Factors promoting vitellogenic competence and yolk deposition in the cockroach ovary: Larval-adult transition

Experimental manipulation of juvenile hormone and ecdysone during larval-adult transition in Periplaneta americana provided evidence that during this period the absence of juvenile hormone and the presence of ecdysone is required for ovarian competence.Inhibition of follicle cell DNA synthesis or juvenile hormone administration during larval-adult transition precluded vitellogenic competence, suggesting that follicle cell DNA synthesis is required for ovarian follicles to later engage in yolk deposition, and that the requisite DNA synthesis occurs only when ecdysone is present in the absence of juvenile hormone.     

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.     

Role of follicle stimulating hormone in the induction of hyperplasia during compensatory ovarian hypertrophy

In adult rats, removal of one ovary leads to an acute albeit transient rise in serum follicle stimulating hormone and an increase in the weight of the remaining ovary. In an attempt to correlate the high titre of endogenous follicle stimulating hormone with the changes taking place at the macromolecular level, the phenomenon of compensatory ovarian hypertrophy was studied for one cycle after hemiovariectomy at metoestrus in the adult, cycling female rats derived from the Holtzman strain. The significant finding with respect to hormonal changes was an acute follicle stimulating hormone surge commencing 6h post-unilateral ovariectomy, reaching a maximum at 12 h and declining thereafter, hitherto not reported in the Holtzman strain. Serum luteinizing hormone, prolactin, oestradiol-17β and testosterone remained unaltered while progesterone showed a decline at 6 h after surgery. There was an increase in the number of healthy class III (> 350 μm) follicles with a concomitant drop in atretic class III follicles 24 h post-unilateral ovariectomy. Analysis for DNA, RNA and protein content showed that all three constituents registered a continuous rise in the hypertrophying ovary up to 120h after surgery. When expressed as ?g/mg ovarian weight, the increase in DNA reached a maximum at 24 h and declined thereafter. The kinetics of DNA synthesis was followed by pulse labelling with [³H] thymidine at 18, 24, 36 and 48 h after unilateral ovariectomy. Maximum incorporation occurred at 36 h. Autoradiographic studies showed that the granulosa cells of healthy follicles preferentially incorporated the label. In an extension of this study, it was found that labelling index registered a significant increase following ovariectomy, the maximum being reached at 24 h especially in classIII follicles. The results clearly point out the crucial role of hyperplasia in the response of the contralateral ovary to the surgery and implicate the rise in follicle stimulating hormone as the primary signal for initiation of such a response. This raises the question whether in compensatory ovarian hypertrophy follicle stimulating hormone has a mitogenic role     

RNA SYNTHESIS IN THE MOUSE OOCYTE

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [³H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles.     

Follicle cell polyploidy in Leucophaea maderae: Regulation by juvenile hormone

Microspectrophotometric analysis of Feulgen-stained nuclei of the terminal follicle cells in the cockroach Leucophaea maderae showed that during maturation the follicle cells became polyploid. In virgin females, the follicle cell nuclei were diploid. After mating, and during vitellogenesis, the ploidy of the follicle cells increased from 2 C to 32 C with a small percentage of 64 C nuclei. There was no further increase in the ploidy levels during the chorionic stage of development.Injections of juvenile hormone III into decapitated virgin females elevated the ploidy levels in the follicle cells. The DNA content of these nuclei at 96–120 h after injection of juvenile hormone III increased from 2 C to 4 C. Such polyploidization of nuclei was dose-dependent with the highest DNA content occurring in response to 25–50 μg juvenile hormone III. The juvenile hormone-induced increase in DNA content correlated with an increase in the rate of [³H]thymidine incorporation into DNA.Our data suggest that the role of juvenile hormone in follicle cell development during the vitellogenic period, whether direct or indirect, is to promote selectively a large increase in the DNA content of the cells. This may facilitate the next stage of follicle cell development, choriogenesis.     

