Protein tyrosine phosphorylation is associated with sperm capacitation and the acrosome reaction in several mammalian species. Changes in phosphorylation of a 95‐kDa protein in human, mouse, and ...domestic cat spermatozoa are known to be influenced by capacitation and exposure to zona pellucida (ZP) proteins. We previously reported diminished phosphorylation of 95‐ and 160‐kDa proteins in spermatozoa from teratospermic cats, compared with normospermic domestic cats. To determine if these proteins and mechanisms are present in other species in the phenotypically diverse Felidae family, we examined the relationship between tyrosine‐phosphorylated sperm proteins and sperm morphology in the leopard cat (∼65% normal sperm/ejaculate), tiger (∼65%), clouded leopard (∼15%), and cheetah (∼30%). Furthermore, we investigated the involvement of cyclic adenosine monophosphate (cAMP) in the regulation of sperm protein tyrosine phosphorylation. Specifically, we assessed the following: 1) presence of tyrosine‐phosphorylated proteins in sperm extracts; 2) changes in protein tyrosine phosphorylation after sperm capacitation and swim‐up separation; 3) impact of tyrosine kinase inhibition on leopard cat sperm protein phosphorylation and ZP penetration; and 4) involvement of a cAMP‐dependent pathway in the regulation of protein tyrosine phosphorylation. Immunoblotting analysis with anti‐phosphotyrosine antibody (PY20) indicated that a 95‐kDa protein was present in all four species. Additional phosphorylated proteins were detected in the leopard cat (145‐ and 175‐kDa proteins), tiger (185‐kDa protein), clouded leopard (160‐ and 190‐kDa proteins), and cheetah (115‐ and 155‐kDa proteins). Sperm capacitation in vitro increased phosphorylation of one or more proteins in the leopard cat, tiger and clouded leopard, but not in the cheetah. Although swim‐up separation increased the proportion of morphologically normal spermatozoa in the clouded leopard and cheetah, no changes were observed in phosphorylation of the 95‐kDa sperm protein. Thus, phosphorylation of the 95‐kDa protein appeared to be related to the condition of teratospermia. Exposing leopard cat spermatozoa to the tyrosine kinase inhibitor, tyrphostin, reduced (P < 0.05) phosphorylation of the 95‐and 145‐kDa proteins, as well as ZP penetration, without affecting sperm motility. Similarly, when spermatozoa were incubated in the presence of cAMP analogs or active and inactive stereoisomers of cAMP, phosphorylation of sperm proteins was either stimulated or inhibited. Together, these data suggest that protein tyrosine kinase mechanisms appear conserved within the family Felidae and are regulated by a cAMP/protein kinase A pathway.
This study assessed the impact of various cryoprotectant (CPA) exposures on nuclear and cytoplasmic maturation in the immature
cat oocyte as a prerequisite to formulating a successful ...cryopreservation protocol. In experiment 1, immature oocytes were
exposed to 0, 0.75, 1.5, or 3.0 M of 1,2-propanediol (PrOH) or 1,2-ethanediol (EG) at room temperature (25°C) or 0°C for 30
min. After CPA removal and in vitro maturation, percentage of oocytes reaching metaphase II (MII) was reduced after exposure
to 3.0 M PrOH at 0°C or 3.0 M EG at both temperatures. All CPA exposures increased MII spindle abnormalities compared to control,
except 1.5 M PrOH at 25°C. In experiments 2 and 3, immature oocytes were exposed to CPA conditions yielding optimal nuclear
maturation that either had caused spindle damage (0.75 M PrOH, 1.5 M EG, and 3.0 M PrOH at 25°C) or not (1.5 M PrOH at 25°C).
After maturation and insemination in vitro, oocytes were cultured for 7 days to assess treatment influence on developmental
competence. CPA exposure did not affect fertilization, but the high incidence of MII spindle abnormalities resulted in a low
percentage of cleaved embryos. Blastocyst formation and quality were influenced by both CPA types (EG was more detrimental
than PrOH) and concentration (3.0 M was more detrimental than 1.5 M). Overall, cat oocytes appear to be highly sensitive to
CPA except after exposure to 1.5 M PrOH at 25°C, a treatment that still allowed â¼60% of the oocytes to reach MII and â¼20%
to form blastocysts.
Tyrosine phosphorylated proteins recently have been found in mouse and human spermatozoa. Our objectives were to (1) determine if domestic cat spermatozoa also express tyrosine phosphorylated ...proteins, and (2) examine the changes in protein phosphorylation between normospermic and teratospermic domestic cats following sperm capacitation, swim‐up separation and exposure to zona pellucida (ZP). Membranes from cat spermatozoa contained two phosphorylated proteins of molecular weights 160 kDa and 95 kDa (designated as p160 and p95) that immunoreacted with monoclonal antibodies to tyrosine phosphate. The p95 protein was distinct from sperm‐specific hexokinase. Following capacitation, the extent of phosphorylation of p95 was increased (P < 0.05) 3‐fold in normospermic cats compared to only 1.75‐fold in teratospermic cats. Similarly, phosphorylation of p160 also increased (P < 0.05) 2.4‐ fold in normospermic compared to 1.84‐fold in teratospermic cats. Although swim‐up separation increased the percentage of normal spermatozoa in teratospermic ejaculates, phosphorylation of p95 in swim‐up aliquots was increased (P < 0.05) only 1.95‐fold in teratospermic cats compared to 2.9‐fold in normospermic counterparts. Likewise, phosphorylation of p160 was lower (P < 0.05) in teratospermic (1.5‐fold) compared to normospermic cats (2.0‐fold) cats. Phosphorylation also was influenced by exposure to cat ZP proteins (P < 0.05). Solubilized cat ZP bound to the sperm proteins of apparent molecular mass 120, 95, 50, 42, 30, 27, 23 and 20 kDa, suggesting a direct binding interaction between p95 and the ZP. Overall, these findings (1) indicate the presence of tyrosine phosphorylated proteins in the domestic cat spermatozoon that directly interact with homologous ZP glycoproteins; (2) demonstrate that cat sperm hexokinase is not phosphorylated on tyrosine residues; and (3) suggest that the diminished phosphorylation efficiency of sperm from teratospermic cats may result in a compromise in capacitation and the acrosome reaction.
