Refrigerated storage of red deer epididymal spermatozoa in the epididymis, diluted and with vitamin C supplementation.

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 x 10(6) sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 mm vitamin C. The remaining epididymides and the diluted samples were stored at 5 degrees C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.

Translation and assembly of CABYR coding region B in fibrous sheath and restriction of calcium binding to coding region A.

CABYR is a highly polymorphic, sperm flagellar calcium-binding protein that is tyrosine as well as serine/threonine phosphorylated during capacitation. Six alternative splice variants of human CABYR (I-VI) have previously been identified, involving two coding regions, CR-A and CR-B, separated by an intervening stop codon. It is presently unknown if proteins encoded by the predicted coding region B of CABYR are translated during spermiogenesis, where they localize, or which CABYR isoforms bind calcium. Immunofluorescent and electron microscopic studies using polyclonal antibodies generated to the recombinant c-terminal 198 aa CABYR-B localized the isoforms containing CABYR-B to the ribs and longitudinal columns of the fibrous sheath in the principal piece of the flagellum. Antisera to recombinant CABYR-A and CABYR-B proteins recognized distinct populations of CABYR isoforms encoded by either CR-A alone and/or CR-B as well as a common population of CABYR isoforms. Only the recombinant CABYR-A and not the CABYR-B bound calcium in vitro, which is consistent with the hypothesis that CABYR-A is the only form that binds calcium in sperm. These observations confirmed that, despite the presence of the stop codon in CR-A, splice variants containing CR-B are expressed during spermiogenesis and assemble into the fibrous sheath of the principal piece; however, calcium binding occurs only to those CABYR isoforms containing CABYR-A.

Tubulin glycylation and glutamylation deficiencies in unconventional insect axonemes.

Though the 9+2 axonemal organization has generally been conserved throughout metazoan evolution, insect spermatozoa possess a substantial variety in axoneme ultrastructure, displaying different axonemal patterns. Therefore, insects provide a wide range of models that may be useful for the study of the mechanisms of axoneme assembly. We have used antibodies specific for glutamylated, monoglycylated, and polyglycylated tubulin to investigate the tubulin isoform content expressed in the unorthodox sperm axonemes of four insect species belonging to both of the superorders Palaeoptera and Neoptera. Each one of these axonemal models exhibits distinctive structural features, either showing the typical radial organization endowed with a ninefold symmetry or consisting of an helical arrangement with up to 200 microtubular doublets, but in all cases these axonemes share the absence of a microtubule central pair. Our results showed that all these atypical patterns are characterized by a dramatic decrease in both tubulin glycylation and glutamylation levels or even lack of both polymodifications. These data provide the first examples of a simultaneous extreme reduction or even absence of both polymodifications in axonemal tubulin. Given the unrelated positions of the analyzed species in the insect phylogenetic tree, this common feature is probably not due to evolutionary relationships. Therefore, our findings support the hypothesis of the existence of a correlation between the low level of polymodifications and the lack of a microtubule central pair in these peculiar insect flagellar axonemes, similarly as was previously proposed for cilia of Tetrahymena glycylation site mutants.

Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni.

The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme. Our results have shown that the unorthodox structure and motility of the Asphondylia axoneme are concomitant with: (1). a very low glutamylation extent in the alpha-tubulin subunit, (2). a high level of glutamylation in the beta-subunit, (3). an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in alpha- or in beta-tubulin, (4). the presence of a strong labeling of glutamylated tubulin isoforms at the proximal end of the axoneme, and (5). a uniform distribution of glutamylated as well as glycylated isoforms along the rest of the axoneme. Thus, our data indicate that tubulin molecular heterogeneity is much lower in the Asphondylia axoneme than in the conventional 9+2 axoneme with regard to both isoform content and isoform distribution along the axoneme.

Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats.

In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.

Ultrastructural changes induced by leaves of Azadirachta indica (Neem) in the testis of albino rats.

The present work was designed to study the effect of Azadirachta indica (Neem) powder on rat testis using the electron microscope. Male albino rats received 100 mg each A. indica leaf powder orally (by gavage). On alternate days, a second group of rats received 0.125 mg testosterone dipropionate intramuscularly. A third group received both A. indica leaf powder by gavage and testosterone dipropionate intramuscularly. Suitable controls were maintained. After autopsy, ultrastructural analysis of the testis revealed that animals treated with testosterone dipropionate showed well-developed Sertoli cells and germ cells with well-developed cytoplasmic organelles. By contrast, in A. indica-treated rats, intracellular spaces and vacuolization were observed in Sertoli cells; whereas in Leydig cells, cytoplasmic inclusions appeared diminished, and the configuration of granular endoplasmic reticulum appeared as a single unbranched tubule. In late spermatids, defects were observed in the mitochondrial sheath. The ultrastructural changes seen in the A. indica-treated group provide a clue that A. indica leaves might affect spermatogenesis through antispermatogenic and antiandrogenic properties.

