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.     

Germ cell removal after induction of cryptorchidism in adult rats

Cryptorchidism is associated with male infertility due to germ cell loss in response to elevated temperature. However, there is a great deal of contradictory information prevalent on the status of germ cells and their process of removal in the cryptorchid testis. In the present study, we investigate the cell removal from cryptorchid rat testis by the methods of morphology and stereology. The testis weight is reduced according to previous reports after surgical induction of cryptorchidism. Interestingly, the epididymal weight is significantly increased in 7 days after surgery, and the caput epididymis tubules show filling with countless round germ cells. We found that the elongating spermatids (steps 10-13), newborn spermatids (step 1) and the dividing spermatocytes are the most susceptible cells to elevated temperature, and are the first disappeared cells from the seminiferous tubules after surgery. Germ cell removal followed the order, starting first with elongating spermatids and newborn spermatids, followed by round spermatids and elongated spermatids and later extending to spermatocytes.     

Appearance of the oocyte activation ability of mouse round spermatids cultured in Vitro

This study was undertaken to determine the expression time of fertilization and oocytes activation abilities of spermatids in the mouse. When elongating or elongated spermatids isolated from fresh testes of adult males (B6D2F1) were injected into mature mouse oocytes, both spermatids could activate the mature oocytes and occur fertilization. On the one hand, the round spermatids could not activate mature oocytes, when microinjected into oocytes. In some experiments, recovered round spermatids were cultured under co-culture systems using Sertoli cells as a feeder cell. Under the co-culture system, developed elongating spermatids could stimulate and fertilized mature oocytes. These results indicate that the start of oocyte activation appearance is between the stage of round spermatid and elongating spermatids and the activation ability increases with the advance of spermiogensis. On the other hand, round spermatids isolated from males of ICR strain mouse already have the oocyte activation ability and the fertilizing ability. The result obtained suggests that the expression time of the oocyte activating ability is difficult between the mouse strain.     

Poly(A) shortening accompanies the activation of translation of five mRNAs during spermiogenesis in the mouse

I have compared the quantity and the length of the poly(A) tracts of five haploid-expressed mRNAs in the polysomal and nonpolysomal fractions of round and elongating spermatids in mice: transition proteins 1 and 2, protamines 1 and 2, and an unidentified mRNA of about 1050 bases. Postmitochondrial supernatants of highly enriched populations of round and elongating spermatids (early and late haploid spermatogenic cells) were sedimented on sucrose gradients, and the size and amount of each mRNA in gradient fractions were analyzed in Northern blots. In round spermatids, all five mRNAs are restricted to the postpolysomal fractions, but in elongating spermatids about 30-40% of each mRNA is associated with the polysomes. The distribution of these mRNAs in sucrose gradients suggests that all five mRNAs are stored in a translationally repressed state in round and early elongating spermatids, and that they become translationally active in middle and late elongating spermatids. The translationally repressed forms of all five mRNAs are long and homogenous in size, whereas the polysomal forms are shorter and more heterogenous due to shortening of their poly(A) tracts. The relationship between translational activity and poly(A) size exemplified by these five mRNAs may be typical of mRNAs which are translationally repressed in round spermatids and translationally active in elongating spermatids.     

How is the flagellar length of mature sperm determined? II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 mum, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 mum, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.     

The aberrant spermatogenesis of the Haplothrips simplex (Buffa) (Thysanoptera): ultrastructural study

The aberrant spermatogenesis of the haploid insect Haplothrips simplex (Thysanoptera) is described. The process, which occurs in the pupal instars, is characterized by two mitotic divisions, the second of which gives rise to two different-sized spermatids: the larger spermatids have a nucleus with diffuse chromatin and proceed into spermiogenesis, while the small spermatids have pycnotic nuclei and degenerate. Both types of spermatids contain two centrioles parallely rather than orthogonally oriented. The occurrence of two centrioles supports a close relationship between Thysanoptera and Phthyraptera. Before the beginning of spermiogenesis, however, the functional spermatids show the unusual presence of a third parallel centriole which is formed by the duplication of one of the two pre-existing centrioles.     

