Yolk Granules Of Oocytes Research Articles

NanoSIMS Imaging of Bioaccumulation and Subcellular Distribution of Manganese During Oyster Gametogenesis

Many bivalve mollusks display remarkable sex differentiation of gonadal accumulation of manganese (Mn), but the underlying processes responsible for such differences have seldom been explored. In this study, the accumulation of Mn in male and female gonads during the reproductive cycle of oysters was first examined, and the distributions of Mn in oocytes and sperm cells at different developmental stages were imaged by the nanoscale secondary ion mass spectrometry (NanoSIMS) at the subcellular level. We found that the distribution and accumulation of Mn during oogenesis were closely associated with the formation and translocation of cortical granules. This is the first time that the enrichment of Mn was directly visualized in cortical granules, which was identified as the major storage site of Mn in oocytes of oysters. Yolk granules were revealed as another storage pool of Mn in oyster oocytes with lower accumulation. In contrast, Mn was mainly distributed in the nucleus of sperm cells with accumulation levels much lower than those in cortical and yolk granules of oocytes. These results demonstrated great differences of the subcellular localization and accumulation capacity of Mn between oocytes and sperm cells in oysters, implying the sex differentiation in susceptibility of reproductive response to Mn stress. Our study also highlights the importance of gender difference in future biomonitoring and ecotoxicological studies of Mn in marine bivalves.

Using clustering algorithms for identification of fish oocyte cohorts based on the characteristics of cytoplasmic structures

In batch spawning fish, secondary growth oocytes (SGO) are recruited and spawned in successive cohorts, and multiple cohorts co-occur in spawning-capable females. So far, histological features such as the prevalence of cortical alveoli or yolk granules are conservatively used to distinguish oocytes in different developmental stages which do not necessarily correspond to different cohorts. In this way, valuable information about spawning dynamics remains unseen and consequently misleading conclusions might be drawn, especially for species with high spawning rates and increased overlapping among oocyte cohorts. We introduce a new method for grouping oocytes into different cohorts based on the application of the K-means clustering algorithm on the characteristics of cytoplasmic structures, such as the varying size and intensity of cortical alveoli and yolk granules in oocytes of different development. The method allowed the grouping of oocytes without the need of using oocyte diameter, and thus, a crucial histological bias dealing with the cutting angle and the orientation of reference points (e.g. nucleus) has been overcome. Using sardine, Sardina pilchardus, as a case study, the separation of cohorts provided new insight into the ovarian dynamics, indentifying successive recruitment of up to five oocyte cohorts between SGO recruitment and spawning. These results verified previous histological indications of the number of cohorts in sardine. Altogether, this method represents an improved tool to study species with complex ovarian dynamics.

Loss of Inhibin Advances Follicle Activation and Female Puberty Onset but Blocks Oocyte Maturation in Zebrafish.

Inhibin was first characterized in mammals as a gonadal dimeric protein that inhibited pituitary follicle-stimulating hormone (FSH) secretion. As in mammals, the inhibin-specific α subunit (INHA/Inha/inha) has also been characterized in teleosts; however, its functions and physiological importance in fish reproduction remain unknown. Using CRISPR/Cas9 method, we generated an inha-deficient zebrafish line and analyzed its reproductive performance. As expected, pituitary expression of fshb increased significantly in both the young and the adult inha mutant. The expression of lhb also increased in the mutant, but only in sexually mature adults. Interestingly, the expression of activin βA (inhbaa) increased significantly in both the ovary and the testis of inha mutant, and the expression of ovarian aromatase (cyp19a1a) also increased dramatically in the mutant ovary. The juvenile female mutant showed clear signs of early follicle activation or precocious puberty onset. However, the adult female mutant was infertile with follicles arrested at the full-grown stage without final oocyte maturation and ovulation. Although follicle growth was normal overall in the mutant, the size and distribution of yolk granules in oocytes were distinct and some follicles showed granulosa cell hypertrophy. In contrast to females, inha-null males showed normal spermatogenesis and fertility. As reported in mammals, we also found sporadic tumor formation in inha mutants. Taken together, our study not only confirmed some conserved roles of inhibin across vertebrates, such as inhibition of FSH biosynthesis and tumor formation, but also revealed novel aspects of inhibin functions such as disruption of folliculogenesis and female infertility but no obvious involvement in spermatogenesis in fish.