Deleterious effects of khat addiction on semen parameters and sperm ultrastructure.

The semen parameters and sperm ultrastructural morphology have been described in semen samples from two groups of Yemeni subjects. The first 'exposed' group comprised 65 khat addicts, while the second control group included 50 non-khat addict subjects. The mean age was 39.94 +/- 13.85 and 35.72 +/- 11.35 years in the exposed and control groups respectively, without a significant difference. The mean duration of khat addiction among the addicts was 25.34 +/- 12.96 years (range 6.00-48.00). Statistically significant differences were detected between the semen parameters of the two groups. Such parameters, including semen volume, sperm count, sperm motility, motility index and percentage of normal spermatozoa, were lower among addicts. Significant negative correlation was also found between the duration of khat consumption and all semen parameters (r ranged from -0.30 to -0.74). At the transmission electron microscopy level, a counting system was incorporated to compare the numbers of normal spermatozoa with deformed and dead spermatozoa in ultrathin plastic sections. The total mean percentage of deformed spermatozoa was approximately 65%. Different patterns of sperm deformation were demonstrated, and included both the head and flagella in complete spermatozoa, aflagellate heads, headless flagella and multiple heads and flagella. Deformed heads showed aberrated nuclei with immature nuclear chromatin and polymorphic intranuclear inclusions; these were associated with acrosomal defects. The deformed flagella demonstrated numeric aberrations of the axonemal 9 + 2 configuration and structural defects of their associated elements. Persistent cytoplasmic droplets were observed frequently. This study has shown for the first time the deleterious effects of khat addiction on semen parameters in general and sperm morphology in particular of all addicts, especially those who have consumed khat for longer periods of time.

Hyperactivated motility is coupled with interdependent modifications at axonemal and cytosolic levels in human spermatozoa.

Whether the motility characteristics of hyperactivated spermatozoa were determined by stable changes at the axonemal level and whether the presence of cytosolic factors was required for the expression of these changes was investigated. Different degrees of sperm hyperactivation were produced in Percoll-washed spermatozoa after incubation for 1 hour to 3 hours at 37 degrees C in Ham's F-10 supplemented with human blood plasma or fetal cord serum. Decomplemented fetal cord serum induced the highest percentage of hyperactivation (19 +/- 3%), followed by human plasma (13 +/- 2%). Fetal cord serum that was not decomplemented did not induce a level of hyperactivation (1.7 +/- 0.2%) significantly different from control levels (0.9 +/- 0.2%). Dialyzed fetal cord serum induced intermediate levels of hyperactivation (6 +/- 1%). The motility characteristics of demembranated sperm models of hyperactivated spermatozoa induced by decomplemented fetal cord serum and nonhyperactivated spermatozoa were compared by videomicroscopy and computer-assisted digital image analysis. After demembranation with Triton X-100 and reactivation of motility by Mg. adenosine triphosphate (Mg.ATP), hyperactivated and nonhyperactivated spermatozoa showed similar motility characteristics. However, hyperactivated spermatozoa that were demembranated and reactivated in cytosolic extracts from hyperactivated spermatozoa had significantly higher (P less than 0.05) linear velocity (33 +/- 4 mu/sec) and lower linearity (0.23 +/- 0.04) than control spermatozoa that were demembranated and reactivated in control cytosolic extracts (velocity = 24 +/- 1 mu/sec; linearity = 0.32 +/- 0.02). The data suggest that the expression of hyperactivated motility requires interdependent changes at the axonemal and cytosolic levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure sperm defects in addicts.

Addiction is a major problem confronting the whole world today. Disruption of interpersonal relationships, economic loss, and crimes against property are frequent consequences. Harm to the individual himself extends to all physiological systems. In the present study, we examined semen samples of six addicts by light and electron microscopy. Oligoasthenospermia was demonstrated in five patients, and necrospermia was observed in one patient. Severe degenerative changes of the sperm heads were noted. Granular condensation of the chromatin with nuclear vacuoles was demonstrated. Persistent cytoplasmic droplets were frequently observed. Degenerated tails showing fragmentation of the plasma membranes and numerical aberrations of the 9 + 2 configuration were also present, together with thickened and disorganized fibrous sheaths. These results confirm the deleterious effects of addiction on the entire sperm structure.