Morphometric and ultrastructural features of the mare oviduct epithelium during oestrus

Morphometric, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) investigations have displayed regional differences in the mare oviductal epithelium. The entire mucosa of the oviduct was lined with a pseudostratified epithelium, which consisted of two distinct cell types, ciliated and non-ciliated. Ciliated cells were predominant in the three different segments of the oviduct and their percentage increased from fimbriae to ampulla and significantly decreased in the isthmus. SEM revealed in the infundibulum finger-like mucosal folds, some of them interconnected, in the ampulla numerous and elaborated branched folds of the mucosa, whereas the isthmus displayed a narrow lumen, short and non-branched mucosal folds. In the ampulla and isthmus the majority of non-ciliated cells showed apical blebs provided or not of short microvilli. TEM displayed different ultrastructural features of ciliated and non-ciliated cells along the oviduct. Isthmus ciliated cells presented a more electron-dense cytoplasm than in infundibulum and ampulla cells and its cilia were enclosed in an amorphous matrix. The non-ciliated cells of infundibulum did not contain secretory granules but some apical endocytic vesicles and microvilli coated by a well developed glycocalyx. Non-ciliated cells of ampulla and isthmus contained secretory granules. Apical protrusions of ampulla displayed two types of secretory granules as well as occasional electron-lucent vesicles. Isthmus non-ciliated cells showed either electron-lucent or electron-dense cytoplasm and not all contained apical protrusions. The electron-dense non-ciliated cells displayed microvilli coated with a well developed glycocalyx. Three types of granules were observed in the isthmus non-ciliated cells. The regional differences observed along the epithelium lining the mare oviduct suggest that the epithelium of the each segment is involved in the production of a distinctive microenvironment with a unique biochemical milieu related to its functional role.     

Effects of azadirachtin on oogenesis in Aedes aegypti

ABSTRACT. . Azadirachtin in blood fed to adult female Aedes aegypti through an artificial membrane does not cause feeding inhibition over a wide dose range (0–200 ng/female), and high doses of ingested azadirachtin fail to inhibit or delay oviposition. However, significant, transient retardation of oocyte growth is observed for up to 72 h after feeding. Immature oocytes are observed in 86% of azadirachtin-fed females decapitated 10 h after a blood meal, whereas 96% of decapitated control females contain maturing oocytes. This suggests that azadirachtin delays the release of one or more factors from the head that regulate oogenesis. We propose that adult females overcome the effect of azadirachtin by rapid metabolism rather than by excretion of the compound, since by 2 h after a blood meal, only 0.1% of ingested azadirachtin was recovered from excreta and 5% recovered from the body.     

Immunocytochemical localization of catechol-O-methyltransferase in the oviduct and in macrophages in corpora lutea of rat

Summary Catechol-O-methyltransferase (COMT) (EC 2.1.1.6) was localized in rat ovary, oviduct, and uterus using immunocytochemical methods. Immunoreactive deposits were found in the cytoplasm of macrophages in the ovary, epithelial cells of the oviduct, and glandular epithelial cells of the non-pregnant uterus. The pattern of localization observed in the extraneuronal elements suggests that enzyme may function in extraneuronal inactivation of catechols in the ovary, oviduct, and uterus.     

Chemosterilant (apholate)-induced ultrastructural changes during oogenesis in Aedes aegypti.

The effect of chemosterilant, apholate, on-oogenesis has been studied in Aedes aegypti. Treatment of larvae to 20 ppm of the chemical induced ultrastural changes in the presumptive and primary follicles of the adult ovary. These changes comprised condensation of chromatin, disruption of nuclear envelope and extensive degeneration as evidenced by numerous myelin figures and residual bodies. In some primary follicles, where cellular degeneration was restricted to epithelial cells, no arrest in development was observed. However, in comparison with controls, these follicles also were retarded. Larval treatment with 30 ppm apholate completely supressed ovariolar development. High incidence of autophagy was observed in tissues at both dose levels.     

