Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

The ageing phenomenon of turbot spermatozoa: effects on morphology, motility and concentration, intracellular ATP content, fertilization, and storage capacities

The deleterious effect of the ageing phenomenon of turbot spermatozoa was investigated in relation to the sampling date. Spermatozoa with a low or highly condensed chromatin and a middle piece containing numerous or a few vesicles were observed simultaneously 80 and 47 days before the beginning of the spawning period of the females. The middle piece of spermatozoa contained few vesicles, 39 days after the end of the reproductive period. At the same date, some spermatozoa appeared in which the plasma membrane was broken. Sperm motility, assessed just after collection in terms of arbitrary motility scores from 0 to 5, was significantly increased both at 10 and 60 s post-activation, for samples collected 18 days after, 25 days before and 9 days after the beginning of the spawning period of the females, respectively compared to samples collected 6 days before, 55 days after and 88 days after the end of this period. A lower short-term storage capacity was recorded at 10 and 60 s post-activation for sperm samples collected 6 days before and 88 days after the end of the reproductive period, respectively compared to 18 days and 9 days after the beginning of the spawning period. At 60 s post-activation, a higher motility of thawed spermatozoa was observed for samples collected 5 days before the beginning of the spawning period (motility recovery index: 86.4 ± 19.4%) compared to 71 days after the end of this period (55.0 ± 12.0%). The fertilizing capacity of sperm samples collected 61 days after the end of the spawning season (66.1 ± 14.6%) was significantly lower than that recorded for samples collected 34 days after the beginning of the spawning period (75.2±9.6%). On the contrary, there was no significant decrease in endogenous ATP content (31 days after the beginning of the spawning period, 14.53 ± 0.84; 48 days after the end of this period, 10.75 ± 5.26 nmol 10^{? 8} spermatozoa). Furthermore, sperm concentration significantly increased between the same dates (respectively 3.3 ± 0.8–9.4±4.8×10⁹ spermatozoa ml^?1).     

Use of negative staining technique and electron microscopy for the study of structural anomalies of outer dense fibres of human flagellum

Motility disorders due to tail defects are often seen in clinical andrology. Sperm motility should be assessed with regard to the morphology of the flagellum. Since suitable longitudinal sections are rarely obtained by routine transmission electron microscopy (TEM) and in view of the importance of dense fibres in modulating sperm motility and providing tensile strength, a detailed, study of human sperm flagellum by negative staining andTEM was attempted. The study was undertaken in two groups of men (I) fertile and (II) asthenozoospermic. The study revealed that outer dense fibres extend to 50–60% of the principal piece. Normal dense fibres were seen in 83% sperms and 23% sperms in groupsI andII respectively. The characteristics seen were variation in diameter, breakage or degradation with lacking or extended endpiece. The negative staining method provides an easy and useful analytical tool for identifying the defects of dense fibres and quantifying them.     

Fossilized spermatozoa preserved in a 50-Myr-old annelid cocoon from Antarctica

The origin and evolution of clitellate annelids—earthworms, leeches and their relatives—is poorly understood, partly because body fossils of these delicate organisms are exceedingly rare. The distinctive egg cases (cocoons) of Clitellata, however, are relatively common in the fossil record, although their potential for phylogenetic studies has remained largely unexplored. Here, we report the remarkable discovery of fossilized spermatozoa preserved within the secreted wall layers of a 50-Myr-old clitellate cocoon from Antarctica, representing the oldest fossil animal sperm yet known. Sperm characters are highly informative for the classification of extant Annelida. The Antarctic fossil spermatozoa have several features that point to affinities with the peculiar, leech-like ‘crayfish worms'' (Branchiobdellida). We anticipate that systematic surveys of cocoon fossils coupled with advances in non-destructive analytical methods may open a new window into the evolution of minute, soft-bodied life forms that are otherwise only rarely observed in the fossil record.     