How is the flagellar length of mature sperm determined? : II. Comparison of tubulin synthesis in spermatids between newt and Xenopus in vitro

In order to elucidate mechanisms that control flagellar length of mature sperm, we studied in synchronous cell suspension cultures flagellar growth, tubulin pool, and tubulin synthesis in round spermatids of Xenopus laevis and the newt Cynops pyrrhogaster. The average final length of flagella in Xenopus round spermatids was 35 μm, almost the same length as that in mature sperm, whereas in the newt round spermatids, the length was 210 μm, almost half that of mature sperm. Kinetics of flagellar growth showed that the rate and period of flagellar growth in the newt spermatids were two to threefold those in Xenopus spermatids. The tubulin pool size in newt spermatids was estimated to be about 10-fold greater than that in Xenopus spermatids. But even if all of the pool was used for flagellar growth, it could support only about a seventh to a tenth of the flagellar length in mature sperm in either species. Thus, the possibility that the tubulin pool primarily determines flagellar length was excluded. Since the tubulin pool size did not change throughout the culture period, the possibility that the termination of flagellar growth is due to the exhaustion of the tubulin pool was also excluded. Tubulin synthesis declined over the culture period but continued in newt spermatids longer than in Xenopus spermatids. The period of flagellar elongation almost coincided with the period of tubulin synthesis. The amount of rRNA did not decrease, excluding the possibility that the decline of tubulin synthesis was due to cytoplasmic shedding which might result in the loss of ribosomes. Tubulin synthesis and the amount of rRNA in newt spermatids was more than threefold greater than that in Xenopus spermatids, which may explain the difference in growth rates of their flagella.     

Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.     

Cellular content of ubiquitin and formation of ubiquitin conjugates during chicken spermatogenesis.

Ubiquitin was purified from chicken testis and its content, biosynthesis and formation of conjugates was determined in germinal cells at successive stages of spermatogenesis. Free ubiquitin increased markedly during spermatogenesis, reaching its maximum level in early spermatids. High levels of ubiquitin were still present in late spermatids but were not detectable in mature spermatozoa. Biosynthesis of ubiquitin occurred in vitro in a fraction containing meiotic and pre-meiotic cells, and during spermiogenesis, in early and late spermatids. The cellular content of free ubiquitin increased after ATP depletion, especially in early spermatids. Lysates of chicken testis cells, particularly those obtained from spermatids, were able to form nuclear (24 and 27 kDa) and extranuclear (55-90 kDa) ubiquitin conjugates in vitro. The presence of increasing levels of ubiquitin and ubiquitin conjugates in chicken spermatids may suggest a possible involvement of this protein in the marked changes of protein turnover, chromatin structure and cell-cell interactions that spermatids undergo during spermiogenesis.     

Synacrosomal formation after cell fusion of round spermatids of Xenopus laevis

Cell fusion was induced by hypotonic medium in pairs of spermatids which were derived from single secondary spermatocytes. In a pair of fused spermatids, a single acrosome (synacrosome) eventually formed whenever the cell fusion was induced during the course of acrosomal formation. Direct observation of the process of synacrosomal formation was made on pairs of fused spermatids which had completed acrosomal formation. Two patterns occurred, namely, fusion of two acrosomes or enlargement of one with diminution of the other. The total volume of the two acrosomes before synacrosomal formation almost equaled the volume of the coalesced synacrosomes in fused spermatids. Neither colchicine nor cytochalasin B prevented synacrosomal formation in spermatids which were fused after each had completed acrosomal formation. These results indicate that neither microtubules nor microfilaments seem to play a role in the formation of a synacrosome in pairs of fused spermatids. However, cycloheximide did inhibit acrosomal formation when present during the early stage of acrosome differentiation in pairs of spermatids which had been fused just after second meiotic division. This fact indicates that acrosomal formation is mediated by some protein(s) which are synthesized during the initial period of acrosomal formation.     

La spermatide, cette méconnue

The use of microinsemination of round or elongated spermatids into ovocytes, in certain cases of male infertility, requires re-examination of the sequence of morphological and functional changes that occur throughout spermiogenesis. This paper reviews essential findings on morphogenesis of spermatids, genome expression during sperm differentiation and cellular interactions between spermatids themselves and between spermatids and Sertoli cells. Round and elongated spermatids appear to represent two classes of structuraly and functionnaly different cells. One question remains: on what criteria can one claim that a round spermatid functions normally when spermiogenesis is blocked or impaired?     