Functional morphology of the midgut of Aedes aegypti L. (Insecta,Diptera) during blood digestion

Morphometric analysis of the epithelial lining of the stomach of A. aegypti suggests that digestion of the first blood meal in the stomach of this species can be viewed as a series of phases that can be correlated with physiological data from the literature. In phase Ia (0-10 h after blood meal [abm]) the whorls of the rough endoplasmic reticulum unfold, the Golgi zones increase, and the basal labyrinth is enlarged. This coincides with processes of synthesis and secretion (e.g., peritrophic membrane, esterases and lipases) and transport by the stomach epithelium. In phase Ib (10-20 habm) the cellular parameters measured further increase, indicating high synthetic and secretory activities (e.g., digestive enzymes). In phase Ic (20-30 habm) cell structures involved in synthesis and secretion still exhibit high values coinciding with maximal activity of proteases in the gut. Enhanced surface area of microvilli, prominent lipid inclusions, and appearance of glycogen deposits in the gut epithelium suggest increased absorption, storage, and transport functions of the stomach cells. In phase II (30-36 habm) structural alteration points to a gradual shift from synthesis and secretion to absorption, partial storage, and transport of nutrients. In phase III (36-72 habm) the cellular apparatus is reduced concomitant with the ending of the digestive cycle. Lipid inclusions and glycogen deposits disappear from the stomach epithelium.     

Sequential deposition of yolk components during oogenesis in an insect,Aedes aegypti (Diptera: Culicidae)

Vitellogenesis in Aedes aegypti of uniform body size was followed at 27 degrees C in narrow time intervals throughout their first reproductive cycle by measuring the length, diameter, and volume of follicles and oocytes, the latter as an expression of the yolk mass (vitellus). Independent of all experimental conditions, a two-step process of elongation was recognized for both follicle length and yolk length, so that growth curves were consistently composed of two linear regressions with different slopes against time. Follicle lengths started to increase immediately after the blood meal, while oocytes took up to 6 h to show a measurable increase in yolk length. The first linear phase continued until 30 h, when yolk length reached 268+/-22 micro m. At this point, a transition occurred where the linearity shifted sharply for the next 6 h to 2-4-times higher slopes for both regressions. This second growth phase represented a 40% elongation of oocytes and follicles. Then, both curves leveled off at their final size, characteristic of mature ovaries: 462+/-10 micro m for oocytes, 489+/-11 micro m for follicles. These values remained constant until oviposition.The first linear growth phase was associated with an equicaloric and synchronous protein and lipid incorporation into the oocytes; levels of these substances reached their maximum by the end of this first phase and remained constant until oviposition. The second linear growth phase was characterized by rapid glycogen incorporation into oocytes from 20 to 100% of the maximum. Subsequently, the surface pattern of the exochorion became visible, marking the end of yolk incorporation. Since eggs are always laid on moist substrates, within 2-3 h of oviposition they double in volume and fresh weight, driven by more than tripling of their water content.When blood-fed females were exposed to five different temperatures between 17 and 37 degrees C, the distinction between the two linear growth phases persisted, but the slopes of the respective regressions, and therefore their durations, were affected. Eggs still matured at 37 degrees C but never hatched and at 12 degrees C only 18% hatched, whereas at all the intermittent temperatures hatching was 80-90%. Oogenesis appears to be limited to the range between 12 and about 32 degrees C.The effects of age, maternal body size and the source of the blood on vitellogenesis were also examined. These parameters affected the onset and/or extent of oogenesis in various ways.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Histological changes and transglutaminase 2 expression in the oviduct of advanced pregnant cows

The oviduct is a dynamic organ that has not been assigned specific functions during advanced pregnancy. However, since changes in the oviductal epithelium during the estrous cycle are attributed mainly to variations in estradiol (E2) levels, and E2 levels increase along pregnancy, we hypothesized that advanced pregnant cows should present changes in the oviductal epithelium. In advanced pregnant cows, the oviducts showed higher leaf-like folds and lower mucosa width and epithelium height than those of cycling animals. Also, PAS-positive apical protrusions and TUNEL-positive extruded cytoplasmic material were observed in advanced pregnant cows. Oviductal fluid from advanced pregnant cows showed lower protein concentration than that from cycling cows. Transglutaminase 2 (TG2) was detected exclusively in oviductal fluid of pregnant cows but not in cells from any stage, whereas its mRNA was detected in different amounts in cells from all stages. This protein was identified by LC/MS-MS and its identity was corroborated by Western blot. The observations in histology of the epithelium and the presence of TG2 in oviductal fluid correlate with high levels of E2 in serum. In conclusion, important histological changes in the oviductal epithelium and secretion of TG2 to the oviductal fluid appear to be triggered by the high E2 levels exclusive of advanced pregnancy.     

