The incorporation of macromolecules into the germ cells of male mice via electroporation and dimethyl sulfoxide

Electroporation (incorporation of macromolecules into the living cells by means of electric pulses) provides inclusion of plasmid 14C-DNA into immature cells of spermatogenic epithelium. The highest level of foreign DNA incorporation into spermatocytes and spermatids has been induced by 8kV electric pulses applied 3 times with 20 sec intervals. Meanwhile, mature sperms are found to be exclusively resistant to exogenous DNA irrespective of the voltage level, the number of pulses and Ca++ uptake (contents). Incubation of mature sperms for two hours in the medium with Ca++ (10 mM) and dimethylsulfoxide--(DMSO, 33%) provides highly reliable incorporation of plasmid 14C-DNA into sperm heads. The sperm cells with foreign DNA incorporated by means of Ca++ and DMSO treatment still remain alive and mobile. The possibilities of mature sperms loaded with foreign DNA for the creation of transgenic mammals are discussed.     

An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water

Dielectrophoresis (DEP), the motion of a particle caused by an applied electric field gradient, can concentrate microorganisms non-destructively. In insulator-based dielectrophoresis (iDEP) insulating microstructures produce non-uniform electric fields to drive DEP in microsystems. This article describes the performance of an iDEP device in removing and concentrating bacterial cells, spores and viruses while operated with a DC applied electric field and pressure gradient. Such a device can selectively trap particles when dielectrophoresis overcomes electrokinesis or advection. The dielectrophoretic trapping behavior of labeled microorganisms in a glass-etched iDEP device was observed over a wide range of DC applied electric fields. When fields higher than a particle-specific threshold are applied, particles are reversibly trapped in the device. Experiments with Bacillus subtilis spores and the Tobacco Mosaic Virus (TMV) exhibited higher trapping thresholds than those of bacterial cells. The iDEP device was characterized in terms of concentration factor and removal efficiency. Under the experimental conditions used in this study with an initial dilution of 1 x 105 cells/ml, concentration factors of the order of 3000x and removal efficiencies approaching 100% were observed with Escherichia coli cells. These results are the first characterization of an iDEP device for the concentration and removal of microbes in water.     

Phenotype and fertilizing capacity of spermatozoa of chimaeric mice produced from two strains that differ in sperm quality

Spermatozoa of males from the inbred mouse strains, KE (albino) and CBA (agouti), are distinguishable by head shape and differ in quality: CBA spermatozoa show a lower percentage of abnormal heads and higher efficiency of fertilization. Aggregation chimaeras were produced to investigate whether these differences are intrinsic or extrinsic to spermatogenic cells. Among 24 overt chimaeras, 14 were males: 1 was sterile, 8 produced either KE or CBA spermatozoa, as recognized by shape and by progeny testing, and 5 (21%) were germ-line chimaeras. Both the level of abnormal sperm heads and the efficiency of fertilization of CBA and KE spermatozoa produced by chimaeric males (except two subfertile ones) were within the range characteristic for the respective strain. All 5 germ-line chimaeras, irrespective of their coat colour composition, produced about 98% KE and only 2% CBA spermatozoa, which indicated strong selection against CBA germ cells. However, mature CBA spermatozoa showed high competitive ability, because the proportion of agouti progeny (from the CBA component) sired by those males was significantly higher than the proportion of CBA spermatozoa, estimated from vaginal plug preparations after every mating. The fact that this difference was particularly striking for one chimaera with a preponderance of CBA somatic component may suggest some influences extrinsic to spermatogenic cells. We conclude from this study that sperm head shape, the level of sperm abnormalities and fertilizing capacity are determined largely autonomously by genes acting in the germ cells. The internal environment created by foreign somatic cells exerts only minor modifications, unless there has been deterioration beyond the range of 'normality'.     