Synacrosomal formation after cell fusion of round spermatids of Xenopus laevis

Cell fusion was induced by hypotonic medium in pairs of spermatids which were derived from single secondary spermatocytes. In a pair of fused spermatids, a single acrosome (synacrosome) eventually formed whenever the cell fusion was induced during the course of acrosomal formation. Direct observation of the process of synacrosomal formation was made on pairs of fused spermatids which had completed acrosomal formation. Two patterns occurred, namely, fusion of two acrosomes or enlargement of one with diminution of the other. The total volume of the two acrosomes before synacrosomal formation almost equaled the volume of the coalesced synacrosomes in fused spermatids. Neither colchicine nor cytochalasin B prevented synacrosomal formation in spermatids which were fused after each had completed acrosomal formation. These results indicate that neither microtubules nor microfilaments seem to play a role in the formation of a synacrosome in pairs of fused spermatids. However, cycloheximide did inhibit acrosomal formation when present during the early stage of acrosome differentiation in pairs of spermatids which had been fused just after second meiotic division. This fact indicates that acrosomal formation is mediated by some protein(s) which are synthesized during the initial period of acrosomal formation.     

Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat

The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.     

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.     

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.     

Fertilization of oocytes and birth of normal pups following intracytoplasmic injection with spermatids in mastomys (Praomys coucha)

The mastomys is a small laboratory rodent that is native to Africa. Although it has been used for research concerning reproductive biology, in vitro fertilization (IVF) and intracytoplasmic sperm injection are very difficult in mastomys because of technical problems, such as inadequate sperm capacitation and large sperm heads. The present study was undertaken to examine whether mastomys spermatids could be used to fertilize oocytes in vitro using a microinsemination technique, because spermatids are more easily injected than mature spermatozoa into oocytes. Most mastomys oocytes (80%-90%) survived intracytoplasmic injection with either round or elongated spermatids. Round spermatids had little oocyte-activating capacity, similar to those of mice and rats, and exogenous stimuli were needed for normal fertilization. Treatment with an electric pulse in the presence of 50 microM Ca2+ followed by culture in 10 mM SrCl2 led to successful oocyte activation. After injection of round spermatids into preactivated oocytes, 93% of oocytes were normally fertilized (male and female pronuclei formed), and 100% of cultured oocytes developed to the 2-cell stage. However, none reached term after transfer into recipient females. Elongated spermatids, which correspond to steps 9-11 in rats, activated oocytes on injection without additional activation treatment. After embryo transfer, five offspring (6% per transfer) developed to term. These results indicate that microinsemination with spermatids is a feasible alternative in animal species that are refractory to IVF and sperm injection and that using later-stage spermatids may lead to increased production of viable embryos that can develop into normal offspring.     

Immunocytochemical localization of protamine in the boar testis

Protamine was specifically demonstrated in spermatids and spermatozoa of the boar by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera, and three different direct or indirect labelling techniques. The two isomers of the protamine could not be labelled separately. The protamine is present in the cytoplasm of elongating spermatids and it enters the nuclei throughout the elongation process after possible storage in the cytoplasm or in the nuclear envelope of spermatids, or both. These findings differ from previous observations in other species.     

How is the flagellar length of mature sperm determined? III. Comparison of initial growth rate of flagella in spermatids from Cynops and Xenopus in the presence and absence of cycloheximide.

Previous studies on flagellar growth in round spermatids from Cynops and Xenopus in vitro have shown that the period and rate of flagellar growth are greater in Cynops than in Xenopus. The present study shows, however, that during the initial phase of flagellar growth (for the first 12 h following the second meiotic division), the growth rate is very similar in both Cynops and Xenopus (0.5-0.6 microns/h at 22 degrees C). The difference in the growth rate between Cynops and Xenopus was observed beyond 12 h following the second meiotic division. When round spermatids in both species were inoculated with 10 microM cycloheximide, flagella grew at the same rate as in the absence of cycloheximide for the first 12 h following the second meiotic division. Beyond 12 h, however, cycloheximide suppressed flagellar growth in round spermatids in both species. These results indicate that the initial flagellar growth in round spermatids is provided for by flagellar protein pools which were present just after the second meiotic division; the growth beyond 12 h in round spermatids is contributed by newly synthesized flagellar proteins.