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

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

Assembly of spermatid acrosome depends on microtubule organization during mammalian spermiogenesis

The acrosome is a secretory vesicle attached to the nucleus of the sperm. Our hypothesis is that microtubules participate in the membrane traffic between the Golgi apparatus and acrosome during the first steps of spermatid differentiation. In this work, we show that nocodazole-induced microtubule depolarization triggers the formation of vesicles of the acrosomal membrane, without detaching the acrosome from the nuclear envelope. Nocodazole also induced fragmentation of the Golgi apparatus as determined by antibodies against giantin, golgin-97 and GM130, and electron microscopy. Conversely, neither the acrosome nor the Golgi apparatus underwent fragmentation in elongating spermatids (acrosome- and maturation-phase). The microtubule network of round spermatids of azh/azh mice also became disorganized. Disorganization correlated with fragmentation of the acrosome and the Golgi apparatus, as evaluated by domain-specific markers. Elongating spermatids (acrosome and maturation-phase) of azh/azh mice also had alterations in microtubule organization, acrosome, and Golgi apparatus. Finally, the spermatozoa of azh/azh mice displayed aberrant localization of the acrosomal protein sp56 in both the post-acrosomal and flagellum domains. Our results suggest that microtubules participate in the formation and/or maintenance of the structure of the acrosome and the Golgi apparatus and that the organization of the microtubules in round spermatids is key to sorting acrosomal proteins to the proper organelle.     

Sperm ultrastructure and a new type of spermiogenesis in two species of Pimelodidae, with a comparative review of sperm ultrastructure in Siluriformes (Teleostei: Ostariophysi)

During spermiogenesis, the spermatids of the pimelodid species Pimelodus maculatus and Pseudoplatystoma fasciatum show a central flagellum development, no rotation of the nucleus, and no nuclear fossa formation, in contrast to all previously described spermatids of Teleostei. These characteristics are interpreted as belonging to a new type of spermiogenesis, named here type III, which is peculiar to the family Pimelodidae. In P. maculatus and P. fasciatum, spermatozoa possess a spherical head and no acrosome; their nucleus contains highly condensed, homogeneous chromatin with small electron-lucent areas; and a nuclear fossa is not present. The centriolar complex lies close to the nucleus. The midpiece is small, has no true cytoplasmic channel, and contains many elongate and interconnected vesicles. Several spherical to oblong mitochondria are located around the centriolar complex. The flagellum displays the classical axoneme (9+2) and no lateral fins. Only minor differences were observed among the pimelodid species and genera. Otherwise, spermiogenesis and spermatozoa in the two species of Pimelodidae studied exhibit many characteristics that are not found in other siluriform families, mainly the type III spermiogenesis.     

A missense mutation in the Capza3 gene and disruption of F-actin organization in spermatids of repro32 infertile male mice

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific “capping protein (actin filament) muscle Z-line, alpha 3” gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse.     

Delta-tubulin is a component of intercellular bridges and both the early and mature perinuclear rings during spermatogenesis

Mammalian spermatogenesis involves drastic morphological changes leading to the development of the mature sperm. Sperm development includes formation of the acrosome and flagellum, translocation of nucleus-acrosome to the cell surface, and condensation and elongation of the nucleus. In addition, spermatogenic cell progenies differentiate as cohorts of units interconnected by intercellular bridges. Little is known about the structural components involved in the establishment of conjoined spermatogenic cells and the mechanism of nuclear shaping of the male gamete. We identified two isoforms of delta-tubulin and found that the long isoform is predominantly expressed in testis, while the short isoform is expressed in all tissues examined. We also found that delta-tubulin forms intercellular bridges conjoining sister spermatogenic cells. In addition, delta-tubulin is a component of the perinuclear ring of the manchette, which acts on translocation and elongation of the nucleus. Furthermore, small rings clearly distinct from the intercellular bridges, which might mature to perinuclear ring of the manchette in later stages of spermatogenesis, were detected on the cell surface of round spermatids. These results suggest that delta-tubulin is a component of two types of ring, the intercellular bridges and the perinuclear rings, which may be involved in morphological changes of spermatid to mature sperm.     

Process of nuclear envelope reduction in spermiogenesis of a mosquito,Culex tigripes

When the Culex tigripes spermatid begins to elongate, the nucleus exhibits on its surface invaginations of the nuclear envelope. These invaginations have a uniform diameter of 0.3 μm. They separate from the envelope of the nucleus and form spherical intranuclear vesicles. In the old spermatids these vesicles are imprisoned in the condensed chromatin. The spermatozoon also possesses these vesicles which are then ovoid in shape. This process of vesiculation permits the diminution of the surface of the nucleus when it decreases in volume during spermiogenesis. © 1993 Wiley-Liss, Inc.     

大鼠精核蛋白的纯化

用大鼠睾丸制备长形精子细胞核及用附睾制备的附睾精子核,用盐酸胍法提取总碱性蛋白（Ｔｏｔａｌ　ｂａｓｉｃ　ｐｒｏｔｅｉｎ,ＴＢＰ）。通过Ｓｅｐｈａｄｅｘ　Ｇ－１００分子筛层析,其第三峰用５％ＴＣＡ沉淀,得到纯大鼠精核蛋白（Ｒａｔ　ｐｒｏｔａｍｉｎｅ,ＲＰ）。ＲＰ也可用ＴＢＰ通过ＨＰＬＣ反相柱得到纯化,ＲＰ位于２６％－２８％乙腈梯度。