The fertilising ability of spermatogenic cells derived from cultured mouse immature testicular tissue

There are many reports about the in vitro culture of spermatogenic cells, but no-one has succeeded in inducing the differentiation from spermatogonia to intact sperm. Also the study of in vitro testicular tissue culture has hardly advanced. We studied the culture of mouse immature testicular tissue derived from 5-day-old mice. We aimed to achieve the differentiation of spermatogenic cells in order to observe spermatogenesis in testicular tissue in vitro. We also froze mature testicular tissue and immature testicular tissue cultured for 2 weeks. Furthermore, spermatogenic cells differentiated by culturing were injected into metaphase II oocytes to determine whether these differentiated cells and frozen-thawed testicular tissue have fertilising and developmental ability. Under the culture conditions employed, secondary spermatocytes and a few round spermatids differentiated from spermatogonia were observed in the immature testicular tissue cultured for 2 weeks. When spermatogenic cells derived from cultured immature testicular tissue, cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were injected into ooplasm, the oocytes were fertilised and fertilised oocytes developed to the 8-cell stage. We suggest that spermatogenic cells derived from cultured immature testicular tissue have fertilising and developmental abilities equivalent to that of sperm. Also these abilities of spermatogenic cells obtained from cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were better than those of the same cells before freezing.     

Cell Culture of Spermatogenic Cells from Amphibians

Cell culture of spermatogenic cells from the primary spermatocyte stage to the early, midspermatid stage has been established for the Japanese newt, Cynops pyrrhogaster (1–4, 10, 11) and for Xenopus laevis (2, 3, 5, 17, 18). The results of many investigations suggest that sperm maturation in vitro for each species proceeds along the same pathway and under the same temporal control as in vivo . Since the shape and size of mature sperm of these two species are very different, comparative studies would enable us to elucidate the mechanisms by which sperm morphology is determined. The results obtained from in vitro studies of spermatogenesis in Cynops and Xenopus demonstrate that there are common and species-specific features.     

生精细胞凋亡相关基因

细胞凋亡(apoptosis)是一种基因控制的细胞生理性自杀行为,用以维持细胞数量的相对恒定,可由某种刺激或抑制剂的移除而激活。在哺乳动物精子发生过程中,各级生精细胞都会发生相应的凋亡,通过严格调控以确保成熟精子生成的数量和质量。生精细胞的凋亡是一个许多基因参与的复杂的不可逆过程,其中Bcl-2/Bax基因族、p53基因、Fas-Fasl基因、C-myc基因、CREM基因、HSP基因族、c-Kit/SCF基因、Insl3基因、iNOS基因、BMP8B基因、TR基因和存活蛋白(survivin)基因等发挥了重要作用。研究哺乳动物睾丸生精细胞凋亡相关基因,有利于了解生精细胞凋亡机制,为进一步阐明精子发生的调控机制,预防和治疗精子发生相关疾病提供重要的理论依据。     

The soluble mammalian sperm factor protein that triggers Ca2+ oscillations in eggs: evidence for expression of mRNA(s) coding for sperm factor protein(s) in spermatogenic cells

At fertilisation in mammals the sperm initiates a series of Ca2+ oscillations that activate development. One theory of signalling at fertilisation suggests that the sperm contains a soluble protein factor that causes these Ca2+ oscillations by entering the egg after sperm-egg membrane fusion. This theory is supported by the finding that, in some species, injection of sperm protein extracts into eggs triggers a pattern of Ca2+ oscillations similar to those seen at fertilisation. So far, all the direct evidence for a sperm factor has been based upon the injection of soluble proteins from mature sperm. Here, we demonstrate that injection of mRNA extracted from hamster spermatogenic cells also leads to generation of prolonged Ca2+ oscillations in mouse eggs. The ability of spermatogenic cell mRNA to induce Ca2+ oscillations is dependent upon translation into protein and also appears to be specific to spermatogenic cells since injection of mRNA isolated from somatic tissues into eggs was ineffective. These data support the hypothesis that sperm contain a soluble, cytosolic protein factor that induces Ca2+ oscillations in eggs at fertilisation. These data are discussed in the light of our recent findings that suggest that the sperm factor possesses a phospholipase C activity.     