Cephalodiscus reproductive biology (Pterobranchia,Hemichordata)

Sexually mature adults and embryos and larvae of Cephalodiscus nigrescens and C. gracilis were studied by light and electron microscopy. Contrary to claims in the literature, individual coenecial cavities are inhabited by colonies of up to 15 joined zooids and not by single individuals, which is important for the interpretation of the mode of life of the related fossil group the graptolites. Some aspects of the reproductive apparatus and reproduction in Cephalodiscus are reported. The ultrastructure of the spermatozoon is described for the first time. Coelom formation is by schizocoely. The structure of the larva at several developmental stages is illustrated. Not all fertilised eggs are destined to become motile larvae and some develop into zooids omitting the motile stage. The lumen of the oviduct is much larger than previously supposed. Spermatozoa are shed into the cavity of the coenecium. It is proposed that fertilisation takes place within the coenecium. The ultrastructure of the enigmatic black ‘Comma Body’ is described and a reproductive function is proposed. Budding takes place from a base common to several zooids. This base probably also serves as an attachment foot. Large masses of yolk have been discovered within the coelom of some zooids and muscle stalks. It is inconceivable that a colony of Cephalodiscus nigrescens could survive unless it spent most of its life outside the coenecium.     

Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean (), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, α-zearalenol, and β-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Florida goats

The aims of this study were to test the presence of discrete sperm subpopulations in Florida goat ejaculates using a computer-assisted sperm analysis (CASA) system and to establish the relationship between the distribution of the subpopulations found and individual buck, total motility, and sperm concentration. Clustering methods and discriminant analysis were applied to identify motile sperm subpopulations within the semen samples. Principal component analysis revealed that three principal components represented more than the 88% of the variance. After the cluster analysis was performed four motile sperm subpopulations were identified. Subpopulation 1 consisted of rapid and linear sperm (39.84%), Subpopulation 2 consisted of slow but linear spermatozoa (33.23%), Subpopulation 3 consisted of rapid, high ALH but non-linear spermatozoa (14.63%), and Subpopulation 4 consisted of slow and non-linear spermatozoa (12.31%). There were significant differences in the distribution of the four subpopulations (P < 0.001) as well as in the percentage of total motility and the overall sperm concentration (P < 0.05) in fresh ejaculates among the four bucks tested. In conclusion, four well-defined motile sperm subpopulations were identified in Florida goat ejaculates. The relationship between the distribution of the sperm subpopulations and individual buck, total motility, and sperm concentration shows that the spermatozoa of each have different motility patterns. Therefore, the study of discrete subpopulations of motile spermatozoa could lead to a substantial increase in information acquired during caprine semen analysis.     

Fine structure of spermatozoa of Chondrostoma nasus and Rutilus meidingerii (Teleostei, Cyprinidae), as revealed by scanning and transmission electron microscopy

Fürböck, S., Patzner, R.A. and Lahnsteiner, F. 2008. Fine structure of spermatozoa of Chondrostoma nasus and Rutilus meidingerii (Teleostei, Cyprinidae), as revealed by scanning and transmission electron microscopy. — Acta Zoologica (Stockholm) 91 : 88–95
The fine structure of spermatozoa of sneep or nase, Chondrostoma nasus , and lake chub, Rutilus meidingerii , was investigated by means of scanning and transmission electron microscopy. The uniflagellate spermatozoa of C. nasus lacked an acrosome. The flagellum contained the conventional nine peripheral doublets and one central pair of microtubules (9 + 2 pattern) and lacked lateral fins. The uniflagellate spermatozoa of R. meidingerii were made up of a head, also without an acrosome. For both species the sperm tail was covered by a plasma membrane. The midpiece of C. nasus contained five or six mitochondria on average, vesicles and glycogen granules, whereas the midpiece of R. meidingerii had seven mitochondria of a spherical or ovoid shape. The centriolar complex was located caudolaterally with respect to the nucleus. In C. nasus , the centrioles were orientated at an angle of 125° to each other, whereas the centrioles of R. meidingerii were at an angle of 110°. The fine structure of C. nasus and R. meidingerii spermatozoa showed species-specific differences in the position of the proximal centriole relative to the distal centriole, the position and number of mitochondria, size of the head and the length of the flagellum. (Correction added on 11 June 2009, after first online publication: The word 'axoneme' was deleted from the sentence 'The flagellum contained the conventional nine peripheral doublets and one central pair of microtubules (9 + 2 pattern) axoneme and lacked lateral fins.')     

