Reproductive capacity of round spermatids compared with mature spermatozoa in a population of azoospermic men

The present study aims to evaluate the injection of testicular round spermatids from patients with complete failure of spermiogenesis compared with that of mature epididymal and testicular spermatozoa. Over a period of 8 months, 188 azoospermic patients were evaluated with a view to their inclusion in our intracytoplasmic sperm injection (ICSI) programme. All patients had had a previous testicular biopsy; 38 had pure obstructive azoospermia, while 150 had non-obstructive azoospermia. Mature spermatozoa were found in 93 patients, whereas spermatozoa were entirely absent, with a predominance of round spermatids in 87. In eight patients, spermatids could not be found and therefore their cycles were cancelled. There was an early appearance of the two pronuclei stage in the round spermatid group compared with the mature spermatozoa group of patients (10.2 and 16 h respectively). The fertilization rate was also significantly lower (P = 0.00001) in the round spermatid group. The numbers of embryos developed and of embryo transfers in the round spermatid injection group were significantly lower compared with the mature spermatozoa injection group (P = 0.05 and 0.0001 respectively). No pregnancies resulted from round spermatid injection, while 18 pregnancies were achieved from the injection of mature spermatozoa. In conclusion, injection of round spermatids from patients with complete failure of spermiogenesis resulted in a significantly lower fertilization rate and a higher developmental arrest compared with injection of mature spermatozoa. With no pregnancies achieved, one may question the unusual variability of reported success rates and stress the need for further research in order to improve the outcome of this novel technique.     

Spermiation and sperm maturation in the marmoset

The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostral edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hook-shaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is an Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates.     

Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter's syndrome

BACKGROUND: Klinefelter's syndrome is the most frequent chromosomal abnormality in infertile men. In this study, the chromosomes of round spermatids and spermatogonia/primary spermatocytes from men with non-mosaic Klinefelter's syndrome were examined, together with the Sertoli cell secretory function and sperm morphometry. METHODS: Twenty-four men with non-mosaic Klinefelter's syndrome and nine men with obstructive azoospermia underwent therapeutic testicular biopsy. When spermatozoa in the final filtrate were present, they were processed for sperm morphometry or ICSI. Sperm morphometry was evaluated by the maximal length and width of the sperm head, the length of the midpiece and the ratio of the acrosomal region to the total surface area of the head. When round spermatids were present, they were processed for fluorescent in-situ hybridization (FISH). FISH was also applied to fragments of seminiferous tubules. Sertoli cell secretory function was measured by the amount of androgen binding protein (ABP) secreted in vitro. RESULTS: More than 93% of the evaluated round spermatids were normal. The proportions of 24,XY and of 24,XX round spermatids to the total number were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Men with Klinefelter's syndrome who had spermatozoa in their testicular tissue (n = 12) were positive for both 46,XY and 47,XXY spermatogonia in their seminiferous tubules. In contrast, men with Klinefelter's syndrome without spermatozoa in their testicular tissue (n = 12) were positive for 47,XXY spermatogonia but negative for 46,XY spermatogonia in their seminiferous tubules. ABP profiles were significantly smaller in men with Klinefelter's syndrome who were negative for spermatozoa compared with men who were positive. Four pregnancies were achieved and five healthy babies were born. CONCLUSIONS: This study suggests that few 46,XXY spermatogonia undergo meiosis with an XX pairing and a Y univalent type of pairing. Hyperhaploid round spermatids (24,XY and 24,XX) may be produced by meiosis of 47,XXY spermatogonia. Men with Klinefelter's syndrome who are negative for testicular spermatozoa have a greater degree of Sertoli cell secretory dysfunction compared with men with Klinefelter's syndrome who are positive for spermatozoa. There are several defects in sperm morphometry with functional significance in men with Klinefelter's syndrome.     

Characterization of Sertoli cell-germ cell junctional specializations in dissociated testicular cells

To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spermatids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.     

Characterization of Sertoli cell-germ cell junctional specializations in dissociated testicular cells.

To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spematids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.     