Rat testicular dolichols: distribution and accumulation with age

Dolichols, linear isoprenoids essential in the biosynthesis of N-glycosylated glycoproteins, are abundant in testicular tissue. This study investigated the distribution of dolichols among testicular cell and subcellular fractions. In addition, the accumulation of dolichol within the rat testis as a function of age was investigated. Dolichol content expressed either as total dolichol/testicle or as dolichol/mg protein exhibited a marked and continuous increase between 14 and 60 days of age. The 4-, 6-, 9-, and 12-mo-old animals exhibited only minor increases in testicular dolichol content. Mean value for retired breeders was 279 ng dolichol/mg protein. Although previous studies have suggested that dolichol synthesis occurs primarily within the spermatogenic cell, elutriation-purified spermatogenic cell fractions showed very low concentrations of dolichol. Pachytene spermatocyte and round spermatid fractions contained 25.8 and 36.5 ng dolichol/mg protein, respectively. Washed epididymal sperm also had a very low dolichol content (18.8 ng dolichol/mg protein). Recovery studies during elutriation purification of spermatogenic cells showed that the majority of dolichol was contained within the Sertoli-rich tubular fragments. Microsomal fractions isolated from whole testis exhibited a small enrichment (1.6-fold) in dolichol content, whereas Golgi apparatus fractions exhibited a large (12-fold) enrichment over that of the initial homogenate. These studies suggest that, although dolichols may be synthesized within the spermatogenic cell, they accumulate within the Sertoli cell.     

Spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs

Starting from the period of testis differentiation, the Sertoli cell plays a pivotal role in the development of a functional testis. FSH is the major mitotic factor for Sertoli cells. Because the supporting capacity of Sertoli cells is relatively fixed for each species, their total number per testis, established just before puberty (approximately 4 months in pigs), dictates the potential for sperm production. In contrast to Sertoli cells that are still undifferentiated, mature Leydig cells are already present at birth in pigs. Spermatogenesis lasts from 30 to 75 days in mammals, and this time period is under the control of the germ cell genotype. In boars, each spermatogenic cycle and the entire spermatogenic process lasts 8.6-9.0 and approximately 40 days, respectively. The sperm transit through the epididymis takes approximately 10 days in pigs and this is within the range cited for most mammals. Germ cell loss occurs normally during spermatogenesis, mainly during the spermatogonial and meiotic phases. In pigs, significant germ cell loss also takes place during spermiogenesis. In mammals in general, including pigs, only 2-3 out of a possible 10 spermatozoa are produced from each differentiated type A1 spermatogonium. The high supporting capacity of Sertoli cells and the short duration of the spermatogenic cycle are the main factors responsible for the comparatively high spermatogenic efficiency of pigs.     

Phagocytosis of sperm by follicle cells of the carnivorous sponge Asbestopluma occidentalis (Porifera, Demospongiae)

During spermatogenesis of the carnivorous sponge Asbestopluma occidentalis, follicle cells that lined the spermatocysts phagocytosed unreleased mature sperm. Such follicle cells are part of the complex envelope that limits spermatocysts of A. occidentalis, which is also comprised of a collagen layer, a thick layer of intertwined cells, and spicules. Follicle cells showed vesicles containing single phagocytosed spermatozoa within their cytoplasm. Additionally, lipids and other inclusions were observed within the cytoplasm of follicle cells. It is likely that follicle cells recapture nutrients by phagocytosing spermatozoa and use them to form lipids and other inclusions. Such sperm phagocytosis is usually performed in higher invertebrates and vertebrates by Sertoli cells that are located in the testis wall. While Sertoli cells develop a wide range of functions such as creating a blood-testis barrier, providing crucial factors to ensure correct progression of spermatogenesis, and phagocytosis of aberrant, degenerating, and unreleased sperm cells, sponge follicle cells may only display phagocytotic activity on spermatogenic cells.     

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.     

Chromatin and chromosomal fine structure in spermatogenesis of some species of amphibians

Spermatogenesis is a complex differentiation process which is characterised, among other features, by conspicuous stage-specific nuclear events such as the pairing of homologous chromosomes coupled with the formation of synaptonemal complexes, the replacement of histones with sperm-specific proteins during spermiogenesis and, as a result, chromatin condensation and its inactivation in sperm cells. The chromatin of spermatogenic cells undergoes dramatic conformational changes upon differentiation from spermatogonia to mature spermatozoa. During the haploid stage of spermatogenesis, histones are gradually replaced, firstly by transition proteins and later by sperm-specific proteins. As a result of the high degree of condensation and inactivation of spermatid and sperm chromatin, Sertoli cells are responsible for the nourishment of germ cells with ribosomal RNA and nutritive substances.     

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.     

Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain–containing protein, develop normally but manifest male sterility. Pih1d3^−/− sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.     