Effect of progestins on serum hormones,semen production,and agonistic behavior in the gerenuk (Litocranius walleri walleri)

The use of progestins to suppress endogenous testosterone production to reduce agonistic behavior and prevent semen production was studied in gerenuk. Five male gerenuk (20 months to 3 years of age), housed as a bachelor group, were treated with 3 monthly injections of medroxyprogesterone acetate (MPA; 2.5–20 mg/kg), followed by a melengestrol acetate implant (MGA; 0.3 g/kg) for 2 months. Blood samples collected monthly were assayed for serum testosterone and cortisol using enzyme‐linked immunoassays. Quantitative behavioral data were collected for 30 min 3/week starting 1 month before treatment. Body weight, testes volume, and semen traits were measured before treatment, after MPA treatment, and after MGA treatment. Results showed lower (P<0.05) mean serum testosterone concentrations after MPA (4.34 pg/ml) and MGA (5.02 pg/ml) treatment compared to pre‐treatment values (65.9 pg/ml) in four of five gerenuk. The remaining sub‐adult gerenuk had low testosterone initially (4.9 pg/ml) that did not decrease further with treatment (1.4 and 7.8 pg/ml for MPA and MGA, respectively). Mean serum cortisol concentrations decreased markedly after treatment with MPA (6.0±3.7 ng/ml) and MGA (0.8±0.3 ng/ml). Cortisol concentrations were regained rapidly post‐treatment (42.8±4.8 ng/ml) and were not significantly different from the pre‐treatment value (60.6±12.6 ng/ml; P>0.05). The mean incidence of combined aggressive/dominant behaviors (horning, sparring, supplanting, threat) was not different before and after treatment. Body weight, total numbers of spermatozoa produced per ejaculate, percent motility, and percent normal spermatozoa declined maximally 8 months after treatment. Mean testes volume decreased (P<0.05) after MGA treatment (10.53 cm³vs. 11.96 cm³ pre‐treatment). Elevated hepatic enzymes and bile acids were seen in three of five animals after progestin treatment and anorexia was noted in two males after MGA implant removal, however two of three males had elevated liver enzymes before progestin treatment began. Results show that reducing serum testosterone concentration does not seem to modify agonistic behavior in bachelor gerenuk groups. Zoo Biol 26:245–257, 2007. © 2007 Wiley‐Liss, Inc.     

Seminal and endocrine characteristics of male Pallas' Cats (Otocolobus manul) maintained under artificial lighting with simulated natural photoperiods

Pallas' cats (Otocolobus manul) have a pronounced reproductive seasonality controlled by photoperiod. Previous studies of reproduction in captive Pallas' cats exposed to natural light showed a breeding season of December–April. This study evaluated the impact of artificial lighting timed to simulate natural photoperiods on male reproductive seasonality of four Pallas' cats housed indoors. Semen evaluation, blood collection, and body weight measurements were conducted every 1–2 months from November 2000–June 2001. Fecal samples were collected from each male twice weekly to assess testosterone and corticoid concentrations. Mean values for reproductive traits (sperm attributes, testicular volume) were highest from February–April, the defined breeding season. Fecal testosterone concentrations were highest from mid‐January to mid‐March. Male Pallas' cats managed indoors under simulated photoperiods experienced a delayed onset of the breeding season by 1–2 months and a decreased length of the breeding season. Over the course of the study, fecal corticoid concentrations did not seem to differ among seasons. Although mating attempts during this study were unsuccessful, subsequent pairings of male and female Pallas' cats in the same research colony during the 2002 and 2003 breeding seasons produced viable offspring. These results suggest that male Pallas' cats, housed indoors under simulated photoperiods, exhibit distinct reproductive cyclic patterns, characterized by a delayed and truncated breeding season. Adrenocortical activity varied among individuals, but did not adversely affect reproductive parameters. Housing Pallas' cats indoors under simulated photoperiods may represent a viable strategy for maintaining breeding success while limiting disease exposure. Zoo Biol 0:1–13, 2007. © 2007 Wiley‐Liss, Inc.