Control of follicular epithelium development and vitelline envelope formation in the mosquito; role of juvenile hormone and 20-hydroxyecdysone

Using microsurgical manipulations, hormone applications, and transmission electron microscopy we have investigated the regulation of differentiation of the follicular epithelium and formation of the vitelline envelope (VE) in primary follicles in the ovary of the mosquito, Aedes aegypti. During the first 3 days after eclosion, the primary follicle grows, and cells of the follicular epithelium differentiate, their content of mitochondria, rough endoplasmic reticulum, and Golgi complexes increases significantly. Growth and differentiation of the follicular epithelium appear to be under the control of juvenile hormone (JH), because they are blocked by removal of corpora allata in newly closed adult females and can be restored by either implantation of corpora allata or application of JH III. In insects, including mosquitoes, VE is the first layer of the eggshell to be deposited. It is formed from the secretory products of the follicle cells and its deposition coincides with yolk accumulation by developing oocytes. Only follicle cells adjacent to the oocyte deposit VE. In decapitated females, given a blood meal by enema and injected with picogram doses of 20-hydroxyecdysone (20-HE), follicle cells synthesize the VE precursors and deposit morphologically normal VE, in contrast to saline injected controls which deposit no VE. We conclude that 20-HE, as well as factors originating from the blood meal and the oocyte, are required for the normal formation of VE in the mosquito follicles.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Distribution of yolk polypeptides in the follicle cells during the differentiation of the follicular epithelium in Sarcophaga bullata egg follicles

In S. bullata, the ovaries contribute to the synthesis of yolk polypeptides. A specific antiserum for yolk polypeptides was used to visualize the presence of yolk polypeptides in the follicle cells during their differentiation. After vitellogenesis has started, all follicle cells contain yolk polypeptides. The squamous follicle cells covering the nurse cells and the border cells lose yolk polypeptides before mid-vitellogenesis, whereas the follicle cells over the oocyte contain yolk polypeptides until after late vitellogenesis. All follicle cells are immunonegative afterwards. In vitro translation of poly(A)+ RNA demonstrated that the presence of yolk polypeptide mRNA correlates well with follicle cell immunopositivity for yolk polypeptides. This suggests that the follicle cells synthesize the ovarian yolk polypeptides. Differences in cellular and nuclear morphology, total and poly(A)+ RNA synthesis and the rate of yolk polypeptide synthesis were shown to be correlated with the presence or absence of yolk polypeptides in the differentiating follicular epithelium. The possible relationship between these different aspects of follicle cell differentiation, follicle cell polyploidy and the extracellular current pattern around follicles are discussed.     

Ultrastructural observations of previtellogenic ovarian follicles of dove

Dove ovarian follicle is a complex structure composed of oocyte surrounded by a somatic compartment consisting of theca externa, theca interna and granulosa. The structure of ovarian follicle (1 and 2 mm) of dove was studied by electron microscopy. The granulosa was pseudostratified in the 1-mm-diameter follicles and stratified with two or three irregular rows of cells in the 2-mm-diameter follicles. In the larger follicle indentations between oocyte and granulosa cells become more numerous and the microvilli of granulosa cell elongated to form a zona radiata with similarly elongated oocyte microvilli. Lining bodies were present at the tips of granulosa microvilli and in the cortical region of the oocyte. In the oocyte cortex were observed coated pits, coated vesicles, dense tubules, multivesicular bodies and primordial yolk spheres. Primordial yolk spheres may contain lining bodies and were observed fused with dense tubules and multivesicular bodies or associated with smooth cisternae.     

Nutrient-mediated juvenile hormone secretion in mosquitoes

Our objective was to determine whether environmental conditions affect the juvenile hormone (J.H.) regulated phase of ovarian development of mosquitoes. Primary ovarian follicles of nutrient-deprived adult Aedes aegypti reared in non-crowded cultures remain in an early, teneral stage of development. After a meal of sucrose or blood, follicles of crowded mosquitoes develop to a stage competent to deposit yolk directly following blood ingestion; a topically-applied analogue of J.H. stimulates the ovaries to develop to a similar stage. A. aegypti collected in nature are of a size suggesting that their follicles would remain in the teneral stage when adults are nutrient-deprived.     