棕色田鼠睾丸及附睾胚后发育的形态学变化

通过组织学方法,对产后1 d、10 d、25 d、45 d、60 d及70 d的棕色田鼠Lasiopodomys mandarinus睾丸和附睾发育进行了观察,以探讨其精子发生特点.结果 发现,1 d棕色田鼠的生殖细胞主要是生殖母细胞和前精原细胞;10 d出现大量精原细胞,睾丸间质细胞明显;25 d出现精子细胞;45 d有少量精子出现;60 d和70 d具有各级生精细胞,睾丸生精小管和附睾内出现大量成熟精子.睾丸生精小管管径和生精上皮厚度随日龄增加,于60 d达到最大;附睾管腔直径和附睾上皮厚度也于60 d达到最大.这些结果表明,棕色田鼠在生后45 d左右进入青春期,60 d左右达到性成熟,精子的产生及成熟与附睾的发育同步.     

TMF/ARA160: A key regulator of sperm development

TMF/ARA160 is a Golgi-associated protein to which several cellular activities have been attributed. These include, trafficking of Golgi-derived vesicles and E3 ubiquitin ligase activity. Here we show that TMF/ARA160 is required for the onset of key processes which underlie the development of mature sperm in mammals.TMF/ARA160 is highly expressed in specific spermatogenic stages. While the protein is not detected in the spermatogenic progenitor cells — spermatogonia, it accumulates in the Golgi of spermatocytes and spermatids but then disappears and is absent from spermatozoa and epididymal sperm cells. Mice that are homozygous null for TMF develop normally are healthy and the females are fertile. However, the males are sterile and their spermatids suffer from several developmental defects. They lack homing of Golgi-derived proacrosomal vesicles to the perinuclear surface, resulting in spermatozoa and epididymal sperm cells which lack acrosome. In a later developmental stage, the cytoplasm is not properly removed, thus resulting in spermatids which bare the nucleus with tightly packed DNA, surrounded by a cytoplasm. Finally, the spermatozoa of TMF^−/− mice also suffer from misshaped heads, tails coiling around the sperm heads, and lack of motility. Taken together our findings portray TMF/ARA160 as a key regulator which is essential for the onset of key events in the differentiation and maturation of mammalian sperm and whose absence severely compromises their ability to fertilize ova.     

Morphology of the male system and spermatophores of the arrowworm Ferosagitta hispida (Chaetognatha)

Transmission electron microscopy reveals that the somatic testicular tissues and sperm ducts are elaborations of the epithelial lining of the tail coelom. The testes consist of closely packed spermatogonia embedded between specialized lateral field cells. These cells contain few organelles and appear to function mainly as a compartment boundary. Masses of spermatogenic cells are released into the tail coelom from the anterior end of the testes. The sperm ducts, lined by simple cuboidal ciliated epithelium, collect mature spermatozoa from the tail coelom and convey them to the blindly ending seminal vesicles. The sperm ducts also modify coelomic fluid entering them along with the spermatozoa. The seminal vesicles consist of a simple glandular lining epithelium embedded in the stratified epidermis. Secretions of the lining epithelium surround the enclosed sperm mass and correspond in position to a noncellular spermatophore coat visible by light microscopy around released sperm masses. Spermatophores leave the seminal vesicles through a temporary split that forms between microfilament-containing suture cells. Maturation of spermatozoa and filling of the seminal vesicles is cyclical, occurring late each day. © 1994 Wiley-Liss, Inc.     

Transformation of the Hprt gene with DNA from spermatogenic cells

DNA-mediated transformation of hypoxanthine guanine phosphoribosyl transferase (HPRT)-deficient cells was used to assess the state of the X chromosome Hprt gene in spermatogenic cells. It had been shown previously that DNA from the inactive X chromosome of somatic cells functions poorly or not at all in HPRT transformation, indicating that DNA modification is involved in somatic cell X chromosome inactivation (XCI). In contrast, DNA from mature sperm does function in HPRT transformation suggesting that DNA modification may not be the basis of XCI in mature sperm. In this paper, transformation of HPRT^– mouse and hamster cells has been performed to test the nature of XCI during earlier stages of spermatogenesis. DNA from these developing murine germ cells was shown to be capable of HPRT transformation, extending the observation that XCI in sperm does not appear to involve a DNA modification. We also show here that DNA from mature sperm of marsupials functions in HPRT transformation, a result consistent with a role for sperm XCI in the evolution of somatic X inactivation.