Electron tomography and computer visualisation of a three-dimensional 'photonic' crystal in a butterfly wing-scale.

A combination of transmission electron tomography and computer modelling has been used to determine the three-dimensional structure of the photonic crystals found in the wing-scales of the Kaiser-I-Hind butterfly (Teinopalpus imperialis). These scales presented challenges for electron microscopy because the periodicity of the structure was comparable to the thickness of a section and because of the complex connectivity of the object. The structure obtained has been confirmed by taking slices of the three-dimensional computer model constructed from the tomography and comparing these with transmission electron microscope (TEM) images of microtomed sections of the actual scale. The crystal was found to have chiral tetrahedral repeating units packed in a triclinic lattice.

Expression of glucosamine trisaccharides on the rat uterine surface is altered by clomiphene citrate. II. Combination with ovarian hormones.

We used a single administration of clomiphene citrate (CC), a synthetic oestrogen that is prescribed for infertility treatment, in combination with either a single administration of oestradiol 17beta (E2) or progesterone (P4) to assess the combined effects of these hormones on the uterine surface. The aim of these experiments was to investigate how CC in combination with these hormones affected both expression of oligosaccharides on the uterine surface and membrane architecture further elucidating CC's agonistic/antagonistic properties. Ovariectomized sexually mature rats were given combinations of E2 and CC (E2 + CC) or P4 and CC (P4 + CC) or P4 and E2 (P4 + E2) and were killed 24 h later. Uterine tissue was labelled with the lectin Phytolacca americana conjugated with avidin and subsequently labelled with biotinylated ferritin and prepared for transmission electron microscopy. Results of the administration of these hormone combinations indicate that CC, when administered in conjunction with E2, had the ability to downregulate expression of oligosaccharides on the membrane surface caused by E2. When administered with P4, CC had the ability to upregulate the effects of P4. Thus, when combined with E2, CC has an antagonistic effect but when combined with P4, CC has an agonistic effect.

Superficial endometrial vascular fragility in Norplant users and in women with ovulatory dysfunctional uterine bleeding.

The aim of this study was to develop an objective test for superficial endometrial vascular fragility at hysteroscopy, and to apply this test to women using Norplant((R)) for contraception and to women with ovulatory dysfunctional uterine bleeding (DUB). A prospective observational study was carried out in 34 Norplant users and 20 women with menorrhagia due to ovulatory DUB. Superficial endometrial vascular fragility was assessed at hysteroscopy by observing the source and extent of frank and subepithelial bleeding during controlled collapse and redistension of the uterine cavity. Superficial endometrial blood vessels were more fragile in Norplant users compared to women with DUB (chi(2) = 11.60, P = 0.02). Superficial endometrial petechiae (chi(2) = 37.9, P < 0.0001) and ecchymoses (chi(2) = 42.2, P = 0.0001) were more frequently observed in the Norplant users than in the menorrhagia group. In Norplant users, superficial endometrial vascular fragility was increased in those with frequent bleeding or spotting during the past 30 days (chi(2) = 6.15, P = 0.01), and in those who were examined during a bleeding episode (chi(2) = 5.3, P = 0.02). Fragility was increased in the menorrhagia group during the perimenstrual period (days 24 to 05; chi(2) = 12.83, P = 0.01). There was no obvious relationship between subepithelial bleeding and circulating concentrations of oestradiol and progesterone.

The measurement of endometrial perfusion in norplant users: a pilot study.

The use of progestogens without oestrogen is commonly associated with irregular menstrual bleeding. Oestrogens and progestogens are both thought to influence endometrial perfusion; changes in endometrial perfusion may contribute to vascular fragility and breakdown. In this study, endometrial perfusion was measured using laser-Doppler fluxmetry in women in the secretory phase of the menstrual cycle before and 4-6 weeks after insertion of the low-dose long-acting levonorgestrel contraceptive implant system, Norplant. Endometrial perfusion was also measured in women exposed to Norplant for up to 19 months. There was no significant difference between endometrial perfusion in control cycles (27.2 flux units +/- 5.5, SEM) and at 4-6 weeks after Norplant insertion (16.3 flux units +/- 5.0), a time when irregular bleeding and spotting are common. Endometrial perfusion was no different from controls after longer periods of Norplant exposure (35.7 flux units +/- 7.2). No direct relationships between endometrial perfusion and plasma concentrations of ovarian steroid hormones were demonstrated. Short-term endometrial vasomotion was largely abolished during Norplant exposure.

