Contents Sperm cryopreservation facilitates the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. The cryopreservation process can damage sperm and compromise functionality. Several cryobiological studies have found that the physical and biological factors that affect sperm survival at low temperatures during the cryopreservation process often involve the integrity of sperm membrane. In this review, the behaviour of the sperm membrane against cooling, cold shock, ice crystal formation, oxidative stress, osmotic changes, reorganization of the lipid bilayer and addition of cryoprotective agents (CPA) is discussed. In addition, the phenomenon of reactive oxygen species (ROS) and its relationship with the cryopreservation process is also described. Semen cryopreservation techniques have progressed slowly in past years, and the current performance, measured as post‐thawed survival, is not very different compared to past decades. Recent advances in understanding the structure of the cell membrane, its function and metabolism have driven to new conservation systems, including lyophilization and vitrification. However, none of these technologies is commercially available, although its future appears very promising.

The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.Our knowledge of lipid metabolism in Bacillus subtilis is scant. Although B. subtilis is often considered the paradigmatic gram-positive organism, the mechanisms involved in lipid biosynthesis have been little studied, and much is argued by analogy with Escherichia coli (5). Recent studies indicate that fatty acids might act as signalling molecules that are important for cellular differentiation in B. subtilis (19), which prompted us to identify genes involved in fatty acid synthesis. Since the first enzymatic step in a metabolic pathway is often rate limiting, we sought to isolate and characterize the genes encoding subunits of the acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), which is the enzyme catalyzing the first committed step of fatty acid synthesis, i.e., the ATP-dependent carboxylation of acetylCoA to malonyl-CoA (12, 13). In E. coli, carboxylation of acetyl-CoA proceeds through two distinct reactions and involves an enzyme composed of four subunits: biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyl transferase (CT), a tetramer composed of two nonidentical subunits (12, 13). Cloning of prokaryotic ACCs subunits in the gram-negative bacteria E. coli (12, 13), Anabaena species (8), and Pseudomonas aeruginosa species (2) and in mycobacteria (16) has been reported; however, no acc genes from Bacillus species or other gram-positive species have been reported. The genes studied encode protein products similar to those of the E. coli BCCP and BC subunits. However, these gene arrangements have been reported to differ. In E. coli (12) and P. aeruginosa (2), the BCCP and BC genes (accB and accC, respectively) form a two-gene operon, whereas in Anabena species, the genes encoding BCCP and BC are unlinked (8). In contrast, genes from Mycobacterium leprae and Mycobacterium tuberculosis encode biotinylated proteins that in these organisms have both BCCP and BC functions (16), which is an arrangement that is also seen in the ␣ subunit of the mammalian mitochondrial propionyl-CoA carboxylase (3, 11). The genes coding for the CT ␣ and ␤ subunits (accA and accD, respectively) have been cloned only for E. coli (13).Here, we report that a DNA fragment containing the gene encoding the BCCP subunit of B. subtilis complements an E. coli strain with a mutant BCCP subunit. Moreover, we present the nucleotide sequence of an approximately 2.5-kb fragment that includes the B. subtilis accB and accC genes, and we demonstrate that the accB and accC genes form part of an operon located 220Њ downstream of spoIIIA (in the sin-ahrC interval) of the B. subtilis chromosome.Cloning of B. s...

The interaction between sperm and oviduct results in the selection of sperm with certain qualities. Porcine oviductal deleted in malignant brain tumor 1, DMBT1 (previously called sperm-binding glycoprotein, SBG), has been proposed to be implicated in sperm selection through acrosome alteration and suppression of motility of a subpopulation of sperm that have begun capacitation prematurely. It produces in vitro acrosome alteration and decrease of motility of boar sperm, concomitant with tyrosine phosphorylation of a 97 kDa sperm protein (p97). We hypothesized that the phosphorylation of p97 may be a link between DMBT1 sensing by a subpopulation of boar sperm and its biological effect. In this work, p97 was identified by mass spectrometry and immunoprecipitation as a porcine homologue of AKAP4. Pro-AKAP4 was localized by immunofluorescence and subcellular fractionation to the periacrosomal membranes and was shown to be tyrosine phosphorylated by DMBT1 regardless of the presence of calcium or bicarbonate, and of cAMP analogs, protein kinase A inhibitors, or a protein kinase C inductor. A processed w80 kDa form of AKAP4 was also detected at the tail of boar sperm, which was not tyrosine phosphorylated by DMBT1 under the conditions tested. Immunohistochemistry of testis showed presence of AKAP4 in boar sperm precursor cells. The evidence presented here supports the involvement of AKAP4 in the formation of the fibrous sheath on boar precursor sperm cells and implicates the phosphorylation of pro-AKAP4 as an early step in the signal transduction pathway gated by DMBT1 that leads to sperm selection through acrosome alteration.

The oviduct is a dynamic organ which modulates gamete physiology. Two subpopulations of sperm have been described in the oviduct of sows, a majority with normal appearance in the deep furrows and a minority, centrally located, and showing damaged membranes. Sperm-oviduct interaction provides the formation of a sperm storage and allows the selection of sperm with certain qualities. Pig (Sus scrofa) oviductal sperm binding glycoprotein (SBG) binds to sperm and exposes Gal beta1-3GalNAc. This disaccharide may be recognized by boar spermadhesin AQN1, which seems to be involved in sperm interaction with the oviduct. SBG is present at the apical surface of the epithelial cells that surround the lumen of the oviduct rather than at the bottom of the crypts. These characteristics imply it could be involved in sperm interaction with this organ. In this study, we evaluate the effect of SBG over boar sperm. We show that the presence of SBG produces alterations of the acrosome morphology of sperm only when they are incubated in capacitating conditions. SBG binds to the periacrosomal region of sperm undergoing capacitation. Its presence induces an increase on the tyrosine-phosphorylation of a polypeptide of apparent molecular mass 97 kDa, as occurs with a 95 kDa protein in other mammalian sperm upon acrosomic reaction. Altogether, these results suggest that SBG might be involved in sperm selection by alteration of the acrosome of sperm that have already begun the capacitation process when they arrive to the oviduct.