Activin A and follicle-stimulating hormone control tight junctions in avian granulosa cells by regulating occludin expression

Within the avian ovarian follicle, the oocyte is surrounded by a monolayer of granulosa cells, which exhibit pronounced epithelial properties. Here we demonstrate the presence of the major tight junction protein occludin in granulosa cells. As shown by immunohistochemistry, occludin localizes to the oocyte-facing granulosa cell surface. Occludin and thus tight junctions are dynamically regulated in a developmental stage-specific manner. Small white follicles, which have not yet started yellow yolk incorporation, show pronounced occludin expression in vitro and in vivo. By contrast, yellow yolk-incorporating small yellow follicles exhibit much lower levels of occludin, and hierarchical, preovulatory follicles are virtually devoid of this essential tight junction component. Using a primary granulosa cell culture system, we demonstrate that concerted action of two well-established ovarian growth regulators, follicle-stimulating hormone and activin A, leads to strong induction of occludin expression in vitro. We suggest that the stage-dependent decrease in the granulosa cell growth factor responsiveness triggers the disruption of tight junctions, enabling rapid and high capacity transport of macromolecules into the oocyte through a paracellular pathway. Such a high-capacity transport for yolk components may represent a crucial prerequisite for rapid oocyte growth once follicles have entered the follicular hierarchy.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

A follicle cell contribution to the yolk spheres of moth oocytes

The incorporation of H(3)-histidine and H(3)-glucosamine by ovarian follicles of cecropia moths during incubation in female blood was followed autoradicgraphically. Labeling was most prominent in the follicle cells, the spaces between these cells, and the nascent yolk spheres in the oocyte cortex. Results of puise-chase experiments and the fact that viable follicle cells were required for normal yolk sphere labeling indicated that the endogenous component of the yolk was provided by the follicle cells via the intercellular spaces. The material was accumulated by the oocyte in the absence of blood proteins, suggesting an independent role in yolk formation.     

Morphology of mouse egg cylinder development in vitro: a light and electron microscopic study.

The endocrine control of yolk deposition in Drosophila melanogaster was studied by ligation and transplantation techniques. Endocrine events associated with the initiation of vitellogenesis were found to be synchronized with eclosion rather than the completion fo adult development. Decapitation experiments showed that a cephalic event occurring at about the time of eclosion is necessary for each animal to initiate vitellogenesis. The morphogenetic effect of the head could be replaced by a juvenile hormone analog (JHA). In addition to the cephalic event, a thoracic factor is required for each follicle to initiate vitellogenesis, since preparation of isolated abdomens before 16 hours after eclosion prevented vitellogenesis. In abdomens isolated after this time, no early vitellogenic stages were formed. The suppression of vitellogenesis in isolated abdomens was reversed by implanting corpora allata or by treating these preparations with JHA, but not by implanting corpora cardiaca. Ovaries that were artificially induced to mature by treating isolated abdomens with JHA still displayed the normal complement of ovarian proteins after electrophoresis in polyacrylamide gels. These results show that a circadian clock triggers vitellogenesis via a cephalic signal at eclosion, which in turn triggers events in the thorax or abdomen. The cephalic signal can be superseded by juvenile hormone, whose presence is necessary for each follicle to become vitellogenic.     

Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (bactrocera) oleae (Diptera: Tephritidae)

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the species Dacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.     

A diet rich in n-3 polyunsaturated fatty acids reduced prostaglandin biosynthesis, ovulation rate, and litter size in mice

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to > 2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.     

