The study of sperm head vacuoles using deep learning algorithm and its correlation with protamine mRNA ratio

BackgroundAs regards the routine semen analysis is not sufficient to assess male fertility status, is it necessary to use other morphological sperm examination that may be more relevant in regard to the promotion of assisted reproduction outcomes? This study was designed for examination of sperm vacuole characteristics, its association with other sperm parameters and protamine 1 to protamine 2 ratio, and predict assisted pregnancy outcomes. Methods 98 Semen samples from subfertile men were classified based on Vanderzwalmen's criteria as follows: grade I, no vacuoles; grade II, ≤ 2small vacuoles; grade III, ≥ 1 large vacuole; grade IV, large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscope methods. The chromatin integrity (toluidine blue staining), condensation status (aniline blue), viability and acrosome integrity (triple staining), and protamination status (CMA3 staining) was evaluated for vacuolated samples. Protamine 1 and protamine 2 gene expression was analyzed by quantitative real-time PCR. The assisted reproduction outcomes were also followed for each cycle. Results The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (p<0.05), and protamine-deficient (p<0.05). The percentage of reacted acrosomes was significantly higher in spermatozoa with grades III and IV compared with normal group (p<0.05). A high protamine mRNA ratio ( prm-2 was underexpressed) was observed in the vacuolated spermatozoa with grade IV (p<0.01). The sperm head vacuole was negatively associated with the fertilization rate (p<0.01) under IVF cycles. This association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P<0.05). Conclusions The results of our study highlight the importance of follow up of more sperm parameters such as sperm head vacuole characteristics, because may reflect protamine-deficient and poor IVF/ICSI outcomes.

The epithelial cytotypes of hepatopancreas in the Chinese mitten crab Eriocheir sinensis (Decapoda, Varunidae)

The Chinese mitten crab,Eriocheir sinensis, is an economically important crustacean. However, the hepatopancreas structure of the crab has not been fully disclosed. Here, we employed light microscopy and transmission electron microscopy to identify the epithelial cytotypes of the hepatopancreas. The hepatopancreas consisted of four epithelial cytotypes, which were B (blister-like), R (resorptive), F (fibrillar), and E (embryonic) cells. B cells had club-shaped nuclei, scant cytoplasm, and contained some small vacuoles and one extremely large vacuole. Both R and F cells were columnar, and F cells were separated by two to three serial R cells. R cells contained small vacuoles, but had no extremely large vacuole in the cytoplasm. Two neighbouring R cells established contact by septate junctions. Many microvilli were present on the surface of the plasmalemma of R cells. Endocytotic vesicles were frequently observed in apical regions of the R cells. F cells were hyperbasophilic and argyrophilic, and contained abundant rough endoplasmic reticulum. E cells were small and contained scant cytoplasm and we rarely observed organelles in these cells. In addition, many vesicae were present in the lumen of the hepatopancreatic tubules.

Osmotic stress–induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P2) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P2 via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P2 synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P2 levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P2 are 18–28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2. After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P2 rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P2 plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P2 continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P2 and provide insight into why PtdIns(3,5)P2 levels rise in response to osmotic stress.

Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii.

The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70-100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.

Differential localization of Acanthamoeba myosin I isoforms.

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.

Embryo sac development in soybean: ultrastructure of megasporogenesis and early megagametogenesis

The soybean ovule is bitegmic with the megasporocyte three to four cell layers beneath the nucellar epidermis. The megasporocyte is much larger than the surrounding nucellar cells, is connected to the nucellus by plasmodesmata, and at this stage exhibits a cytoplasmic density comparable with cells of the nucellus. After meiosis, the chalazal megaspore becomes functional in megagametogenesis. It alone retains plasmodesmatal connections to the nucellus. Chalazal megaspore expansion is accompanied by development of many small vacuoles having a uniform distribution. The first megaspore mitosis results in two nuclei lying on an axis parallel to the longitudinal axis of the embryo sac. Ultimately, these two nuclei are separated by a large vacuole. Numerous Golgi vesicles and proteinlike bodies are observed along the periphery of vacuoles in the 1-, 2-, and 4-nucleate embryo sacs. As the contents of vesicles and proteinlike bodies are observed deposited in vacuoles, it is probable that they both add osmotica to the vacuoles, thus promoting a water flux. We believe that the production of Golgi vesicles and putative protein bodies may be important in the formation and expansion of the large vacuole that appears to drive embryo sac expansion during early megagametogenesis in soybean. It is also believed that the timing to this vacuole's development has important developmental consequences.

Infection of algae-free Paramecium bursaria with symbiotic Chlorella sp. isolated from green paramecia

Algae-free Paramecium bursaria was exposed to cells of Chlorella sp. for 30s (pulse) and then incubated in algae-free medium for periods between 0 and 15 min. During this chase the fate of the vacuoles formed during the exposure to algae was followed in order to reveal the moment of perialgal vacuole (PV) formation. PVs are characterized by always enclosing an individual algal cell and thus differ from digestive vacuoles (DVs). PVs did not appear immediately after the pulse, but were found only in cells chased for at least 3 min. In those ciliates the algae-containing vacuoles were more than 3 min old and located in the middle part of the cell. These results showed that PVs were not formed directly at the cytopharynx, but many algae were at first enclosed together in large DVs. After the release from the cytopharynx DVs undergo a sequence of fusion events during their cyclosis: fusion with acidosomes apparently occurs at the cell's posterior end, not later than 2 min, and fusion with lysosomes in the middle region of the cell at the earliest at about 7 min, after the pinching off of a DV from the cytopharynx. Thus, PVs appeared to develop from condensing DVs after acidosomal but before lysosomal fusion. As the first step, part of the DV enclosing an individual algal cell must detach from the large vacuole. Further steps and the implications of the mechanism of PV formation resulting in the re-establishment of endosymbionts in P. bursaria are discussed.

Ultrastructural studies of ascospore liberation in Pyronema domesticum

Ascospores of Pyronema domesticum contain three distinct spore wall layers. The liberation of ascospores presumably commences immediately after the three spore wall layers are formed. This is evidenced by the fact that vesiculation of the investing membrane was observed at the time when three wall layers could be distinguished. Vesiculation continues until the total disappearance of the perispore. Concurrently the epiplasm of the ascus degenerates and converts into a large vacuole within the ascus. Spores are violently ejected through the apical pore that is surrounded by a weakened apical ring. Presumably eight ascospores are discharged at the same time but do not adhere as a single unit. The operculum is generally not hinged to the main body of the ascus and an ascus without ascospores degenerates.

Fine structure during ontogeny of xylem transfer cells in the rhizome of Hieracium floribundum

A number of cytological changes occur in rhizome transfer cells with age, the most striking being the appearance of microbodies each with a crystalline nucleoid and the presence of unusual plastids. Plastids in older transfer cells develop one or more electron-translucent regions and lack a defined thylakoid system. The number and size of vacuoles increases until ultimately one large vacuole is formed in old transfer cells. Accompanying these cytological changes in the cytoplasm the wall ingrowths change from being highly involuted and reaching a considerable distance into the cytoplasm of the cell to becoming thicker and less numerous, and finally form a rather uniformly thickened wall layer. The orientation of microfibrils in the thickened cell wall, resulting from the joining of the original wall projections adjacent to the tracheary elements, is random, while the wall thickenings away from the tracheary elements have more orderly arrangements of cellulose microfibrils.