Immunohistochemical observations of vitellin synthesis and accumulation processes in ovary of Ezo abalone Haliotis discus hannai

Ovarian follicle cells are the site of yolk protein synthesis in the Ezo abalone Haliotis discus hannai. In this study, histological observations of ovarian follicle cells were conducted in H. discus hannai by immunocytochemical and in situ hybridization methods focusing on their function of yolk protein synthesis. An antibody raised against purified yolk protein (vitellin, Vn) recognized yolk granules in oocytes, and ovarian follicle cells adjacent to the oocytes under yolk accumulation were stained positively with both anti-Vn antibody and an antisense probe for vitellogenin (the precursor for vitellin, Vtg) mRNA. These results indicate that the abalone Vtg gene is transcribed and translated in the ovarian follicle cells. In oocytes in the early phase of yolk accumulation, positive reactions with the antibody appeared first in the stalk part, and the follicle cells adjacent to the stalk were also stained positively. These observations imply local transport of yolk protein from the follicle cells to the oocyte through an extracellular space around the oocyte stalk. Ovarian follicle cells changed their morphological characteristics and reactivity to the antibody in close relation to the stages of the adjacent oocyte, which suggests the presence of functional interactions between follicle cells and oocytes in the course of yolk protein synthesis and accumulation.

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of R. (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data, suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens.

Direct molecular interaction of a conserved yolk granule protein in sea urchins.

The regulation of yolk storage in oocytes and subsequent utilization in embryos is critical for embryogenesis. In sea urchins, the major yolk protein is made in the intestines, transported to the ovaries and accumulated in developing oocytes within membrane-bound vesicles comprising approximately 10% of the mass of an egg. Here, a non-yolk protein that accumulates specifically in yolk granules is reported. This protein was identified by cDNA cloning and, by use of antibodies to the recombinant protein, it was shown that this molecule is stored selectively in yolk granules of oocytes and embryos. No accumulation was seen in the accessory cells, testis, or intestines. In situ ribonucleic acid (RNA) hybridizations showed that the transcript accumulated only in oocytes, and was more highly concentrated in young oocytes. However, later in oogenesis, the messenger ribonucleic acid (mRNA) levels decreased significantly so that no signal was detectable in mature haploid eggs or at any later stage in development. However, by immunofluorescence and western blot analysis, the 30 kDa band was present throughout development. The predicted sequence of this protein shows that it is a member of the bep, HLC-32, EBP family of sea urchin proteins, but as it does not accumulate at the cell surface, nor in the hyaline layer in the two species studied here, as do other members of the family, it has been referred to as YP30 (30 kDa protein of the yolk platelet). To address its potential function, yeast two-hybrid analysis was performed to screen for proteins that potentially interact with YP30. It was found that it binds itself, and forms strongly interacting dimers. It is hypothesized that YP30 participates in the packaging and storage of major yolk protein during oogenesis, or in the utilization of the major yolk protein in development.

Identification and partial characterization of vitellin from the barnacle Balanus amphitrite

The major protein in occytes of the barnacle Balanus amphitrite has been identified as vitellin (Vt) and its biochemical properties partially characterized. SDS-PAGE of protein extracts from different developmental stages of occytes and embryos of B. amphitrite revealed that a 170 kDa polypeptide was abundantly present in fully matured oocytes. Its amount increased with occyte maturation and decreased with embryonic development after fertilization. Antiserum, prepared against 170 kDa polypeptide, was found to recognize only 170 kDa among proteins in ovary extract (OE) by immunoblot analysis. Immunohistochemical study indicated localization of 170 kDa polypeptide in yolk granules of oocytes, oviducts and embryos. Combination of native electrophoresis with agarose gel and SDS-PAGE displayed that the main protein in OE was lipophilic, contained 85 kDa polypeptide in addition to 170 kDa, and was positive in PAS reaction. The main peak eluted from OE contained 170 kDa and 85 kDa polypeptides, and corresponded to 200 kDa when filtrated through superose 6 gel. Conclusively, vitellin of B. amphitrite is a lipophilic glycoprotein consisting of two subunits, a heavy chain with 170 kDa and a light chain with 85 kDa. Two other barnacles, Megabalanus rosa and Tetraclita squamosa japonica, also contained proteins similar to B. amphitrite.