Requirement for ADAMTS-1 in extracellular matrix remodeling during ovarian folliculogenesis and lymphangiogenesis

Murine ovarian folliculogenesis commences after birth involving oocyte growth, somatic cell differentiation and structural remodeling of follicle stromal boundaries. The extracellular metalloproteinase ADAMTS-1 has activity against proteoglycans and collagen and is produced by the granulosa cells of ovarian follicles. Mice with ADAMTS-1 gene disruption are subfertile due to an unknown mechanism resulting in severely reduced ovulation. Here we show that ADAMTS-1 is necessary for structural remodeling during ovarian follicle growth. A significant reduction in the number of healthy growing follicles and corresponding follicle dysmorphogenesis commencing at the stage of antrum formation was identified in ADAMTS-1-/- ovaries. Morphological analysis and immunostaining of basement membrane components identified stages of follicle dysgenesis from focal disruption in ECM integrity to complete loss of follicular structures. Cells expressing the thecal marker Cyp-17 were lost from dysgenic regions, while oocytes and dispersed cells expressing the granulosa cell marker anti-mullerian hormone persisted in ovarian stroma. Furthermore, we found that the ovarian lymphatic system develops coincidentally with follicular development in early postnatal life but is severely delayed in ADAMTS-1-/- ovaries. These novel roles for ADAMTS-1 in structural maintenance of follicular basement membranes and lymphangiogenesis provide new mechanistic understanding of folliculogenesis, fertility and disease.     

Exclusion of plasma lipoproteins of intestinal origin from avian egg yolk because of their size

Increasing the fat content of the diet increases the proportion of large triglyceride-rich (TGR) lipoproteins (portomicrons) in laying-hen plasma, but has no effect on the size distribution of yolk TGR-lipoproteins. Electromicrographs of the ovarian follicle walls of hens fed a high-fat diet show the presence of numerous portomicron-like particles in the lumen of the thecal capillaries, in the pericapillary spaces and in the theca interna, but portomicrons were absent from the basal lamina, between the granulosa cells and in newly deposited yolk. Most of the lipoprotein lipase activity in the ovarian follicles is associated with the granulosa cells, but total activity in the follicle is very small compared to heart or adipose tissue. The results indicate that the ovarian follicle of the laying-hen specifically excludes lipoproteins of intestinal origin from yolk, most probably because they are too large to pass through the connective tissue matrix of the basal lamina. The low lipoprotein lipase activity of the ovarian follicle, together with its distribution within the follicle wall, indicates that the ovarian follicles make little contribution to catabolism of circulating portomicrons.     

Corpus allatum control of ovarian development in Aedes aegypti

Ovarian follicles of Aedes aegypti normally pass through a potential stage of arrest 1 day after adult ecdysis and after blood feeding. Removal of the corpora allata or decapitation within an hour of these events interrupts normal development, and development is restored by reimplantation of active allata or by administration of synthetic analogues of juvenile hormone. Only follicles in the normal stage of arrest deposit yolk when stimulated by exogenous ecdysone or after the female takes a meal of blood. Follicles that ceased development as a result of allatectomy or decapitation respond to these oögenic stimuli, but degenerate without depositing yolk.     

Primordial follicle activation and follicular development in the juvenile rabbit ovary

Of all the stages of mammalian folliculogenesis, the primordial to primary follicle transition is the least understood. In order to gain new insights into this process, we have conducted a comprehensive morphological, morphometric and molecular study of ovarian organisation and early follicle development in the rabbit. The structure of ovaries collected from rabbits aged from 2–12 weeks (a period encompassing primordial follicle formation, activation and the first wave of folliculogenesis in this species) has been analysed by light microscopy and the follicles present have been measured and scored for their developmental stage. To establish useful molecular markers of activation, we have further classified follicles according to their expression of the proliferative marker, proliferating cell nuclear antigen, and the zona pellucida protein, ZPB. The activation of primordial follicles is initiated immediately following their formation in the rabbit ovary and is characterised by oocyte growth, granulosa cell morphogenesis and increased granulosa cell mitosis. Enhanced ZPB protein expression at the oolemma is also associated with follicle activation and development. Few primordial follicles in the juvenile rabbit ovary are lost by atresia, as assessed by the TUNEL assay. The appearance of apoptotic granulosa cells is however coincident with the development of antral follicles. This study thus describes the temporal and spatial regulation of early follicular development in the post-natal rabbit ovary and, for the first time, shows that the primordial to primary transition in the juvenile rabbit is a highly ordered process occurring within quantifiable parameters.K.J.H. was supported by the Pest Animal Control CRC and Post Graduate scholarships from the Australian National University.