Failure to assemble the peri-nuclear structures in GOPC deficient spermatids as found in round-headed spermatozoa

Deletion of the GOPC gene encoding mouse GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) causes infertile round-headed spermatozoa, which have acrosome-less round heads and deformed tails (Yao et al, 2002). This study investigated how GOPC deficient spermatids fail to assemble the peri-nuclear structures in round-headed spermatozoa during spermiogenesis in GOPC knockout mouse testes. In step 1-8 spermatids, Golgi-derived proacrosomal vesicles that are transported to the perinuclear region formed acrosome-like vesicles of various sizes, called pseudoacrosomes. The marginal ring of the acroplaxome, which is generally formed between the descending edge of a developing acrosome and nuclear envelope in a wild spermatid, was poorly formed between the pseudoacrosome and nuclear envelope. In step 9-11 elongating spermatids, a majority of pseudoacrosomes were detached from the nucleus and disappeared from the perinuclear region by spermiation. Concomitantly, several failures occurred on the nucleus, manchette, postacrosomal sheath (perinuclear theca), and posterior ring. Ectoplasmic specializations were poorly formed, and did not always associate with developing spermatids. Consequently, spermatid nuclear elongation to form round-headed spermatozoa developed was impaired. In addition to these sequential failures, the posterior ring deficiency was attributed to the tail deformation destined to occur during epididymal maturation as reported in an accompanying paper (Suzuki-Toyota et al, 2004 in this issue), its eventual phenotype being reminiscent of the round-headed spermatozoa of human infertile globozoospermia.     

Three-dimensional reconstruction of a rat stage V Sertoli cell: III. A study of specific cellular relationships

Specific Sertoli–Sertoli and Sertoli–germ-cell contacts and/or junctions were investigated employing micrographs used to reconstruct serially a model of a rat stage V Sertoli cell. The Sertoli–Sertoli junctional contact areas occurred in a belt-like arrangement near the base of the Sertoli cell. This configuration is consistent with their proposed function as a sealing element limiting the passage of materials toward the tubular lumen. Sertoli ectoplasmic specializations also formed a continuous belt, or band, around the reconstructed cell at the junctional contact area. Eighteen Sertoli–Sertoli tubulobulbar complexes were found; some (12 in number) invaginated the reconstructed cell, while others (6) emanated from it. Of 37 round germ cells that were sectioned in their entirety and adjoined the reconstructed cell, 23 displayed desmosome–gap junctions with either the reconstructed cell or an adjoining cell. Since there were multiple junctions connecting some germ cells to Sertoli cells, the total number of junctions was much greater (35). Desmosome–gap junctions of the Sertoli cell were numerous connecting pachytene spermatocytes, less numerous connecting type B spermatogonia, and even less numerous connecting step 5 spermatids; and none was seen joining Sertoli cells with elongate spermatids. Most desmosome-gap junctions join germ cells to the body of the Sertoli cell at its basal aspect. Their numbers and position indicate that they play a role in the maintenance of the integrity of the seminiferous epithelium and may provide a route for cell-to-cell communication. Ectoplasmic specializations of the reconstructed cell were seen facing only 3 of 37 round germ cells, and 7 ectoplasmic specializations from adjoining Sertoli cells faced these germ cells, all of which were step 5 spermatids. That there were no ectoplasmic specializations facing pachytene cells indicates that ectoplasmic specializations are not acquired as these cells pass through Sertoli–Sertoli junctions, but are acquired later in spermatogenesis.     

Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: an electron microscope study

Affinity purified antibodies prepared against proteins isolated from fibrous sheath (FS) and outer dense fibers (ODF) were utilized in an immunocytochemical study of spermatids at various steps of spermiogenesis. This study, using the immunogold technique, was performed on sections of Epon or Lowicryl embedded tissues examined with the electron microscope. In the case of FS antibodies there was a selective immunoreactivity of the FS itself from step 10 onwards, but no reactivity over the plasma membrane associated FS anlagen. In addition there was a diffuse immunoreaction over the cytoplasmic matrix from step 9 until step 18 of spermiogenesis but no reactivity over the various types of dense bodies (e.g., granulated bodies, reticulated body, etc.) seen in the cytoplasm of these spermatids. In the case of ODF antibodies the ODF were immunolabeled throughout their development from step 11 onward. In addition to a diffuse immunoreactivity of the cytoplasmic matrix of spermatids from step 9 until step 18 of spermiogenesis, there was an immunolabeling of "granulated bodies." These bodies appeared in relation to ER cisternae during steps 10-14, increased in number and size during steps 15-17 and decreased in number thereafter leaving only a few coarsely granulated bodies in the residual cytoplasm which detached from late step 19 spermatids. No other cytoplasmic structures were labeled with the ODF antibody-gold complexes. Thus the granulated bodies appeared to serve as a transitory storage site for some proteins destined to form ODF, a major cytoskeletal element of the tail of rat spermatozoa.     

Germ cell apoptosis in men with complete and incomplete spermiogenesis failure

Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-V binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane's outer surface. Both methods were combined, in double- fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception.      

Genetic approach to male meiotic division deficiency: the human macronuclear spermatozoa.