Expression of glucosamine trisaccharides on the rat uterine surface is altered by clomiphene citrate.

We have studied histochemically the effects of clomiphene citrate on the expression of oligosacchrides on the apical plasma membrane of uterine epithelial cells using the lectin Phytolacca americana. Ovariectomized sexually mature rats were given a single injection of either clomiphene in two concentrations or estradiol 17 beta or progesterone and were killed 24 hr later. Uterine tissue was labeled with Phytolacca americana conjugated with avidin and subsequently labeled with biotinalyted ferritin and prepared for transmission electron microscopy. Our results indicate that clomiphene and to a lesser degree progesterone significantly increased lectin binding. However, the increase was not as large as that observed with a single dose of estrodiol 17 beta. When the proportion of lectin positivity in relation to total membrane length was analyzed, treatment with clomiphene and progesterone did not have significantly different effects. Low dose clomiphene did not have a significant effect as compared with controls. Our data show that clomiphene has a dose-dependent adverse effect on lectin binding as compared with ovarian hormones. We suggest that these effects contribute to low pregnancy rates with clomiphene use.

Precise measurements of intrauterine vascular structures at hysteroscopy in menorrhagia and during Norplant use.

Using currently available equipment for panoramic hysteroscopy, the size of images viewed cannot be accurately judged because of the magnifying and distorting effects of the objective lens. This study has demonstrated that magnification by the hysteroscope lens can alter the apparent size of images by up to 27%. An additional effect of lens distortion can alter the apparent size of images viewed by up to 28%, depending on the position of the image in the field of view. These effects are independent and may be additive. Thus, the apparent size of intrauterine structures at hysteroscopy may bear little resemblance to their actual size. Image-correction methods are described which reduce the effects of image magnification on the apparent size of an object viewed through the hysteroscope to 7%, and the effect of distortion to 3 %. This technique can greatly improve the accuracy of measurement at hysteroscopy, and has been utilized in this study for the precise measurement of superficial endometrial vascular diameter (mean+/-SEM) in 34 Norplant users (120+/-11.6 microm) and 20 women with spontaneous menorrhagia (74+/-7.2 microm). It has also confirmed the presence of scattered dilated vessels (up to 777 microm in diameter) on the endometrial surface in some Norplant users.

PIP: As a result of the magnification and distortion caused by the fish-eye lens of the panoramic hysteroscope, the size of images cannot be measured accurately. Magnification can alter the apparent size of images by up to 27%, while lens distortion can produce an alteration of up to 28%. These effects are independent and may be additive. The present study applied a new technique for measuring the diameter of intrauterine structures at hysteroscopy to measurement of the superficial vasculature in 34 Norplant users recruited from a family planning clinic in London, England, and in 20 controls diagnosed at the hysteroscopy clinic with ovulatory dysfunctional uterine bleeding. This technique involved advancement of biopsy forceps into the field of view, use of a grid of known internal size, and computer-assisted transformation of the curved image into a flattened one. The mean diameter of superficial dilated vessels in Norplant users (120 mcm) exceeded that in women with menorrhagia (74 mcm). Scattered dilated vessels up to 777 mcm in diameter were observed on the endometrial surface in some Norplant users. The small endometrial polyps observed in Norplant users appeared to have a single fine vessel in the pedicle and were transparent. The image correction technique reduced the effect of image magnification on the apparent size of an object viewed through the hysteroscope to 7% and the effect of distortion to 3%.

Endometrial vasculature in Norplant users: preliminary results from a hysteroscopic study.

Long-acting progestogenic contraceptives are frequently associated with disturbances of menstrual bleeding patterns. In particular, irregular, frequent and prolonged bleeding are commonly seen. The mechanism of this irregular bleeding is unknown, but changes in the endometrial vasculature are thought to be of importance. In endometrial biopsies from Norplant users, an increase in endometrial microvascular density has been observed after 3-12 months. Morphological changes in endometrial capillaries following progestogen exposure have suggested an increase in vascular fragility. Little is known about the structure and function of the endometrial vasculature in vivo following exposure to exogenous contraceptive steroids. This study has developed techniques for the assessment of vascular fragility by imposing a mechanical stress on the endometrium and observing subsequent bleeding under direct vision. The techniques were used in a preliminary examination between 1 and 9 months after Norplant insertion, and the study identified a number of morphological and functional characteristics of Norplant-exposed endometrium.