Ultrastructural changes in the oviduct of the laying hen during the laying cycle

The ultrastructural changes occurring in the fully functional oviduct of Isa Brown laying hens were studied during various stages of the laying cycle. Hens were killed at different positions of the egg in the oviduct. The oviduct was lined by ciliated and non-ciliated cells (also referred to as granular cells). The granular cells in the infundibulum contributed to secretion during egg formation, whereas ciliated cells showed little evidence of secretion. Ultrastructural changes were recorded in the granular and glandular cells of the distal infundibulum. In the magnum, the surface ultrastructure revealed glandular openings associated with the ciliated and granular cells. Cyclic changes were recorded in the glandular cells of the magnum. With respect to the three observed types of glands, the structure of gland type A and C cells varied at different egg positions in the oviduct, whereas type B cells represented a different type of gland cell containing amorphous secretory granules. The surface epithelium of the isthmus was also lined by mitochondrial cells. Two types of glandular cell (types 1 and 2) were recorded in the isthmus during the laying cycle. Intracisternal granules were found in type 2 cells of the isthmus. A predominance of glycogen particles occurred in the tubular shell gland. The granular cells in the shell gland contain many vacuoles. During egg formation, these vacuoles regressed following the formation of extensive rough endoplasmic reticulum; the reverse also occurred. The disintegrated material found in the vacuoles may have been derived from the disintegrating granules. The Physiology Teaching Unit, University of New England, provided financial support to K. Chousalkar for this study.     

In vitro optimization of the Gallus domesticus oviduct epithelial cells culture

The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells research as this work generates questions about undocumented sources of oviduct progenitor cells.     

Ultrastructure and morphology of midgut visceral muscle in early pupal Aedes aegypti mosquitoes

These studies focus on the pupal Aedes aegypti midgut muscularis for the first 26 h following larval-pupal transition. The midgut muscularis of Ae. aegypti pupae during this first half of the pupal stadium is a grid of both circularly and longitudinally oriented muscle bands, arranged in a manner resembling that of the larvae. While many muscle bands exhibit signs of degeneration during the time period studied, not all bands degrade, nor is this degradation simultaneous. Band deterioration involves destruction of internal elements while the muscle fiber plasma membrane remains intact. Deterioration of contractile elements may involve proteosome-like structures and associated enzymes. Many features of the larval muscularis including cruciform cells, bifurcating circular bands, and bifurcating longitudinal bands of muscle are retained during the time period investigated. Neuromuscular junctions along some muscle bands are retained through at least 16 h into the pupal stadium. The selective nature of muscle fiber degradation, coupled with the retention of larval features and neural input, may allow for limited functionality of the muscularis during metamorphosis. Evidence of sexual dimorphism in the midgut muscularis of male and female Ae. aegypti pupae was not observed during the time period studied.     

Ultrastructural changes in the fat body of adult female Aedes aegypti in relationship to vitellogenin synthesis

Summary The ultrastructure of the fat body of Aedes aegypti was followed from emergence through a blood meal. Changes in the volume of protein granules and lipid droplets were also examined. The relationship of these events to the known endocrinology of vitellogenin synthesis in mosquitoes is discussed.Massachusetts Experiment Station Paper No. 2130. Supported by the Massachusetts Agricultural Experiment Station, Project n° 356, and by NIH grant n° AI-11909     

