An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle

We have cloned and functionally characterized a novel protein, BmVMP30, which is synthesized by the cells of the follicular epithelium of the ovarian follicles of the domesticated silkworm Bombyx mori, secreted from them and associated with the vitelline membrane. BmVMP30 is a 30 kDa protein that bears limited structural features reminiscent of other insect vitelline membrane proteins. Although BmVMP30 does not share pronounced similarities or signature motifs with other reported proteins, its temporal and spatial expression and its behavior throughout oogenesis suggest that it is a novel member of the insect vitelline membrane protein family. The protein is expressed exclusively in the cells of the follicular epithelium during stages -15 to -1 of vitellogenesis, secreted from them and, ultimately, localized at the junction between the oocyte and the eggshell, where the vitelline membrane is located. Treatment of follicles with an antisense oligonucleotide that encompasses the translation initiation codon results in the production of an N-terminally truncated protein and disruption of the integrity of the follicular epithelium. Antisense oligonucleotide treatment, however, has no effect on the implementation of the developmental program that directs the autonomous progression of ovarian follicles through the last stages of vitellogenesis and choriogenesis.     

BmECM25, from the silkworm Bombyx mori,is an extracellular matrix protein

BmECM25 (previously reported as BmVMP25) was previously predicted as a gene encoding the vitelline membrane protein in silkworm, Bombyx mori. In this study, we investigated the detail temporal and spatial patterns of BmECM25 protein. Western blot results showed that BmECM25 was expressed in the follicular epithelium cells from stages −6 to +1, and was then secreted into the oocytes. However, the abundance of BmECM25 decreased during the subsequent oogenesis and finally disappeared in the mature follicles. Immunofluorescence detection showed that BmECM25 locates inside the VM layer and forms a discontinuous layer. These features of BmECM25 suggest that it is an oocyte membrane matrix protein, not a vitelline membrane protein.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

Electrotonic junctions in cecropia moth ovaries.

The steady-state potential of the oocyte, resistance between the ooplasm and the medium, and electronic coupling between oocytes in adjacent follicles were examined in vitellogenic ovarioles of Hyalophora cecropia. The steady-state potential had a constant value of ?40 mV throughout the 100-fold volume increase accompanying yolk deposition, while membrane resistance decreased gradually with increasing size. Resistance rose steeply with the onset of chorion deposition, but did not detectably change with either nurse cell collapse or termination of vitellogenesis. Nonrectified electrical coupling was found between oocytes in adjacent follicles, and fluorescein ions injected into the ooplasm moved readily from follicle to follicle. Large surface area and low membrane resistance made coupling difficult to detect electrically between more mature oocytes, but interfollicular fluorescein migration was found to persist until the end of vitellogenesis. Migration of fluorescein from the oocyte to the follicular epithelium could also be visualized and fingers of ooplasm that cross the vitelline envelope and terminate in dome-shaped attachments to the epithelial cells were implicated in this transfer. The termination of interfollicular coupling coincided with the termination of epithelial-oocyte coupling, and is proposed to result from thickening of the vitelline envelope and withdrawal of the ooplasmic processes.     

Specific protein synthesis in cellular differentiation: III. The eggshell proteins of Drosophila melanogaster and their program of synthesis

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with ³H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [³H]proline and [³H]alanine during stages of active vitelline membrane synthesis.     

Ultrastructure and melatonin 1a receptor distribution in the ovaries of African ostrich chicks

Healthy 90-day-old ostrich chicks were used in the present study. The ultrastructure and melatonin 1a receptor (MT1) distribution in the ovaries of ostrich chicks was observed by transmission electron microscope and light microscope. The results showed that the ostrich chick ovary contained primordial follicles, primary follicles and secondary follicles, but no mature follicles. There are some unique ultrastructural characteristics observed in the secondary follicle, such as the cortical granule, which was located in cytoplasm beside the nucleus and appeared first in the oocyte. The zona radiata appeared in the secondary follicle, and there was an obvious vitelline membrane. There were intraovarian rete, connecting rete, and extraovarian rete in the ovaries of ostrich chicks. This is the first study that provides immunohistochemical evidence for the localization of the melatonin MT1 in the ostrich chick ovary. The germinal epithelium, follicular cell layer of every grade of follicle, cytoplasm of the oocyte and interstitial cells all expressed MT1. The expression of positive immunoreactivity materials was the strongest in the follicular cell layer of the primordial follicle and germinal epithelium, was weaker in the follicular cell layer of the primary follicle and secondary follicle, and was weakest in the oocytes of all grades of follicle. In addition, the extraovarian rete displayed strong positive expression of MT1, while there was no positive expression in the intraovarian rete or connecting rete. The positive expression of MT1 immunoreactivity in the ovary was very strong, implying that the ovary is an important organ for synthesizing MT1.     

