primary spermatocytes which do not undergo meiotic divisions. Moreover, only flagella develop in these spermatids and the nuclei remain spherical. Post-diapause resumption of spermatogenesis is thus a complex process in which meiosis-blocking and meiosis-deblocking factors, ecdysteroids, and the ASIF play regulative roles.     

Peculiarities in the organization of testis of Ophidian sp. (Pisces Teleostei). Evidence for two types of spermatogenesis in teleost fish

The walls of lobules in the testis of Ophidion sp. are composed of Scrtoli cells and young germinal cells (spermatogonia and spermatocytes). Spermatocytes are linked by cytoplasmic bridges. The associations of Sertoli cells and spermatocytes constitute true cysts. Meiosis takes place in the cysts. When meiosis is complete, cysts open. Spermatids are released into the lumen of the lobules and the cyloplasmic bridges break down. Spermiogenesis occurs in the lumen. Spermatids at various levels of spermiogenesis are then mixed with ripe spermatozoa. In teleosts we thus recognize two types of spermatogenesis: a cystic type where spermatogenesis is completed within cysts, and leads to synchronous development of germ-cells; and a semi-cystic type, where spermatogenesis occurs partly outside cysts. This may produce asynchronous spermatogenesis.     

Reproductive cycle and sexual maturity of the anglerfish Lophiomus setigerus in the East China Sea with a note on specialized spermatogenesis

The reproductive cycle and sexual maturity of the anglerfish Lophiomus setigerus were examined. Spermatids were released from the germinal cysts into the lumina of the seminal lobules, and both spermatids and spermatozoa were present in the lumina of the seminal lobules and sperm ducts. Spermatogenesis and vitellogenesis occurred throughout most of the year. The testes of males were full of spermatozoa throughout the year, with spawning from May to November. Males and females reached sexual maturity at a mean total length and age of 178 mm, 3.3 years and 303 mm, 6.1 years, respectively. There were clear seasonal cycles in the gonadosomatic index (I_G) and hepatosomatic index ( I _H) in females. The mean I_G of females increased rapidly with ovarian development while the mean I _H decreased from the middle of vitellogenesis to final maturation. Mean values of I _G and I _H in males increased with testicular development.     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids.

DNase I-hypersensitivity of rat spermatogenic cells was analyzed 1) to establish overall patterns of hypersensitivity in individual cell types, 2) to correlate these patterns with known changes in chromatin organization and function, and 3) to provide a foundation for further analyses examining DNase I-hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I-hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell-type specific manner during the course of spermatogenesis, reflective of known stage-dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from "checkerboard" in steps 7-8 round spermatid nuclei to "lamellar" in steps 10-11 elongating spermatids. In steps 12-13 elongating spermatids. DHR were localized throughout the nuclei or in a graded manner--increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12-13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16-19 spermatids and mature spermatozoa did not demonstrate DNase I-hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12-13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12-13 elongating spermatids to nick translation in the absence of exogenous nuclease indicators the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.