Fas ligand in bull ejaculated spermatozoa:A quantitative immunocytochemical study.

Apoptosis, or programmed cell death, provides a way to remove redundant cells at the end of their lifespan and thus acts as a homeostatic mechanism, maintaining the correct number of cells in the body by balancing their production and death. In the testis, this process seemed to play a pivotal role in spermatogenesis. It is generally accepted that Sertoli cells control the germ cell population through one of the best-known apoptotic pathways, the Fas/Fas L paracrine signal transduction system, in which a Fas ligand (Fas L) expressed by Sertoli cells induces apoptosis when it binds with its receptor, Fas, expressed by the germ cells. Recently, we demonstrated the presence of Fas antigen in normal ejaculated spermatozoa from fertile bulls and suggested that this molecule might have a non-apoptotic, defensive role against injuries, especially oxidative stress. We have now investigated whether bull mature, fertile spermatozoa express not only the Fas receptor but also its natural ligand Fas L. Our results indicate that the whole sperm population expresses Fas L. We suggest that Fas L in bull spermatozoa, like in murine spermatozoa, might be able to kill activated lymphocytes and protect the male gamete from damage by the self-immune system or the cytotoxic activity of leukocytes in the female genital tract.

A possible role of Fas antigen in ejaculated spermatozoa of fertile bulls: an immunocytochemical quantitative approach.

The Fas/Fas L system is a widely recognized apoptosis signal transduction pathway in which transmembrane receptor protein (Fas) triggers a programmed cell death when bound by the Fas ligand (Fas L). This system in the testis is believed to be a paracrine signaling system by which Sertoli cells expressing Fas L can initiate killing of Fas-expressing germ cells during spermatogenesis. So far, the presence of Fas antigen in ejaculated spermatozoa was related only to subfertility or infertility conditions. We demonstrated for the first time that normal ejaculated spermatozoa also express Fas antigen. Our data showed that a large percentage of normal ejaculated spermatozoa of fertile bulls are immunocytochemically positive for Fas. Our observations provide further evidence of the "regionalization" of sperm membrane antigens. Furthermore, we suggest that in mature fertile ejaculated spermatozoa the Fas antigen may also provide resistance to programmed cell death, like in some other cells expressing molecules that inhibit the signals induced by Fas or the death program itself. In addition, we suggest that Fas antigen can partly protect the spermatozoa against apoptosis induced by lipoperoxidative damage that can occur spontaneously in the male gamete at various stages in its lifetime.

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C.

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

ILA 147 immunoreactivity of the bull spermatozoa membrane during epididymal maturation.

A cytochemical quantitative study was carried out to detect immunostaining of bull spermatozoa during epididymal maturation using an ILA 147 monoclonal antibody and a standard immunoperoxidase method. This antibody recognizes a bovine panleukocyte determinant. Microdensitometric measurements were made on spermatozoa collected from different sites of the male genital tract (caput, corpus and cauda epididymidis and ductus deferens). On the basis of ILA 147 staining, at least 5 subpopulations of sperm cells from each site of the genital tract were found. These subpopulations showed: 1) immunostaining in the acrosome domain and in the cytoplasmic droplet in a proximal position; 2) immunostaining in the acrosome domain and in the distal cytoplasmic droplet; 3) immunostaining in the acrosomal region and lack of a cytoplasmic droplet; 4) immunostaining only in the cytoplasmic droplet; 5) lack of a cytoplasmic droplet and absence of staining of the head. The possible relationship between the presence of ILA 147 on the sperm head and the maturation process was evaluated, and we suggest that the most significant changes in ILA 147 expression occur in the corpus epididymidis. The absence of immunocytochemical staining in some spermatozoa may be related to plasma membrane damage due to spontaneous peroxidative damage of lipids.

Microdensitometric assay of enzymatic activities in parthenogenetically activated and in vitro fertilized bovine oocytes.

To examine the paternal genome's role in reprogramming metabolic activity in one-cell embryos, we investigated metabolic aspects of bovine oocytes after in vitro maturation and in vitro fertilization and after in vitro parthenogenetic activation with a Ca2+ ionophore and 6-dimethylaminopurine. We assayed succinate dehydrogenase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities by microspectrophotometry in immature oocytes and oocytes after maturation, in vitro fertilization and parthenogenetic activation. Succinate dehydrogenase activity significantly increased after in vitro maturation, significantly decreased after Ca2+ ionophore activation and further decreased after 6-dimethylaminopurine treatment. Lactate dehydrogenase activity showed a significant decrease in bovine oocytes after in vitro maturation, remained unchanged in Ca2+ ionophore-treated oocytes and rose significantly after 6-dimethylaminopurine treatment. This activity was dramatically reduced after in vitro fertilization, reaching absorbance levels that were not different from those in mature and Ca2+ ionophore-treated oocytes. Glucose-6-phosphate dehydrogenase activity was significantly lower in matured oocytes as compared to immature oocytes, was significantly higher after artificial activation with Ca2+ ionophore and remained constant after 6-dimethylaminopurine treatment or after in vitro fertilization. We suggest that metabolic changes involved in parthenogenetic activation are similar to those occurring after fertilization.