Germline and somatic vitelline proteins colocalize in aggregates in the follicular epithelium of Drosophila ovaries

Nasrat and Polehole, two Drosophila proteins related functionally and by sequence, are secreted from the oocyte and incorporated into the vitelline membrane, where they play a role in the integrity of the same and in the activation of embryonic Torso RTK. In addition, they also accumulate in a punctate pattern in the follicular epithelium. Here we show that their accumulation at the follicle cells depends on their gene expression in the germline, indicating that these proteins move from the oocyte to the follicle cells in a process that does not require endocytosis. Finally we used cell markers to examine the distribution of these proteins at the follicle cells and show they accumulated in aggregates with vitelline membrane proteins in close association with the plasmatic membrane. We propose that these aggregates represent spatially restricted sinks for vitelline membrane proteins that fail to be incorporated into vitelline bodies and later on into the vitelline membrane.     

BmCAP,a silkmoth gene encoding multiple protein isoforms characterized by SoHo and SH3 domains: Expression analysis during ovarian follicular development

CAP/ArgBP2/vinexin family proteins, adaptor proteins characterized by three SH3 domains at their C-termini and a SoHo domain towards their N-termini, are known to regulate cell adhesion, cytoskeletal organization, and growth factor signaling. Here we present the isolation and ovarian expression of the BmCAP gene which encodes CAP/ArgBP2/vinexin family proteins in the silkmoth, Bombyx mori. Screening for full-length cDNA clones identified three mRNA isoforms, BmCAP-A1, BmCAP-A2 and BmCAP-B, which show expression throughout ovarian follicular development. Using an antibody raised against a unique region between the SoHo and SH3 domains, BmCAP-A protein isoforms were identified that show specific expression in different compartments of the ovarian follicles. Immunofluorescence staining of the cells of the follicular epithelium establishes a dynamic pattern of BmCAP-A protein localization during choriogenesis. During early choriogenesis, BmCAP-A has a diffuse localization in the cytoplasm but could also be found concentrated at the apical and basal sides at the cell–cell junctions. During late choriogenesis, the diffuse cytoplasmic staining of BmCAP-A disappears while the staining pattern at the apical side resembles a blueprint for the eggshell surface structure. We suggest that BmCAP-A isoforms have important functions during ovarian development, which involve not only the traditional roles in actin organization or cell–cell adhesion but also the regulation of secretion of chorion proteins and the sculpting of the chorion surface.     

The ovaries and changes in their structural components at the end of vitellogenesis and during vitelline membrane formation in the butterfly, Calpodes

The eight ovarioles of Calpodes ethlius are meroistic and polytrophic with seven nurse cells per follicle. The follicles consisting of oocyte, nurse cells and surrounding follicular cells are connected by interfollicular bridges, whose cells are characterized by bundles of microtubules which appear, with some fine filaments, to terminate on or near to the plasma membrane at hemidesmosomes. The ovariolar sheath consists of tightly knit circular and longitudinal muscles which are heavily tracheated. The ovariolar duct consists of more loosely arranged circular and longitudinal muscles and an inner epithelial layer, also tracheated. The vitelline membrane appears to be secreted largely by the oocyte first as an electron-lucent layer which becomes gradually more electron-dense, probably as a result of addition of material from the follicular cells. Overlapping plate-like structures on the outer surface of the fully formed vitelline membrane may provide waterproofing.     