Identification, localization, and functional implications of an abundant nematode annexin.

Cultures of the nematode C. elegans were examined for the presence of calcium-dependent, phospholipid-binding proteins of the annexin class. A single protein of apparent mass on SDS-polyacrylamide gels of 32 kD was isolated from soluble extracts of nematode cultures on the basis of its ability to bind to phospholipids in a calcium-dependent manner. After verification of the protein as an annexin by peptide sequencing, an antiserum to the protein was prepared and used to isolate a corresponding cDNA from an expression library in phage lambda gt11. The encoded protein, herein referred to as the nex-1 annexin, has a mass of 35 kD and is 36-42% identical in sequence to 10 known mammalian annexins. Several unique modifications were found in the portions of the sequence corresponding to calcium-binding sites. Possible phosphorylation sites in the NH2-terminal domain of the nematode annexin correspond to those of mammalian annexins. The gene for this annexin (nex-1) was physically mapped to chromosome III in the vicinity of the dpy-17 genetic marker. Two other annexin genes (nex-2 and nex-3) were also identified in chromosome III sequences reported by the nematode genomic sequencing project (Sulston, J., Z. Du, K. Thomas, R. Wilson, L. Hillier, R. Staden, N. Halloran, P. Green, J. Thierry-Mieg, L. Qiu, et al. 1992. Nature (Lond.). 356:37-41). The nex-1 annexin was localized in the nematode by immunofluorescence and by electron microscopy using immunogold labeling. The protein is associated with membrane systems of the secretory gland cells of the pharynx, with sites of cuticle formation in the grinder in the pharynx, with yolk granules in oocytes, with the uterine wall and vulva, and with membrane systems in the spermathecal valve. The presence of the annexin in association with the membranes of the spermathecal valve suggests a novel function of the protein in the folding and unfolding of these membranes as eggs pass through the valve. The localizations also indicate roles for the annexin corresponding to those proposed in mammalian systems in membrane trafficking, collagen deposition, and extracellular matrix formation.

Large-Scale Preparation of a Ribonuclease fromRana catesbeiana(Bullfrog) Oocytes and Characterization of Its Specific Cytotoxic Activity against Tumor Cells

Rana catesbeianaribonuclease (RC-RNase) is a pyrimidine–guanine sequence-specific ribonuclease found only inR. catesbeiana(bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09MNaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.

Structure of yolk granules in oocytes and eggs of Blattella germanica and their interaction with vitellophages and endosymbiotic bacteria during granule degradation

Oocytes and embryos of the cockroach Blattella germanica were examined by optical and electron microscopy to study yolk granule degradation during embryo development. During vitellogenesis, progressively larger yolk granules are formed in the ooplasm and by chorionogenesis, the mature granules are packed so tightly that their shape is highly distorted. Throughout ovarian development, endosymbiotic bacteria lie at the follicle cell/oocyte interface. Just prior to chorionogenesis the endosymbionts transit the oocyte plasma membrane and cluster at the periphery. Bacteria become more numerous over the ventral region of the egg by day 1 postovulation and begin to invade the interior of the yolk mass from the ventral periphery. At that time, lysis of the nearby yolk granules occurs while those in the central ooplasm remain intact and free of bacteria up to day 4. Vitellophages become evident by day 2 postovulation. These cells are also distributed over the egg's periphery but are most numerous in the ventral region. Vitellophages, in association with the endosymbionts, protrude toward the yolk granules and extend filo- and lamellipodia over the granule surface. Portions of the yolk granules are then engulfed and sequestered as large vacuoles in the vitellophage's cytoplasm. The vacuoles then become vesiculated. As embryo development proceeds, the vesiculated portions partition into smaller multivesicular bodies. This study describes the dynamics of yolk granule-vitellophage interaction in embryos of B. germanica and suggests that yolk utilization entails the cooperative efforts of both vitellophages and endosymbiont bacteria.