Progesterone receptor in the liver and oviduct of the lizard Podarcis sicula

In this paper we report the presence of a (3)H-Progesterone ((3)H-P) binding moiety, which has the characteristics of a true receptor, in the liver of the female of the lizard Podarcis sicula. (3)H-P binding studies show the presence of one type of binding site with an average Kd value of 6.2 +/- 2.0 nM in the cytoplasm and 6.3 +/- 1.1 nM in the nucleus. Competition experiments showed that progesterone (P) was the best competitor, while testosterone, deoxycorticosterone (DOC), corticosterone, 17 alpha-hydroxyprogesterone; R5020; RU486 and RU26988-5 were poor competitors. We have also investigated the immunological characteristics of progesterone receptor (PR) in both the liver and the oviduct of Podarcis sicula, by Western blotting using the monoclonal antibody PR22 raised against the PR isoforms A and B of chicken. One imunoreactive band of about 70 kDa was detected in cytoplasmic and nuclear extracts of both the liver and the oviduct. PR immunoreactivity was present in the liver during the quiescent phase. In the oviduct PR immunoreactivity increased from the recovery to the full grown phase. P treatment of estrogen-primed females did not affect the presence of PR in the liver, while brought about a PR increase in the oviduct. This study suggests that PR is expressed differently in the liver and the oviduct of Podarcis sicula throughout the reproductive cycle. PR might fulfill different requirements in relation to the different physiological functions of the tissue during the reproductive cycle.     

Organization, ultrastructure, and development of midgut visceral muscle in larval Aedes aegypti

The midgut muscularis of larvae of the mosquito Aedes aegypti takes the form of a grid of longitudinal and circular muscle bands. The longitudinal and circular bands overlap at near right angles at many areas of intersection. The longitudinal bands run the length of the midgut. However, some bands of circular muscle, located in the anterior midgut, pass only partway around the gut. An unusual feature was observed at some regions where longitudinal and circular bands of muscle intersect: filaments oriented at near right angles to one another were present in the same membrane-bound fiber. These cruciform regions send contractile elements into both circular and longitudinal bands. The muscularis was fixed in a contracted state, so most of the sarcomeres are represented by complete overlap of myosin and lighter staining actin filaments. Features characteristic of supercontracting muscle, including perforated Z-lines, were seen in sarcomeres of circular muscle bands. Small invaginations resembling transverse tubules were present but a sarcoplasmic reticulum was not observed. While occasional cells that may be neurons or neurosecretory cells were observed, a network that might serve to coordinate the segmentation and peristaltic movement of the muscularis was not apparent.     

Ultrastructural comparison of smooth muscle from ovarian follicle and oviduct of the golden hamster

Summary Ultrastructural characteristics of smooth muscle taken from ovarian follicles and oviducts of hamsters are compared. Differences between the two muscle types are more quantitative than qualitative, thus confirming that follicular muscle is a true smooth muscle with no unique characteristics. While both muscle types contain 50–80 Å filaments, -glycogen deposits, and organelles characteristically found in smooth muscle, the oviductal cells have substantially more sacs, tubular structures, sarcoplasmic reticulum, and mitochondria. Another difference concerns the cellular junctions; the oviductal cells exhibit nexuses, whereas the follicular cells show desmosomelike junctions. Based on ultrastructural differences, follicular smooth muscle seems to be a relatively toneless muscle suited for short, infrequent contractions, whereas oviductal smooth muscle is probably involved in more active tonic contractions.Supported by an Institutional Research Grant from Texas Women's University, by NIH Grant HD 12988, and by the Department of Anatomy at Wright State University     

Changes in ribonuclease activity during development of the mosquito, Aedes aegypti

In the mosquito Aedes aegypti, quantitative and qualitative changes have been detected in ribonuclease activity during development. Ribonuclease activity is particularly high in extracts from larvae, relative to that in extracts from pupae or adults. Larval extract is enriched for a ribonuclease that is heat-labile, has an alkaline pH optimum, and is extremely sensitive to the divalent cation, manganese. Extract from adult females is enriched for a heat-stable component that has an acidic pH optimum and is more active at 56 than at 30 degrees C. Throughout the vitellogenic cycle, no major changes in ribonuclease activity were detected in fat body extracts.