Structure and morphogenesis of the eggshell and micropylar apparatus in the olive fly, Dacus oleae (Diptera: Tephritidae)

The egg of the olive fly, Dacus oleae (Diptera, Tephritidae), is laid inside olives and the larva eventually destroys the fruit. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The chorion covering the main body of the egg outside of the vitelline membrane includes a "wax" layer, an innermost chorionic layer, an endochorion consisting of inner and outer layers separated by pillars and cavities similar to their counterparts in Drosophila melanogaster, as well as inner and outer exochorionic layers. The anterior pole is shaped like an inverted cup, which is chiefly hollow around its base and has very large openings communicating with the environment. Holes through the surface of the endochorion result from deposition of endochorionic substance around follicular cell microvilli. An opening at the apex of the cup provides an entrance for sperm entering the micropylar canal, which traverses the endochorion and continues into a "pocket" in a thickened vitelline protrusion. The micropylar canal is formed by deposition of endochorion and vitelline membrane around an elongated pair of follicular cell extensions. These extensions later degenerate and leave an empty canal about 5 microns in diameter and the narrower pocket about 1 micron in diameter. Respiration is thought to be facilitated by openings at the base of the anterior pole as well as by openings through the "plastron" around the main body of the shell.     

Presence of an aromatase inhibitor, possibly heat shock protein 90, in dominant follicles of cattle.

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 +/- 0.09%) than in the culture medium (1.3 +/- 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp 90 to bovine follicles in vitro depressed aromatase activity by altering the K(m) value (and possibly the Vmax value) of the enzyme. It is proposed that hsp 90 is a functional regulator of follicular maturation through its action on aromatase.     

The N-terminal prodomain of sV23 is essential for the assembly of a functional vitelline membrane network in Drosophila

The vitelline membrane is a specialized extracellular matrix that surrounds and protects the oocyte. Recent studies indicate that it also serves as a storage site for embryonic pattern determinants. sV23, a major vitelline membrane protein, is essential for the morphogenesis of the vitelline membrane as sV23 protein null mutants lay flaccid, infertile eggs. By analyzing a series of sV23 mutant transgenes in the sV23 protein null genetic background, we have shown that sV23 is secreted as a proprotein in functional excess and that C- and N-terminal prodomains are removed successively, following its deposition in the extracellular space. Although a target site for subtilisin-like convertases is essential for N-terminal processing, N-terminal processing is not necessary for the assembly of a functional vitelline membrane layer. While C-terminal truncations were tolerated, the removal of N-terminal sequences lead to the production of flaccid, infertile eggs with a soluble, rather than insoluble, vitelline membrane network. We propose that the hydrophobic N-terminal prodomain plays an early and essential role in aligning molecules within the vitelline membrane network, much like hydrophobic domains within elastin drive the assembly and alignment of molecules within elastin-based extracellular matrices.     

A modified chromatin-immunoprecipitation protocol for silkmoth ovarian follicular cells reveals C/EBP and GATA binding modes on an early chorion gene promoter

Standard Chromatin immunoprecipitation protocols have been designed to suit studies performed on cell line cultures or yeast cells growing in liquid cultures. In these cases cross-linking/fixation takes place directly in the growing medium of the cells by the addition of a general fixation reagent. When applied on whole isolated silkmoth follicles, this procedure results in poor release of follicular cells from the basal membrane and lower yield of cross-linked chromatin. We present a modification to the standard protocol, where detachment of follicular cells from the basal membrane of the egg and nuclei isolation precedes formaldehyde-mediated cross-linking. We also discuss application of the modified method for the identification of distinct BmC/EBP and BmGATAβ binding modes on a chorion gene promoter from the Er1.A/B early gene pair.     

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.     

Regulation of matrix metalloproteinase-19 messenger RNA expression in the rat ovary

Matrix metalloproteinases (MMPs) are instrumental in the constant tissue remodeling in the ovary. An induction of MMP-19 mRNA in periovulatory follicles has been reported in mouse ovaries. However, little is known about MMP-19 expression during the follicular and luteal periods or about the ovarian regulation of MMP-19 mRNA expression. We examined the expression pattern of MMP-19 mRNA during various reproductive phases and the periovulatory regulation of MMP-19 mRNA in the rat ovary. In gonadotropin-primed, immature rat ovaries, levels of MMP-19 mRNA transiently increased during both follicular growth and ovulation. The MMP-19 mRNA was localized to the theca-interstitial layer of growing follicles and to the granulosa and theca-interstitial layers of periovulatory follicles. A similar expression pattern of MMP-19 mRNA in periovulatory follicles was observed in ovaries from naturally cycling adult rats. Accumulation of MMP-19 mRNA was detected in regressing corpus luteum. The regulation of MMP-19 mRNA expression during the periovulatory period was investigated via in vivo studies and through in vitro culture studies on follicular cells. The hCG-induction of MMP-19 mRNA was mimicked by treating granulosa cells, but not theca-interstitial cells, from preovulatory follicles with LH or activators of the protein kinase (PK) A or PKC pathways. Cycloheximide blocked the LH- or forskolin-induced MMP-19 mRNA expression, demonstrating the requirement for new protein synthesis. In contrast, blocking activation of the progesterone receptor or prostaglandin synthesis had no effect on the increase in MMP-19 mRNA expression. In conclusion, the induction of MMP-19 mRNA suggests an important role of this proteinase during follicular growth, ovulation, and luteal regression.     