Human macronuclear spermatozoa (also termed large-headed or macrocephalic spermatozoa) are tetraploid and represent a mammalian model of meiotic division deficiency (MDD). Their genetic origin is strongly suggested by the existence of familial cases. They arise from spermatocytes I with a blockage of organelle displacement at the pachytene stage which disables the assembly of a bipolar meiotic spindle. Spermiogenesis can sometimes be complete, showing that meiotic divisions and spermiogenesis can be decoupled. However, the microtubular manchette is unilateral leading to an irregular sperm nucleus. A severe MDD phenotype also exhibits atrophic flagella. Another MDD phenotype is characterized by arrest at the round spermatid stage, suggesting the existence of factors coordinating meiosis and spermatid differentiation. An attempt is made herein to understand why MDD spermatocytes escape the pachytene and spindle-assembly checkpoints. These human MDD are revisited in the light of Drosophila mutants for cell cycle factors, meiosis division-promoting factors and microtubule components. Several human genes are known to be homologous to genes involved in male MDD in Drosophila mutants, and their number will soon be increased. These candidate genes open the way to investigation of human genes possibly mutated in patients with macronuclear spermatozoa and/or macronuclear spermatids.     

A scanning electron microscopic study of the Sertoli cell and spermiation in the syrian hamster

The scanning electron microscope was used to examine the Sertoli cells in normal and germ-cell-depleted testes. The Sertoli cells appear to attain characteristic configurations in the various stages of the cycle of the seminiferous epithelium. In tubules containing maturation-phase spermatids, Stages I-IV, the Sertoli cells exhibit column-like bases which give rise to lamellae measuring 25–30 μm in width which ensheath the spermatids and residual cytoplasmic bodies. In the Stages VIII-IX long flat sheets of Sertoli cytoplasm rest atop the step-8 and -9 spermatids. These sheets are oriented parallel to the basement membrane of the tubule with their long axis parallel to the long axis of the tubule. In Stages V-VI the head and proximal portion of the tail of the maturation-phase spermatids are ensheathed in sleeve-like Sertoli cell processes. Cuff-like terminations demarcate the terminus of these sleeves that surround maturing spermatids up to spermiation. In tubules undergoing spermiation, the sleeves retract so that only the tip of the spermatid head remains in the sleeve. Appendicular processes extend from the dorsum of the Sertoli cell sleeves in the tubules undergoing spermiation. After spermiation the appendix elongates while the sleeve evaginates until the everted sleeve is a finger-like process that extends into the tubular lumen. In tubules in which the seminiferous epithelium has been depleted with epinephrine injections the Sertoli cells attain two configurations. The first is characterized by having long attenuated lamellar processes that orient perpendicular to the basement membrane, with numerous ramifying processes arising from the lamellae. In the second configuration the Sertoli cells' lamellae orient parallel to the basement membrane of the tubule and lack the elaborate ramifications seen in the first configuration.     

Changes in Spermatozoa due to Large Doses of Pyridoxine (Vitamin B6)

To investigate the changes of spermatozoa by high doses of vitamin B₆, (B6), the alterations in spermatozoa and testis of rats after the administration of high doses of B6 were evaluated quantitatively and morphometrically. Wistar rats of 11 weeks of age were intraperitoneally injected with 63,125,250 and 500 mg/kg of B6 daily 5 times per week for 6 weeks. Using the spermatozoa taken from the epididymis and ductus deferens, the number, motility and nuclear morphology of spermatozoa were examined. After preparing 7 parameters for the nuclear morphology, the morphometry was performed by an IBAS version 2 (Zeiss) image analysis system. The number of spermatocytes and spermatids in representative stages of spermatogenesis was counted per Sertoli cell histologically. Mild deformation of spermatozoa nuclei occurred in the 63 mg or more exposure groups. In the 125 and 250 mg groups, the decrease in number as well as motility of spermatozoa together with slight decrease of spermatids in late maturation phase (mature spermatids) and the delay in spermia-tion appeared. Phagocytosis of mature spermatids by Sertoli cells was clearly increased in the 250 mg group. The alteration and the decreased number of spermatozoa are suggested to have mainly resulted from alteration of mature spermatids and the increased phagocytosis of mature spermatids by Sertoli cells. Computer-assisted morphometry of spermatozoa nuclei was useful not only to evaluate morphological changes objectively but also to discern them early. Acta Pathol Jpn 42: 861–869, 1992.     

Changes in spermatozoa due to large doses of pyridoxine (vitamin B6).

