Ultra‐structural analysis of human spermatozoa by aperture scanning near‐field optical microscopy

Scanning near‐field optical microscopy (SNOM) represents a potential candidate for investigation of ultrastructure in human spermatozoa. It is a noninvasive optical technique that offers two main advantages: minimal sample preparation and simultaneous topographical and optical images acquisition with a spatial resolution beyond the diffraction limit. This enables the combination of surface characterization and information from the inner cellular organization in a single acquisition providing an immediate and comprehensive analysis of the cellular portions. In this work spermatozoa are immobilized on poly‐L‐lysine coated coverslips, fixed according to a standard protocol and imaged by aperture‐SNOM in air. In the SNOM images, all peculiar sperm portions show well‐resolved optical features, which exhibit good similarities with the structures revealed in transmission electron microscopy images, as compared with literature data. The optical features of anomalous spermatozoa are clearly different as respect with those observed for healthy ones. This analysis reveals the potentialities of SNOM and opens to its application to high‐resolution analysis of sperm morphological alterations, which might be relevant in reproductive medicine.     

Fish germinal cells. I. Comparative spermatology of seven Cyprinid species

The spermatozoa of seven fishes belonging to Cyprinid family are examined. They have no acrosome, like all Teleost fishes, a spheroidal or slightly elliptic nucleus, always eccentrically placed on the tail, two variously oriented centrioles, and a postnuclear cytoplasmic region of various size that contains some mitochondria (2 to 10) and surrounds a periaxonemal postnuclear canal. The tail is of moderate length (from 36 to 60 μm) and contains a “9 + 2” axoneme: both dynein arms are present. Comparative examination of the spermatozoa in the seven species shows that significant differences occur among them, even when they belong to the same genus. These concern the tail length; the position of the centrioles, the proximal with respect to the central one and with respect to the nucleus; the number of mitochondria, which is in relationship to the depth of the postnuclear canal. In the uniform general pattern of the ultrastructure of the Cyprinid spermatozoa, each species is characterized by a particular organization of the sperm organelles; in this respect, the two species examined by us, Leuciscus cephalus and souffia, are more closely related, even if easily recognizable one from the other. From a phylogenetic point of view, the comparative spermatology of the Cyprinid fishes suggests that the mitochondrial number is a good character, which enables us to order them in a phylogenetic arrangement.     

Study of DNA integrity in somatic cells and spermatozoa by single-cell gel electrophoresis with silver-nitrate staining

In the present work, genome instability in human and bovine peripheral blood lymphocytes and spermatozoa was studied by the method of DNA microelectrophoresis with subsequent staining of single cells with silver nitrate. A comparative analysis of the types of damage to human and bovine lymphocytes and spermatozoa genomes was performed. In the group of healthy donors, the spontaneous frequency of DNA damage revealed by single cell DNA microelectrophoresis did not exceed 9% and amounted, on average, to 4.8 ± 1.2%. In studying the effect of the duration of cryoconservation on bovine spermatozoa, no significant changes were revealed between the group of bulls whose spermatozoa were stored for less than one year (3.1 ± 0.9%) and the group of animals whose spermatozoa were under conditions of cryoconservation for more than 20 years (4.3 ± 0.5%). From the obtained single-cell DNA microelectropheretic data on the types and frequencies of DNA damage, a conclusion was made regarding the possibility of using a light variant based on cell staining with silver nitrate for the detection of genome instability, not only in somatic, but also in reproductive, cells.     

Changing Male Reproductive Health: A Review of the Clinical Evidence?

Several lines of circumstantial evidence suggest that we may be seeing adverse changes in male reproductive health. A possible decline in semen quality has attracted most attention, but there are stronger indications of a rising incidence of testicular cancer, with increases observed in both Europe and the USA. There are striking geographic variations in both the incidence of testis cancer and in the observed rate of increase, and it is noteworthy that testis cancer is much more common in Denmark, where low sperm counts have been reported, than in Finland, where semen quality seems to be better. Another cause for concern is the rising incidence of congenital malformations of the male genital tract — cryptorchidism and hypospadias. In the UK, for example, rates of cryptorchidism have increased by as much as 65 to 77%. The data are harder to interpret on semen quality. In a meta-analysis, Carlsen et al. (1992) identified significant decreases over time in sperm concentration, corresponding to a fall of almost 50% between 1940 and 1990. Several groups have since examined secular trends in semen quality, with some reporting a downward trend and others no change. However, evidence has emerged of striking regional differences in semen quality, whether due to ethnic, genetic, environmental, or lifestyle factors remains to be determined.     