Structural and biochemical analysis of the Leptinotarsa decemlineata (Coleoptera; Chrysomeloidea) crystalline chorionic layer

The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.     

The vitelline membranes of Aedes aegypti and Drosophila melanogaster: A comparative review

Summary

The insect eggshell provides a model system for the study of gene regulation because several proteins are synthesized in an ordered spatial and temporal pattern within a single tissue, the follicular epithelium. Progress is being made towards an understanding of Aedes aegypti and Drosophila melanogaster vitelline membrane formation. The vitelline membrane is the innermost layer of the eggshells of A. aegypti and D. melanogaster. Genes encoding three A. aegypti and four D. melanogaster vitelline membrane proteins have been cloned and sequenced. Significant similarity is observed between the A. aegypti and D. melanogaster vitelline membrane genes. Both families contain highly conserved regions of 34 and 38 amino acids in A. aegypti and D. melanogaster respectively. The protein composition of the two families are both rich in proline and alanine, but differ in their serine and histidine compositions. The regulation of vitelline membrane gene expression in A. aegypti and D. melanogaster is compared.     

Immunolocalization and expression of the steroidogenic acute regulatory protein during the transitional stages of rat follicular differentiation.

This study was designed to determine the pattern of expression and cellular distribution of the steroidogenic acute regulatory protein (StAR) during the transitional stages of follicular differentiation in rat ovary. Using specific antisera against the StAR, immunohistochemistry, Western blotting, and immunoprecipitation analyses provide evidence confirming the localization and expression of StAR in granulosa cells (GCs) of juvenile rat ovaries before and after PMSG treatment. The results also show that StAR expression occurs in theca intersitial cells surrounding preantral, antral, and larger antral follicles in adult diestrous ovaries. Furthermore, we have demonstrated heterogenous StAR immunoreactivity in the granulosa cell layers and cells of the corpora lutea. A novel finding presented here is that, during ongoing growth and differentiation of the follicle, the immunoreactivity of StAR tends to shift from the GC of early antral follicles to the theca cell layers in the adult. The spatiotemporal changes or shifts in StAR expression and cellular localization also coincide with the appearance of more acidic isoforms of the 30-kD protein, as determined by two-dimensional gel electrophoresis. Although the functional implications of these observations remain unclear, the acute temporal changes in StAR expression and localization may not only reflect the dynamic steroidogenic capacity of follicular cells but may also support a possible role for FSH in the induction of follicular maturation.     

Morphology of ovarian changes in the garden lizard, Calotes versicolor

Ovarian changes during the reproductive cycle of the oviparous garden lizard (Calotes versicolor) are described. It ovulates from last week of June to first week of September but most often in July and August when the monsoon occurs. The number of eggs ovulated vary from 10 to 32. After ovulation, the ovaries are reduced in size. From October to May, the ovaries contain small pre-vitellogenic follicles, which increase in size in June when most of yolk deposition occurs. Several nuclei are seen in the ooplasm of pre-vitellogenic follicles; they are finally absorbed before yolk deposition starts. Follicular atresia generally occurs in follicles with polymorphic granulosae, in post-ovulatory ovaries. Presumably interstitial gland cells are formed by the hypertrophy of the theca interna cells of atretic follicles. Pre-ovulatory follicles have highly vascularized thecae and invaginations of the follicular epithelium. After ovulation, the follicle cells hypertrophy to form the luteal cell mass filling the follicular cavity. Fibroblasts, which appear to arise from the theca interna, invade the luteal cell mass and form septa. Capillaries occur in the luteal cell mass.