To investigate the changes of spermatozoa by high doses of vitamin B6 (B6), the alterations in spermatozoa and testis of rats after the administration of high doses of B6 were evaluated quantitatively and morphometrically. Wistar rats of 11 weeks of age were intraperitoneally injected with 63, 125, 250 and 500 mg/kg of B6 daily 5 times per week for 6 weeks. Using the spermatozoa taken from the epididymis and ductus deferens, the number, motility and nuclear morphology of spermatozoa were examined. After preparing 7 parameters for the nuclear morphology, the morphometry was performed by an IBAS version 2 (Zeiss) image analysis system. The number of spermatocytes and spermatids in representative stages of spermatogenesis was counted per Sertoli cell histologically. Mild deformation of spermatozoa nuclei occurred in the 63 mg or more exposure groups. In the 125 and 250 mg groups, the decrease in number as well as motility of spermatozoa together with slight decrease of spermatids in late maturation phase (mature spermatids) and the delay in spermiation appeared. Phagocytosis of mature spermatids by Sertoli cells was clearly increased in the 250 mg group. The alteration and the decreased number of spermatozoa are suggested to have mainly resulted from alteration of mature spermatids and the increased phagocytosis of mature spermatids by Sertoli cells. Computer-assisted morphometry of spermatozoa nuclei was useful not only to evaluate morphological changes objectively but also to discern them early.     

Comparison of lectin-staining pattern in testis and epididymis of gerbil,guinea pig,mouse, and nutria

The testis and epididymis of gerbil, guinea pig, nutria, and mouse were studied after staining with seven rhodamine-conjugated lectins to disclose the distribution of glycoproteins with different sugar residues. In the testis, the lectins showed a variable affinity for Leydig cells, tubular basement membrane, cytoplasm, acrosome, and plasma membrane of maturing spermatids as well as for Sertoli cell extensions. During acrosomal development, the staining pattern showed characteristic changes with different lectins indicating a gradual processing of the glycoprotein components. The staining in the Sertoli cell extensions displayed a cyclic change linked with the release of spermatozoa. A nuclear staining was prominent in zygotene and pachytene spermatocytes in the mouse, weak in the nutria, but absent in gerbil and guinea pig. The principal cells of epididymis showed a lectin-stained Golgi region as well as a similar staining in the apical surface, microvilli, and tubular contents. This staining was most prominent in the caput/corpus regions with some interspecies differences indicating the epididymal areas active in secretion. Narrow cells active in absorption of testis-derived material were lectin-positive in the initial segment of mouse, gerbil, and nutria epididymis. Large light cells with a strong affinity for some lectins were found in the proximal cauda of gerbil and guinea-pig epididymis. In the nutria, corresponding cells were arranged as islands within the low epithelium. The distal cauda of mouse, gerbil, and nutria was the site for lectin-stained light cells interspersed among the low principal cells. It is concluded that the high and low light cells may be active in the absorption and phagocytosis of residual bodies/cytoplasmic droplets and surplus epididymal secretory material, respectively. Thus, labeled lectins formed a useful tool in the analysis of glycoprotein distribution, processing, secretion, absorption, and degradation in the male reproductive tissues.     

Meiosis and spermiogenesis in the testis of Salamandra salamandra (L.) (Amphibia, Urodela)

Meiosis and spermiogenesis in the fire salamander, Salamandra salamandra, take place in an intermediate zone of the testis between the cephal immature and the more caudal mature part. Primary spermatocytes in zytogene and pachytene are characterized by synaptonemal complexes, flattened vesicles at the periphery of the cytoplasm and mitochondria with dilated cristae. Mitochondria in primary spermatocytes during meiosis, in secondary spermatocytes and early spermatids are typically arranged beneath the plasmalemma. Secondary spermatocytes are provided with pro-acrosomal granules, nucleolus-like bodies and complexes of annulate lamellae. Cytoplasmic parts with numerous vesicles seem to become extruded from secondary spermatocytes and spermatids. In testicular lobules containing pachytene spermatocytes the normally fibroblast-like follicle cells transform into glandular Sertoli cells. Already after the second meiotic division of germ cells lobule boundary cells show morphological features of steroid hormone secreting cells.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis

Sodium-potassium ATPase (Na⁺ K⁺-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na⁺ and K⁺ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na⁺ K⁺-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na⁺ K⁺-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show (1) that rat testis and epididymal Na⁺ K⁺-ATPase share some immunological determinants with the canine enzyme; (2) that the epididymal enzyme is located in the conventional basolateral position; and (3) that the distribution of Sertoli cell Na⁺ K⁺-ATPase is probably apical and lateral rather than basal.