Effects of osmolality stresses on the sperm of normospermic (>60% normal sperm/ejaculate) versus teratospermic (<40% normal sperm) domestic cats and the normospermic leopard cat and the teratospermic ...clouded leopard were studied. Spermatozoa were exposed to various anisotonic solutions in a single step or returned to near isotonic conditions in a single step after exposure to anisotonic solutions. The percentage of sperm motility was measured subjectively, and dual fluorescent stains were used to assess membrane integrity by flow cytometry. The percentage of sperm motility declined (P < 0.05) in domestic cat sperm exposed to osmolalities <200 and >450 mOsm. Spermatozoa from all felines underwent marked (P < 0.05) membrane disruption following a hypotonic stress, but sperm from teratospermic donors experienced greater (P < 0.05) membrane disruption in response to decreased osmolality. While feline spermatozoa appeared to be highly resistant to hypertonic (600, 1200, and 2400 mOsm) conditions, with >85% of the cells maintaining intact membranes, severe membrane disruption occurred when cells were returned to isotonicity in a single step. There was no difference (P > 0.05) between a 1- and 5-min exposure to various anisotonic solutions. Similarly, sperm from normospermic and teratospermic domestic cats responded identically after exposure to ionic or nonionic solute. Results demonstrate that: (1) spermatozoa from teratospermic males are more vulnerable to a hypotonic stress than sperm from normospermic counterparts; (2) in response to small deviations in osmolality, feline sperm experience a more rapid decline in motility than membrane integrity; and (3) an abrupt return to isotonicity after a hypertonic stress causes extensive sperm membrane damage regardless of ejaculate quality.
The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat normospermic versus teratospermic, the normospermic serval and the ...teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1
M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Ham's F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (
P<0.01) sperm motility compared to control spermatozoa maintained in Ham's F10. Exposing sperm from all species to a 1
M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (
P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Ham's F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.
The common domestic cat is an important research model for endangered felids, as well as for studying genetic dysfunctions, infectious diseases and infertility in humans. Especially significant is ...the trait of teratospermia (ejaculation of < 40% morphologically normal spermatozoa) that commonly occurs in about 70% of the felid species or subspecies studied to date. Teratospermia, discovered more than two decades ago in the cheetah, is important: (i) for understanding the significance of sperm form and function; and (ii) because this condition is common in human males. It is apparent from IVF that deformed spermatozoa from teratospermic felids do not fertilize oocytes. However, the inability of spermatozoa from teratospermic males to bind, penetrate and decondense in the cytoplasm of the oocyte is not limited to malformed cells alone. Normal shaped spermatozoa from teratospermic males have reduced functional capacity. IVF results have consistently revealed a direct correlation between teratospermia and compromised sperm function across felid species and populations. The most significant differences between normospermic (> 60% normal spermatozoa per ejaculate) and teratospermic felids include: (i) the time required for sperm capacitation and the acrosome reaction to occur in vitro; (ii) culture media requirements for capacitation in vitro; (iii) phosphorylation patterns of tyrosine residues on sperm membrane proteins during capacitation; (iv) susceptibility to chilling-induced sperm membrane damage; (v) sensitivity to osmotic stress; (vi) stability of sperm DNA; (vii) sperm protamine composition; and (viii) fertilizing ability after intracytoplasmic sperm injection. In conclusion, (i) the felids (including wild species) are valuable for studying the functional significance of both pleiomorphic and normally formed spermatozoa from teratospermic donors, and (ii) the impact of teratospermia is expressed at both macrocellular and subcellular levels.
Glucosidase I initiates the processing of the oligosaccharide, Glc3Man9GlcNAc2, in newly assembled glycoproteins by excising the distal alpha 1,2-linked glucosyl residue in the oligosaccharide. ...Earlier, the enzyme purified from the ER of rat and bovine mammary gland has been found to have M(r) of 85 kDa, as examined by SDS-PAGE along with a domain structure in which a 39 kDa lumenally-oriented region is anchored to the ER through a transmembrane segment and a short cytoplasmic tail. These studies were further extended to include the enzyme from several different tissues of the rat, mouse, guinea pig and bovine mammary glands, sheep liver and pig kidney. Using anti-rat glucosidase I antibody as a probe and several biochemical parameters such as SDS-PAGE analysis, trypsin-catalyzed digestion, ConA-binding, endo H susceptibility and peptide mapping analysis by cleavage of the tryptophanyl peptide linkages within the enzyme, it was found that glucosidase I in all of the tissue sources examined has an M(r) of 85 kDa and is cross-reactive to anti-rat glucosidase antibody. The enzyme is a high mannose glycoprotein, and has domain features in its structure; the enzyme from mouse, rat, guinea pig and bovine mammary glands and sheep liver is sequentially cleaved by trypsin to generate fragments of 69, 55 and 39 kDa. The rate of release of the different fragments differs for different sources, indicating some evolutionary changes in its primary structure. The trypsin-released fragments from pig kidney enzyme are 69, 45 and 29 kDa in size, identical to the same observed earlier for pig liver.
Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for ...aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.
Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for ...aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.
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