Cytoskeletal proteins F-actin and ß-dystrobrevin are altered by the cryopreservation process in bull sperm.

The cryopreservation process has an important impact on sperm structure and physiology. The negative effects have been mainly observed on the plasma membrane, which is directly stabilized by the cytoskeleton. Since cytoskeleton proteins are osmosensitive and thermosensitive, the aim of this study was to evaluate the damage caused to the bull sperm cytoskeleton by cryopreservation (freezing-thawing). Fresh and frozen-thawed bull semen samples were exposed to a treatment with the neutral detergent Brij 36-T. Electron microscopy evidenced important damages at the sperm perinuclear theca after the protein extraction protocol; the perinuclear theca was partially solubilized, the perinuclear theca substructure disappeared in the cryopreserved samples. Furthermore, the sperm head's shape was significantly altered on the cryopreserved samples. Fluorescence analysis showed a decrease of the intensity of actin and dystrobrevin on the frozen-thawed samples. Western blot assays revealed a stronger signal for actin and ß-dystrobrevin in the frozen-thawed sperm samples than in the fresh ones. Our results suggest that the cryopreservation process highly alters the sperm cytoskeleton stability, causing its proteins to become more fragile and therefore more susceptible to be extracted.

Dietary Biotin Supplementation Impairs Testis Morphology and Sperm Quality.

Supplements containing pharmacological concentrations of biotin are commercially available over the counter. Classical toxicity studies have considered biotin administration as harmless; however, recent investigations have shown that biotin supplementation modifies tissue morphology without changes in toxicity markers, raising concerns about the consequences of morphological changes on tissues' functions and the safety of pharmacological concentrations of the vitamin. Testes are very sensitive to toxicants, and testicular histology is a reliable method to study its function. In this work, we investigated the effects of dietary biotin supplementation on testis morphology and spermatogenesis function using an experimental model, in which we have not observed unfavorable effects on other tissue functions or toxicity markers. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 weeks. Compared to the control group, the biotin-supplemented mice presented remarkable testis morphology changes, including increased spermatogonia layers; the cellular mechanism involved is related to increased proliferation. Sperm count and serum testosterone levels were not affected, but spermatozoa motility and morphology were significantly impaired in the biotin-supplemented mice. These results caution against the use of supplements with high concentrations of biotin and indicate that biotin's pharmacological effects on morphology need to be considered in toxicological studies.

Presence, processing, and localization of mouse ADAM15 during sperm maturation and the role of its disintegrin domain during sperm-egg binding.

Successful fertilization requires gametes to complete several stages, beginning with maturation and transport along the male and female reproductive tracts and ending with the interaction between the sperm and the egg. This last step involves sperm-egg adhesion and membrane fusion. ADAMs (disintegrin and metalloprotease domain proteins) are a family of membrane-anchored glycoproteins that are thought to play diverse roles in cell-cell adhesion through their interaction with integrins. This study analyzes the presence, location, processing, and possible role of ADAM15 in mouse sperm. The presence of ADAM15 in mouse spermatozoa was detected by Western blotting, which revealed that ADAM15 is post-translationally processed, during epididymal sperm maturation and the acrosome reaction. The 35 kDa antigen present in the acrosome-reacted sperm is the last proteolytic product of the 110/75 kDa ADAM15 found in non-capacitated sperm. This 35 kDa protein contains the disintegrin domain. By indirect immunofluorescence, ADAM15 was identified in the acrosomal region and along the flagellum of mouse spermatozoa. In acrosome-reacted sperm, ADAM15 was lost from the acrosomal region, but remained diffusely distributed throughout the head and flagellum. Furthermore, the ADAM15 disintegrin domain (RPPTDDCDLPEF) partially inhibited fusion and almost completely inhibited sperm-oolemma adhesion. In conclusion, our data indicate that ADAM15 is present in the testis and in spermatozoa from the caput, corpus, and cauda epididymis, as well as in non-capacitated and acrosome-reacted gametes. Results also indicate that ADAM15 is processed during epididymal maturation and acrosome reaction and that it may play a role during sperm-egg binding.

Actin, myosin, cytokeratins and spectrin are components of the guinea pig sperm nuclear matrix.

The nuclear matrix (NM) of somatic cells is an internal nuclear framework structure, with a structural function and participation in DNA replication and transcription. The NM has been described in mouse, hamster and human spermatozoa. In this study, an NM structural component of the guinea pig sperm nucleus was obtained by removing nuclear proteins and DNA from DTT-CTAB nuclei. Removal was achieved with high ionic strength salt and microccocal nuclease treatments including a heparin treatment to cause a slight swelling of the nucleus and facilitate material extraction. Actin, myosin, cytokeratins and spectrin were detected associated to NM by indirect immunofluorescence, immunogold staining and Western blotting analysis using specific antibodies. The presence of NM in guinea pig sperm nucleus is shown for the first time and some of its components are identified. This is also the first report on cytokeratins and myosin presence in guinea pig sperm. A retarding effect of nuclear decondensation caused by heparin is induced after phalloidin and/or diacetyl-monoxime (a myosin ATPase activity inhibitor) treatment, suggesting a role for F-actin and myosin in the maintenance of nuclear stability in sperm. The actin role was supported by the decondensing effect that citochalasin D and gelsolin had on sperm nuclei.