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.     

Rat Sertoli and spermatogenic cells express a similar gene, and its product is antigenically related to an outer dense fiber-associated protein.

We have previously reported that a heterodimeric protein secreted by rat Sertoli cells is antigenically related to a protein associated with outer dense fibers of the sperm tail. Therefore, we have explored the possibility that Sertoli and spermatogenic cells express a similar gene encoding a homologous protein. A Sertoli cell heterodimeric protein cDNA probe recognizes specific mRNA in pachytene and round spermatids fractionated by centrifugal elutriation; however, this specific mRNA was less prominent than in cultured Sertoli cells. In agreement with these observations, in situ hybridization experiments show that Sertoli cells are predominantly engaged in active heterodimeric protein mRNA synthesis, while meiotic prophase spermatocytes and spermatids also show significant but less abundant specific mRNA. Immunoblotting experiments demonstrate that, while Sertoli cells synthesize a heterodimeric protein consisting of two disulfide-linked components with molecular masses of 45 and 35 kD, both primary spermatocytes and round spermatids synthesize single 30 kD monomers not associated by disulfide linkage but recognized by antisera to Sertoli cell heterodimeric protein. Immunoblotting and immunogold electron microscopic studies show that antisera to Sertoli cell heterodimeric protein recognize a protein associated with outer dense fibers. This immunoreactivity was abolished by a 5-min pronase treatment, without affecting the integrity of outer dense fibers. Results of this study and previous studies demonstrate that both Sertoli and spermatogenic cells express a similar gene and that an antigenically related product encoded by this gene becomes associated with outer dense fibers during their assembly at spermiogenesis.     

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats

Summary Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6–10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal Leydig cells resulting in their disappearance by day 7. Destruction of foetal Leydig cells was followed by an arrest of testicular growth in comparison to testes of intact age-matched control rats. In testes of EDS-treated rats, gonocytes and spermatogonia also degenerated, forming pyknotic bodies within the seminiferous cords. In contrast, interstitial fibroblasts and mesenchymal cells showed proliferative activity, which on days 4 and 5 after treatment resulted in peritubular hyperplasia surrounding each seminiferous cord. Thereafter, on day 21 after EDS administration, the previously depressed serum testosterone levels became markedly elevated coincident with the development of many immature-type Leydig cells, of which the total volume per testis was similar to that of Leydig cells in control testes, despite a four- to five-fold difference in testicular volumes. The results indicate that, although EDS destroys the foetal Leydig cells and impairs spermatogenesis, the interstitial tissue exhibits increased cell growth. The latter probably occurs in response to altered gonadotrophic stimulation and/or disturbances in the interaction between the seminiferous cords and the interstitial tissue.     

Biology and regulation of ectoplasmic specialization, an atypical adherens junction type, in the testis

Anchoring junctions are cell adhesion apparatus present in all epithelia and endothelia. They are found at the cell-cell interface (adherens junction (AJ) and desmosome) and cell-matrix interface (focal contact and hemidesmosome). In this review, we focus our discussion on AJ in particular the dynamic changes and regulation of this junction type in normal epithelia using testis as a model. There are extensive restructuring of AJ (e.g., ectoplasmic specialization, ES, a testis-specific AJ) at the Sertoli-Sertoli cell interface (basal ES) and Sertoli-elongating spermatid interface (apical ES) during the seminiferous epithelial cycle of spermatogenesis to facilitate the migration of developing germ cells across the seminiferous epithelium. Furthermore, recent findings have shown that ES also confers cell orientation and polarity in the seminiferous epithelium, illustrating that some of the functions initially ascribed to tight junctions (TJ), such as conferring cell polarity, are also part of the inherent properties of the AJ (e.g., apical ES) in the testis. The biology and regulation based on recent studies in the testis are of interest to cell biologists in the field, in particular their regulation, which perhaps is applicable to tumorigenesis.     

Transforming growth factor-β1 in reproduction

Expression patterns of TGF-βs during embryogenesis and in adult reproductive organs, as well as the activities of these molecules in in vitro assays of biological processes relating to reproduction and development, have suggested that TGF-βs may play a role in both reproductive function and embryonic development. To investigate the function of TGF-β1 in vivo, the murine TGF-β1 gene was disrupted by gene targeting, and animals that lacked TGF-β1 activity were generated. Homozygous mutant animals were obtained which exhibited a multifocal inflammatory disease. However, the observed numbers of homozygous mutant offspring were less than expected, suggesting the occurrence of some type of prenatal lethality. This paper reviews the proposed role of the TGF-βs in reproductive and developmental processes and discusses observations obtained from the TGF-β1 gene-targeting experiments as they relate to these processes. © 1994 Wiley-Liss, Inc.     