PIP: Endometrial biopsies collected from Norplant users have revealed an increase in endometrial microvascular density after 3-12 months of use. Moreover, morphologic changes in endometrial capillaries after progestogen exposure have suggested increased vascular fragility. In this study, hysteroscopy, and the attendant imposition of mechanical stress on the endometrium, was used to identify in vivo characteristics of the endometrial vasculature in 34 Norplant acceptors from Australia. A total of 24 women presented for hysteroscopy during a bleeding episode. Superficial vascularity appeared to increase as early as 1 month after Norplant insertion. Women with a relatively avascular endometrial surface experienced less bleeding than those with extensive networks of superficial vessels. Common were areas of dense vascularity adjacent to areas that appeared pale and relatively avascular. Dilated superficial vessels, observed in 1/3 of cases, were associated with more breakthrough bleeding in the previous 30 days. Unexpected was the predominance of petechiae and ecchymoses in the endometrium of Norplant users. Profuse bleeding at deflation and reflation of the uterine cavity was observed in most subjects and was also associated with an increase in breakthrough bleeding. Larger vessels appear to be the site of the breakthrough bleeding. The role of mechanical stress in the initiation of bleeding may be important in spontaneous menstrual bleeding, especially when vessels are made fragile by lack of stromal support due to progestogen-induced endometrial atrophy.

Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep.

Development to the blastocyst stage was assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The proportion of cleaved oocytes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal sheep than for those from adult sheep (7.4% and 24.6% respectively). There were no differences in the metabolism of glucose, glutamine or pyruvate between oocytes matured in vivo and in vitro, or of glucose or pyruvate between oocytes from prepubertal and adult sheep. Glutamine metabolism by mature oocytes from prepubertal sheep was significantly lower than that by oocytes from adult sheep. Ultrastructural studies revealed no differences in the morphology of cytoplasmic organelles of oocytes matured in vitro from prepubertal and adult sheep, but differences in the volume fraction and size of mitochondria and cortical granules were observed. These data suggest that mature oocytes from prepubertal sheep do not possess the developmental potential of their adult-derived counterparts, and this phenomenon may be associated with metabolic and ultrastructural anomalies.

Morphometric comparison of uterine glandular epithelium in the early secretory phase from patients treated with different superovulatory drugs on an in vitro fertilization programme.

The glandular structure in the uterine epithelium of three groups of patients on standard superovulatory regimes was studied using a fully automated image analysis system. It was found that one treatment (buserelin stimulation) produced more glandular area than either of the other two. We suggest that buserelin gives the morphometric appearance of the most normal endometrial glands and find that our morphometric analysis is a reliable means to evaluate glandular structure in patients on superovulatory therapy.

Increase in cholesterol in the apical plasma membrane of uterine epithelial cells during early pregnancy in the rat.

Freeze-fracture cytochemistry with the cholesterol-binding antibiotic filipin has been used to examine the plasma membrane of uterine epithelial cells at different stages of pregnancy in the rat. We find many more filipin-induced lesions on day 6 of pregnancy than on day 1 and suggest that this indicates a higher cholesterol content at this time. Since day 6 of pregnancy is the time at which blastocysts implant in the rat uterus, we consider the possible significance of an increased cholesterol content for implantation.

Glycerol increases the cytochemical detectability of cholesterol in the apical plasma membrane of uterine epithelial cells.

The apical plasma membrane of uterine epithelial cells in the rat has been treated with glycerol before fixation and then examined by freeze-fracture cytochemistry using digitonin and filipin. Many more lesions were produced by both cytochemicals following glycerol treatment than in untreated controls, and we suggest that this indicates an increased detectability of cholesterol. We consider the implications of the findings for the way in which glycerol acts on membranes and propose that glycerol promotes increased binding between cholesterol and the cytochemicals.

Calcium ionophore A23187 as a probe for freeze-fracture studies of membrane changes in the head of human spermatozoa.

The membranes of the head of human spermatozoa were examined after incubating the sperm with and without inophore A23187 and calcium ions, using freeze-fracture electron microscopy. After exposure to ionophore and calcium there was a remarkable rearrangement of the intramembraneous particles, especially in the plasma membrane. Buckling of the plasma membrane occurred prior to breaking away from the outer region of the sperm head. The outer acrosomal membrane bubbled and broke down to form vesicles, and blebbing of the inner acrosome membrane also occurred. The nuclear envelope degenerated and often displayed an undulating topography.