Phosphorylated MARCKS: A novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs

AbstractMARCKS (myristoylated alanine-rich C-kinase substrate) is a major substrate for protein kinase C (PKC), a kinase that has multiple functions during oocyte maturation and egg activation, for example, spindle function and cytoskeleton reorganization. We examined temporal and spatial changes in p-MARCKS localization during maturation of mouse oocytes and found that p-MARCKS is a novel centrosome component based its co-localization with pericentrin and γ-tubulin within microtubule organizing centers (MTOCs). Like pericentrin, p-MARCKS staining at the MI spindle poles was asymmetric. Based on this asymmetry, we found that one end of the spindle was preferentially extruded with the first polar body. At MII, however, the spindle poles had symmetrical p-MARCKS staining. p-MARCKS also was enriched in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain. Because phosphorylation of MARCKS modulates its actin crosslinking function, this localization suggests p-MARCKS functions as part of the contractile apparatus during polar body emission. Our finding that an activator of conventional and novel PKC isoforms did not increase the amount of p-MARCKS suggested that an atypical isoform was responsible for MARCKS phosphorylation. Consistent with this idea, immunostaining revealed that the staining patterns of p-MARCKS and the active form of the atypical PKC ζ/λ isoform(s) were very similar. These results show that p-MARCKS is a novel centrosome component and also defines a previously unrecognized subdomain of the actin cap overlying the spindle

Sperm from Hyh Mice Carrying a Point Mutation in αSNAP Have a Defect in Acrosome Reaction

Hydrocephalus with hop gait (hyh) is a recessive inheritable disease that arose spontaneously in a mouse strain. A missense mutation in the Napa gene that results in the substitution of a methionine for isoleucine at position 105 (M105I) of αSNAP has been detected in these animals. αSNAP is a ubiquitous protein that plays a key role in membrane fusion and exocytosis. In this study, we found that male hyh mice with a mild phenotype produced morphologically normal and motile sperm, but had a strongly reduced fertility. When stimulated with progesterone or A23187 (a calcium ionophore), sperm from these animals had a defective acrosome reaction. It has been reported that the M105I mutation affects the expression but not the function of the protein. Consistent with an hypomorphic phenotype, the testes and epididymides of hyh mice had low amounts of the mutated protein. In contrast, sperm had αSNAP levels indistinguishable from those found in wild type cells, suggesting that the mutated protein is not fully functional for acrosomal exocytosis. Corroborating this possibility, addition of recombinant wild type αSNAP rescued exocytosis in streptolysin O-permeabilized sperm, while the mutant protein was ineffective. Moreover, addition of recombinant αSNAP. M105I inhibited acrosomal exocytosis in permeabilized human and wild type mouse sperm. We conclude that the M105I mutation affects the expression and also the function of αSNAP, and that a fully functional αSNAP is necessary for acrosomal exocytosis, a key event in fertilization

MARCKS protein is phosphorylated and regulates calcium mobilization during human acrosomal exocytosis.

Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis

Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs.Fil: Bello, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Cappa, Andrea Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: de Paola, Maria Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Zanetti, Maria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Fukuda, Mitsunori. Tohoku University; JapónFil: Fissore, Rafael A.. Dep. Veterinary And Animal Science-univ.massachusetts A; Estados UnidosFil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Michaut, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentin

α-SNAP is expressed in mouse ovarian granulosa cells and plays a key role in folliculogenesis and female fertility

Abstract The balance between ovarian folliculogenesis and follicular atresia is critical for female fertility and is strictly regulated by a complex network of neuroendocrine and intra-ovarian signals. Despite the numerous functions executed by granulosa cells (GCs) in ovarian physiology, the role of multifunctional proteins able to simultaneously coordinate/modulate several cellular pathways is unclear. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (α-SNAP) is a multifunctional protein that participates in SNARE-mediated membrane fusion events. In addition, it regulates cell-to-cell adhesion, AMPK signaling, autophagy and apoptosis in different cell types. In this study we examined the expression pattern of α-SNAP in ovarian tissue and the consequences of α-SNAP (M105I) mutation (hyh mutation) in folliculogenesis and female fertility. Our results showed that α-SNAP protein is highly expressed in GCs and its expression is modulated by gonadotropin stimuli. On the other hand, α-SNAP-mutant mice show a reduction in α-SNAP protein levels. Moreover, increased apoptosis of GCs and follicular atresia, reduced ovulation rate, and a dramatic decline in fertility is observed in α-SNAP-mutant females. In conclusion, α-SNAP plays a critical role in the balance between follicular development and atresia. Consequently, a reduction in its expression/function (M105I mutation) causes early depletion of ovarian follicles and female subfertility

αSNAP and NSF expression in the reproductive tract of wild type (wt) and SP hyh (hyh) mice.