Fasciola hepatica: A light and electron microscope autoradiographic study of incorporation of monosaccharides into glycogen and glycoprotein

Incorporation of [³H]galactose and [³H]glucose into the parenchyma, tegument, testis, and muscle of Fasciola hepatica slices was studied by lightand electron-microscope autoradiography. “Accumulation” labeling periods of up to 60 min were used.Both monosaccharides were found to be readily incorporated into glycogen in the parenchymal cells and muscle and [³H]glucose entered the glycogen stores of spermatozoa.No evidence was found for the involvement of any particular cell organelle in glycogenesis, but the demonstration of high synthetic activity in parenchymal evaginations to the base of the surface syncytial tegument supports physiological evidence that glucose enters the fluke mainly across the tegument.Ethylene glycol-dehydrated preparations showed that [³H]galactose was incorporated into glycoprotein by Type I tegumental cells, and perhaps also by sperm morulae. The carbohydrate component seems to be added to the tegumental secretions in the vesicular-lamellar region of the Golgi complex.Following the longest periods of incubation, labeling was observed in the tubules connecting the tegumental cells and syncytium, but not in the surface syncytium itself.     

ELF MAGNETIC FIELD EFFECTS ON FATTY ACID COMPOSITION OF PHOSPHOLIPID FRACTION AND REPRODUCTION OF RATS' TESTES

In this study, we investigated how rat reproductive cells, testosterone, and the fatty acid composition of the phospholipid fraction of rats' testis cells are affected by extremely low frequency magnetic field (ELFMF). The change in fatty acid composition of the membrane phospholipid fraction can be the mechanism for this effect. We used a total of 26 male Wistar Albino rats, 14 experimental, and 12 controls. The experimental group rats were exposed to a magnetic field (0.8 mT) for 5 weeks, 3 hr per day. The control group rats were kept between inactive coils. After 5 weeks, the testis tissues and sperm cells of all rats were histopathologically investigated and sperm counts determined. Epididymal sperm count did not change compared to the control group (p>.05). Besides this, amorphous head, banana-like head, hammer head, coiled tail, abnormal mid-piece and tail, multiple, and cytoplasmic-droplet type cell numbers did not change in either group (p>.05). However, a statistical difference was found between the control and experimental groups with respect to head with lack of hook and isolated head type sperm (p<.05). In addition, testosterone levels were also found to be altered (p<.05). In the histopathologic investigation of testis tissue, decreased spermatogenesis in some seminiferous tubules, congestion in blood vessels of the interstitium, and increases in interstitial edema and Sertoli cells were observed. Leydig cells were found to be normal in appearance. The fatty acid of the testis cell membrane phospholipids was decreased in the experimental group with respect to the control group.     

Health status of the lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) subject to different anthropogenic pressures

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.     

四氯化碳联合二乙基亚硝胺对小鼠精巢的毒性作用

为了研究四氯化碳(CCL4)联合二乙基亚硝胺(DEN)对小鼠(Mus musculus domesticus)精巢的毒性作用。将昆明小鼠随机分为对照组与实验组,各12只个体。实验组每周2次腹腔注射含有15%CCl4的花生油溶液,同时自由饮用0.07‰的DEN溶液,连续诱导8周。对照组每周2次注射花生油,饮用蒸馏水。分别于第3、6、8周处死实验组及对照组小鼠各4只,对其精巢组织进行常规固定、包埋、切片、H.E染色,观察精巢的形态及精子数量的变化。与实验组比较,对照组小鼠体重明显增加,且实验组与对照组差异显著;与对照组相比,实验组睾丸系数以及精子存活率均显著减少。对照组小鼠精巢都具有正常的生精小管结构,管腔中存在大量精子;第3周,实验组小鼠的精巢与对照组类似,生精小管较为完整,生精细胞稍有散乱;第6周,实验组生精小管变形,生精细胞排列疏松,精子数目减少且畸形;第8周,实验组生精小管残缺不全,生精细胞和精子散乱排列,精子数量大大减少且变形更为严重。实验说明,CCl4联合DEN能够造成小鼠精巢组织的损害以及精子数目的减少和畸变。