Cloning in action: can embryo splitting,induced pluripotency and somatic cell nuclear transfer contribute to endangered species conservation?

The term ‘cloning’ refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation.     

Freezability of Iberian ibex (Capra pyrenaica) spermatozoa according to the glycerolization temperature and plasma testosterone concentration

Ibex spermatozoa can be successfully frozen using glycerolated media. However, no information is available regarding the most effective method of glycerol addition in this species. The aim of the present work was to evaluate the effect of the glycerolization temperature on the response to freezing-thawing of ibex spermatozoa collected by electroejaculation. The effect of the interaction glycerolization temperature x plasma testosterone concentration was also evaluated. The spermatozoa used in this work came from six adult ibexes maintained in captivity. Each ejaculate was divided into two aliquots in a Tris-egg yolk-based medium. One fraction was subjected to single step dilution with 5% glycerol at room temperature (23 °C). The other fraction was diluted in two steps, first by dilution at room temperature with an extender identical to that described above but without glycerol, followed by the addition of glycerol after cooling to 5 °C. The glycerolization temperature did not affect any sperm variable after thawing. Heterospecific artificial insemination involving domestic goats, revealed no differences in the fertilization rate for frozen-thawed spermatozoa diluted by the one or two step procedures (18.2% vs. 20.0%). The interaction glycerolization temperature x plasma testosterone concentration had no affect on the freezing-thawing of the sperm cells. The results revealed, however, that high plasma testosterone levels during the pre-rutting season may interfere with the freezing-thawing process, having a negative influence on sperm cryosurvival.     

Effects of semen preservation on boar spermatozoa head membranes

Head plasma membranes were isolated from the sperm-rich fraction of boar semen and from sperm-rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3-5 weeks (frozen-thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm-rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen-thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca⁺⁺ (10 mM) lowered the fluidity of the head plasma membranes from sperm-rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen-thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.     

The three-dimensional nature of the sperm tail wave studied using heterophil head-to-head agglutinins

Head-to-head agglutinates of bull spermatozoa, produced by heterophil agglutinins in human sera, revealed the same phenomenon as described for human spermatozoa: When rotating on the plate all agglutinates rotated counterclockwise. The interpretation lends further support to the hypothesis that the normal direction of the propagation of the overall helix-like tail wave is counterclockwise (ie, opposite the direction of the axonemal arms), corresponding to a clockwise shape of the wave (from base to tip) at a given instant. Heteroagglutinins may be a useful tool in physioanatomical studies of spermatozoa.     

Delta-9-tetrahydrocannabinol and human spermatozoa

Thein vitro effect of δ⁹-tetrahydrocannabinol on adenosine triphosphatase and phosphodiesterase activities as well as on the cyclic-AMP content of human spermatozoa has been studied. At a concentration of 1.0 ▪g, sperm metabolism may be increased as shown by increased cyclic AMP and adenosine-triphosphatase activity while at a higher concentration (10 ▪g tetrahydrocannabinol), it may be reversed.     

Reproduction in mice: Spermatozoa as factors in the development and implantation of embryos

The effects on mouse embryo development in vivo of varying the numbers of spermatozoa used in artificial inseminations was studied. The two criteria used in the evaluation of the progress of embryo development were 1) ability to reach the two-cell stage and 2) success of development from the two-cell stage through implantation. A 44% reduction in the yield of two-cell embryos and a 67% reduction in the number of implants was observed when C3HeB/FeJ females were inseminated with one-twentieth the number of spermatozoa estimated to be present in a typical ejaculate. The reduction in the yield of two-cell embryos was substantially reversed by a second insemination of a large number of heat-inactivated spermatozoa 12 hr after the first insemination. The sperm-dependent reduction in development from the two-cell stage through implantation was prevented only by normal viable (unheated) spermatozoa. These results were rationalized by the hypothesis that in female C3HeB/FeJ mice spermatozoa serve physiological functions beyond the fertilization of ova and that spermatozoa may act to foster early embryo development through modulation of the environments embryos experience as they move through the reproductive tract.