<p>(A) Proteins extracted from testis and cauda epididymidis of wt and SP hyh mice were analyzed by Western blot using an antibody recognizing αSNAP (upper panel) or NSF (middle panel). Signals detected with an anti-actin antibody served as internal controls for equal protein loading (lower panel). Cauda epididymidis extracts were obtained before (Cauda+sperm) and after (Cauda-sperm) sperm were washed out from the organ. Brain was used as a positive control. Blots are representative of 3 or 4 independent experiments. (B, C) Densitometric analysis of Western blot for αSNAP (B) and NSF (C). Black bars (mean±SEM, N = 3 or 4) refer to the relative amount of each protein in hyh samples compared to wt. * p<0.05 (Student's t-test).</p

αSNAP expression and localization in sperm from wild type (wt) and SP hyh (hyh) mice.

<p>(A) Sperm protein extracts obtained from wt and hyh mice were analyzed by Western blot using an antibody recognizing αSNAP (upper panel) or NSF (middle panel). Signals detected with an anti β-tubulin antibody served as internal controls for equal protein loading (lower panel). Blots are representative of seven independent experiments. (B) Densitometric analysis of Western blot for αSNAP and NSF. Black bars (mean±SEM, N = 7) refer to the relative amount of each protein in hyh samples compared to wt (gray bars). * p<0.05 (Student's t-test). (C) αSNAP localizes to the acrosomal region in mouse spermatozoa. Sperm from wild type and hyh mice were fixed, permeabilized and triple-stained with an anti-α/βSNAP antibody (green); TRITC-PNA, a lectin that recognizes the intra-acrosomal content (red); and Hoechst 33258 to visualize the nucleus of the cell (blue). Shown are epifluorescence micrographs of typically stained cells. Scale bar, 10 µm.</p

Sperm from SP hyh mice have a deficient acrosomal reaction.

<p>Sperm from wild type and SP hyh (hyh) mice were collected from the cauda epididymidis, incubated under capacitation conditions for 1 h and stimulated with buffer (control), 10 µM progesterone (Pg) or 10 µM A23187 (A23187) for 15 min at 37°C. The cells were spotted on slides and fixed in ice-cold methanol. Acrosomal status was evaluated in at least 200 sperm by staining with TRITC-PNA. The data represent the mean±SEM of three independent experiments (*, significant differences between same groups for wild type and hyh mice, P<0.001, Student's t test).</p

In vitro fertilization (IVF) assays using sperm from wild type and SP hyh mice.

<p>Metaphase II eggs from wild type (<i>Napa</i>^(+/+)) female mice were incubated with sperm from wild type <i>(Napa</i>^(+/+), wt) or mutant homozygous with the slow progressive phenotype (<i>Napa^(hyh/hyh)</i>, hyh) mice. Following capacitation, sperm from wild type and hyh mice were diluted to 2 or 4×10⁵ sperm/ml and coincubated with eggs from wild type female mice. In experiment “A”, the same number of sperm cells from wt and hyh mice was used (2×10⁵ sperm/ml). In experiment “B” the number of sperm from hyh mice was duplicated (4×10⁵ sperm/ml). (*, p<0.02 or **, p<0.001 for hyh versus wt; Fisher's exact probability test).</p

Reproductive efficiency of wild type and SP hyh mice.

<p>wt: wild type (<i>Napa^(+/+)</i>); het: heterozygous (<i>Napa^(hyh/+)</i>); hyh: mutant homozygous (<i>Napa^(hyh/hyh)</i>) with the slow progressive phenotype.</p>a<p>Matings are considered “productive” if at least one offspring was born.</p>b<p>“Relative fecundity” is obtained as: (productive mating/100)×(litter size)×(number of litters); the value obtained is a measure of the overall fecundity according to the Handbook of Genetically Standardized JAX Mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004963#pone.0004963-Green1" target="_blank">[33]</a>.</p>c<p>The same hyh male had two productive matings.</p